Sample records for tabacum plants transformed
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Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.
2016-01-01
This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217
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Moss is a key nurse plant for reintroduction of the endangered herb, Primulina tabacum Hance.
Hai Ren; Guohua Ma; Qianmei Zhang; Qinfeng Guo; Jun Wang; Zhengfeng Wang
2010-01-01
The rare and endangered plant Primulina tabacum is a calciphilous perennial herb found only at the entrances of a small number of karst cave drainages in southern China. In a conservation effort, we identified potentially suitable habitats and reintroduced P. tabacum plantlets (propagated in vitro) to one historical and two new cave entrances. The transplanted...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Altabella, T.; Chrispeels, M.J.
Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less
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Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.
Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.
1997-08-01
On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential.
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Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.
Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.
1997-01-01
On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential. PMID:12223765
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USDA-ARS?s Scientific Manuscript database
Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...
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Chen, Shih-Cheng; Liu, Hui-Wen; Lee, Kung-Ta; Yamakawa, Takashi
2007-01-01
The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 microM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42 degrees C heat treatment, and the expressed GUS specific activity was 7-26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.
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Mercx, Sébastien; Smargiasso, Nicolas; Chaumont, François; De Pauw, Edwin; Boutry, Marc; Navarre, Catherine
2017-01-01
Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells. PMID:28396675
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Mercx, Sébastien; Smargiasso, Nicolas; Chaumont, François; De Pauw, Edwin; Boutry, Marc; Navarre, Catherine
2017-01-01
Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N -glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker ( bar ), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells.
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Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.
Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc
2016-01-01
Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.
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Ganapathy, Mathangi; Chakravarthi, M; Charles, S Jason; Harunipriya, P; Jaiganesh, S; Subramonian, N; Kaliraj, P
2015-08-01
Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.
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Ryan, Annette C; Hewitt, C Nicholas; Possell, Malcolm; Vickers, Claudia E; Purnell, Anna; Mullineaux, Philip M; Davies, William J; Dodd, Ian C
2014-01-01
Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.
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Golemboski, D B; Lomonossoff, G P; Zaitlin, M
1990-01-01
Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected. Images PMID:2385595
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Colgan, Richard; Atkinson, Christopher J; Paul, Matthew; Hassan, Sally; Drake, Pascal M W; Sexton, Amy L; Santa-Cruz, Simon; James, David; Hamp, Keith; Gutteridge, Colin; Ma, Julian K-C
2010-04-01
Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth
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Zhang, Long; Rylott, Elizabeth L; Bruce, Neil C; Strand, Stuart E
2017-09-01
Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT. The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.
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Giles, Courtney D; Richardson, Alan E; Cade-Menun, Barbara J; Mezeli, Malika M; Brown, Lawrie K; Menezes-Blackburn, Daniel; Darch, Tegan; Blackwell, Martin Sa; Shand, Charles A; Stutter, Marc I; Wendler, Renate; Cooper, Patricia; Lumsdon, David G; Wearing, Catherine; Zhang, Hao; Haygarth, Philip M; George, Timothy S
2018-03-02
Citrate and phytase root exudates contribute to improved phosphorus (P) acquisition efficiency in Nicotiana tabacum (tobacco) when both exudates are produced in a P deficient soil. To test the importance of root intermingling in the interaction of citrate and phytase exudates, Nicotiana tabacum plant-lines with constitutive expression of heterologous citrate (Cit) or fungal phytase (Phy) exudation traits were grown under two root treatments (roots separated or intermingled) and in two soils with contrasting soil P availability. Complementarity of plant mixtures varying in citrate efflux rate and mobility of the expressed phytase in soil was determined based on plant biomass and P accumulation. Soil P composition was evaluated using solution 31 P NMR spectroscopy. In the soil with limited available P, positive complementarity occurred in Cit+Phy mixtures with roots intermingled. Root separation eliminated positive interactions in mixtures expressing the less mobile phytase (Aspergillus niger PhyA) whereas positive complementarity persisted in mixtures that expressed the more mobile phytase (Peniophora lycii PhyA). Soils from Cit+Phy mixtures contained less inorganic P and more organic P compared to monocultures. Exudate-specific strategies for the acquisition of soil P were most effective in P-limited soil and depended on citrate efflux rate and the relative mobility of the expressed phytase in soil. Plant growth and soil P utilization in plant systems with complementary exudation strategies are expected to be greatest where exudates persist in soil and are expressed synchronously in space and time. This article is protected by copyright. All rights reserved.
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Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike
2016-04-01
Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.
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Nicotiana tabacum as model for ozone - plant surface reactions
NASA Astrophysics Data System (ADS)
Jud, Werner; Fischer, Lukas; Wohlfahrt, Georg; Tissier, Alain; Canaval, Eva; Hansel, Armin
2015-04-01
Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. The ensuing injuries have been related to the uptake of ozone through the stomatal pores and oxidative effects damaging the internal leaf tissue. A striking question of current research is the environment and plant specific partitioning of ozone loss between gas phase, stomatal or plant surface sink terms. Here we show results from ozone fumigation experiments using various Nicotiana Tabacum varieties, whose surfaces are covered with different amounts of unsaturated diterpenoids exuded by their glandular trichomes. Exposure to elevated ozone levels (50 to 150 ppbv) for 5 to 15 hours in an exceptionally clean cuvette system did neither result in a reduction of photosynthesis nor caused any visible leaf damage. Both these ozone induced stress effects have been observed previously in ozone fumigation experiments with the ozone sensitive tobacco line Bel-W3. In our case ozone fumigation was accompanied by a continuous release of oxygenated volatile organic compounds, which could be clearly associated to their condensed phase precursors for the first time. Gas phase reactions of ozone were avoided by choosing a high enough gas exchange rate of the plant cuvette system. In the case of the Ambalema variety, that is known to exude only the diterpenoid cis-abienol, ozone fumigation experiments yield the volatiles formaldehyde and methyl vinyl ketone (MVK). The latter could be unequivocally separated from isomeric methacrolein (MACR) by the aid of a Selective Reagent Ion Time-of-Flight Mass Spectrometer (SRI-ToF-MS), which was switched every six minutes from H3O+ to NO+ primary ion mode and vice versa. Consistent with the picture of an ozone protection mechanism caused by reactive diterpenoids at the leaf surface are the results from dark-light experiments. The ozone loss obtained from the
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Xu, Bingfang; Copolla, Michael; Herr, John C; Timko, Michael P
2007-01-01
The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant
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Vera-Estrella, Rosario; Gómez-Méndez, María F; Amezcua-Romero, Julio C; Barkla, Bronwyn J; Rosas-Santiago, Paul; Pantoja, Omar
2017-09-01
Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H + exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl 2 and ZnSO 4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl 2 or 1-mM ZnSO 4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H + transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd 2+ or Zn 2+ /H + exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.
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Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.
Lyubenova, Lyudmila; Nehnevajova, Erika; Herzig, Rolf; Schröder, Peter
2009-07-01
Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the
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Boonyapookana, Benjaporn; Parkpian, Preeda; Techapinyawat, Sombun; DeLaune, R D; Jugsujinda, Aroon
2005-01-01
The ability of three plant species: Helianthus annuus, Nicotiana tabacum, and Vetiveria zizanioides for phytoaccumulation of Pb was studied. Plants were grown in hydroponic solution containing Pb(NO3)2 at concentration of 0.25 and 2.5 mM Pb in the presence or absence of chelating agents (EDTA or DTPA). Lead (Pb) transport and localization within the tissues of the plant species was determined using scanning electron microscope equipped with energy dispersive X-ray spectrometers (SEM-EDS). The addition of chelators increased Pb uptake as compared to plants grown in solution containing Pb alone. Lead taken up by the plant species were concentrated in both leaf and stem at the region of vascular bundles with greater amounts in the leaf portion. Lead granules were also found in the H. annuus root tissue from the epidermis layer to the central axis. After four weeks of growth a 23-fold increase in shoot Pb content for H. annuus and N. tabacum and 17-fold increase in shoot Pb for V. zizanioides resulted from plants grown in the 2.5 mM Pb-EDTA treatment. The higher Pb treatment (2.5 mM Pb containing EDTA) resulted in higher concentrations of Pb in plant tissue at the fourth week of exposure as compared to Pb treatment containing DTPA. Overall, Pb accumulation potential of H. annuus was greater than that of N. tabacum and V. zizanioides as indicated by the bioconcentration factor (171, 70, and 88, respectively). The highest measured Pb concentrations were found in H. annuus roots, stems, and leaves (2668, 843, and 3611 microg/g DW, respectively) grown in the 2.5 mM Pb-EDTA treatment. The addition of chelators caused some reduction in plant growth and biomass. Results showed that the three plant species tested have potential for use in phytoaccumulation of Pb since the Pb was concentrated in leaf and stem as compared to control plants. H. annuus however best meet the prerequisites for a hyperaccumulator plant and would have the potential for use in the restoration of
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Toussaint, Frédéric; Pierman, Baptiste; Bertin, Aurélie; Lévy, Daniel; Boutry, Marc
2017-05-04
Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1 mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
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Hai Ren; Qianmei Zhang; Zhengfeng Wang; Qinfeng Guo; June Wang; Nan Liu; Kaiming Liang
2010-01-01
The distribution of the rare and endangered perennial herb Primulina tabacum Hance is restricted to eight karst caves in southern China. To conserve P. tabacum and to evaluate possible reintroduction, we studied its historical distribution and conducted field surveys of both its biotic and physical environment. We used detrended...
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Huchelmann, Alexandre; Gastaldo, Clément; Veinante, Mickaël; Zeng, Ying; Heintz, Dimitri; Tritsch, Denis; Schaller, Hubert; Rohmer, Michel; Bach, Thomas J.; Hemmerlin, Andréa
2014-01-01
S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway–dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism. PMID:24367019
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Efficient Plastid Transformation in Arabidopsis.
Yu, Qiguo; Lutz, Kerry Ann; Maliga, Pal
2017-09-01
Plastid transformation is routine in tobacco ( Nicotiana tabacum ) but 100-fold less frequent in Arabidopsis ( Arabidopsis thaliana ), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2 , encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded ACC2 -defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance ( aadA ) gene and gfp , encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops. © 2017 American Society of Plant Biologists. All Rights Reserved.
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NASA Astrophysics Data System (ADS)
Neubert, A.; Kley, D.; Wildt, J.; Segschneider, H. J.; Förstel, H.
The uptake of NO, NO 2 and O 3 by sunflowers ( Helianthus annuus L. var. giganteus) and tobacco plants ( Nicotiana tabacum L. var. Bel W3), using concentrations representative for moderately polluted air, has been determined by gas exchange experiments. Conductivities for these trace gases were measured at different light fluxes ranging from 820 μEm -2s -1 to darkness. The conductivities to water vapor and the trace gases are highly correlated. It is concluded that the uptake of NO, NO 2 and O 3 by sunflowers and tobacco plants is linearly dependent on stomatal opening. While the uptake of NO is limited by the mesophyll resistance, the uptake of NO 2 is only by diffusion through the stomata. Loss processes by deposition to the leaf surfaces are more pronounced for O 3 than for NO and NO 2.
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Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C
1994-01-01
We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.
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Transformation of medicinal plants using Agrobacterium tumefaciens.
Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata
2016-12-20
For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.
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Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens
DOE Office of Scientific and Technical Information (OSTI.GOV)
Muller, A.; Manzara, T.; Lurquin, P.F.
1984-09-17
Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciensmore » T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.« less
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Genetic transformation of mature citrus plants.
Cervera, Magdalena; Juárez, José; Navarro, Luis; Peña, Leandro
2005-01-01
Most woody fruit species have long juvenile periods that drastically prolong the time required to analyze mature traits. Evaluation of characteristics related to fruits is a requisite to release any new variety into the market. Because of a decline in regenerative and transformation potential, genetic transformation procedures usually employ juvenile material as the source of plant tissue, therefore resulting in the production of juvenile plants. Direct transformation of mature material could ensure the production of adult transgenic plants, bypassing in this way the juvenile phase. Invigoration of the source adult material, establishment of adequate transformation and regeneration conditions, and acceleration of plant development through grafting allowed us to produce transgenic mature sweet orange trees flowering and bearing fruits in a short time period.
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Piano, Dario; El Alaoui, Sabah; Korza, Henryk J; Filipek, Renata; Sabala, Izabela; Haniewicz, Patrycja; Buechel, Claudia; De Sanctis, Daniele; Bochtler, Matthias
2010-12-01
Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.
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Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A
2015-01-01
Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.
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Physical methods for genetic plant transformation
NASA Astrophysics Data System (ADS)
Rivera, Ana Leonor; Gómez-Lim, Miguel; Fernández, Francisco; Loske, Achim M.
2012-09-01
Production of transgenic plants is a routine process for many crop species. Transgenes are introduced into plants to confer novel traits such as improved nutritional qualities, tolerance to pollutants, resistance to pathogens and for studies of plant metabolism. Nowadays, it is possible to insert genes from plants evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. The objective of this article is to review the state of the art of the physical methods used for genetic plant transformation and to describe the basic physics behind them.
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Lactoferrin-derived resistance against plant pathogens in transgenic plants.
Lakshman, Dilip K; Natarajan, Savithiry; Mandal, Sudhamoy; Mitra, Amitava
2013-12-04
Lactoferrin (LF) is a ubiquitous cationic iron-binding milk glycoprotein that contributes to nutrition and exerts a broad-spectrum primary defense against bacteria, fungi, protozoa, and viruses in mammals. These qualities make lactoferrin protein and its antimicrobial motifs highly desirable candidates to be incorporated in plants to impart broad-based resistance against plant pathogens or to economically produce them in bulk quantities for pharmaceutical and nutritional purposes. This study introduced bovine LF (BLF) gene into tobacco ( Nicotiana tabacum var. Xanthi), Arabidopsis ( A. thaliana ) and wheat ( Triticum aestivum ) via Agrobacterium -mediated plant transformation. Transgenic plants or detached leaves exhibited high levels of resistance against the damping-off causing fungal pathogen Rhizoctonia solani and the head blight causing fungal pathogen Fusarium graminearum . LF also imparted resistance to tomato plants against a bacterial pathogen, Ralstonia solanacearum . Similarly, other researchers demonstrated expression of LF and LF-mediated high-quality resistance to several other aggressive fungal and bacterial plant pathogens in transgenic plants and against viral pathogens by foliar applications of LF or its derivatives. Taken together, these studies demonstrated the effectiveness of LF for improving crop quality and its biopharming potentials for pharmaceautical and nutritional applications.
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Navarre, Catherine; Smargiasso, Nicolas; Duvivier, Laurent; Nader, Joseph; Far, Johann; De Pauw, Edwin; Boutry, Marc
2017-06-01
Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.
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Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.
2015-01-01
Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504
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NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.
Ducos, Eric; Fraysse, Staffan; Boutry, Marc
2005-12-19
In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family.
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Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L
2014-10-01
Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.
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Srinivasan, C; Liu, Zongrang; Scorza, Ralph
2011-04-01
Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.
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Acharya, Komal P; Shilpkar, Prateek
2016-03-01
Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.
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Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants
Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.
2001-01-01
Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962
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Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik
2016-01-01
Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746
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Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.
Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S
2006-01-01
This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.
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Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik
2016-04-01
Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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Liu, Hongshuo; Marubashi, Wataru
2014-01-01
Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis. PMID:24806486
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Pavlíková, Daniela; Zemanová, Veronika; Procházková, Dagmar; Pavlík, Milan; Száková, Jiřina; Wilhelmová, Naďa
2014-02-01
Increased endogenous plant cytokinin (CK) content through transformation with an isopentyl transferase (ipt) gene has been associated with improved plant stress tolerance. The objective of this study is to determine amino acid changes associated with elevated CK production in ipt transgenic tobacco (Nicotiana tabacum L., cv. Wisconsin 38). Nontransformed (WT) and transformed tobacco plants with ipt gene controlled by senescence-activated promoter (SAG) were exposed to zinc soil contamination (tested levels Zn1=250, Zn2=500, Zn3=750 mg kg(-1) soil). The Zn effect on plant stress metabolism resulted in changes in levels of selected free amino acids playing an important role in adaptation to stress and plant senescence (alanine, leucine, proline, methionine and γ-aminobutyrate) and differed for transformed and nontransformed tobacco plants. Analyses of amino acids confirmed that SAG tobacco plants had improved zinc tolerance compared with the WT plants. The enhanced Zn tolerance of SAG plants was associated with the maintenance of accumulation of proline, methionine and γ-aminobutyrate. The concentrations of leucine and alanine did not show significant differences between plant lines. © 2013 Published by Elsevier Inc.
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Plants having modified response to ethylene by transformation with an ETR nucleic acid
Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.
2001-01-01
The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.
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Enhancing fire safety at Hydro plants with dry transformers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Clemen, D.M.
Hydroelectric plant owners and engineers can use dry-type transformers to reduce fire hazards in auxiliary power systems. The decision to replace a liquid-immersed transformer with a dry-type product has a price: higher unit cost and a need to be more vigilant in detailing transformer specifications. But, whether the change affects only one failed transformer or is part of a plant rehabilitation project, the benefits in safety can be worth it. Voltages on hydroelectric plant auxiliary power systems can range from a 20 kV medium-voltage system to the normal 480-208/120 V low-voltage system. Dry transformers typically are used in such systemsmore » to reduce the fire hazard present with liquid-filled transformers. For a hydro plant owner or engineer seeking alternatives to liquid-filled transformers, there are two main kinds of dry-type transformers to consider: vacuum pressure impregnated (VPI) and cast coil epoxy resin. VPI transformers normally are manufactured in sizes up to 6,000 kVA with primary voltage ratings up to 20 kV. Cast coil transformers can be made in sizes from 75 to 10,000 kVA, with primary voltage ratings up to 34,500 V. Although the same transformer theory applies to dry transformers as to liquid-filled units, the cooling medium, air, required different temperature rise ratings, dielectric tests, and construction techniques to ensure reliability. Consequently, the factory and field tests for dry units are established by a separate set of American National Standards Institute (ANSI)/Institute of Electrical and Electronics Engineers (IEEE) standards. Cast coil transformers have several important advantages over VPI units.« less
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Infection of non-host model plant species with the narrow-host-range Cacao swollen shoot virus.
Friscina, Arianna; Chiappetta, Laura; Jacquemond, Mireille; Tepfer, Mark
2017-02-01
Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically. © 2016 BSPP AND JOHN WILEY & SONS LTD.
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Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.
Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P
1999-01-01
Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829
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Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins
Bally, Julia; Job, Claudette; Belghazi, Maya; Job, Dominique
2011-01-01
Background Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. Methodology/Principal Findings Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. Conclusions/Significance The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation. PMID:21966485
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Wound-Induced Deposition of Polyphenols in Transgenic Plants Overexpressing Peroxidase 1
Lagrimini, L. Mark
1991-01-01
Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H2O2 via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue. ImagesFigure 1Figure 2Figure 3 PMID:16668224
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Classification of explosives transformation products in plant tissue
DOE Office of Scientific and Technical Information (OSTI.GOV)
Larson, S.L.; Jones, R.P.; Escalon, L.
Explosives contamination in surface or groundwater used for the irrigation of food crops and phytoremediation of explosives-contaminated soil or water using plant-assisted biodegradation have brought about concerns as to the fate of explosives in plants. Liquid scintillation counting, high-performance liquid chromatography, and gel permeation chromatography were utilized to characterize explosives (hexahydro-1,3,5-trinitro-1,3,5-triazine and trinitrotoluene) and their metabolites in plant tissues obtained from three separate studies. Analyzing tissues of yellow nutsedge (Cyperus esculentus), corn (Zea mays), lettuce (Lacuta sativa), tomato (Lyopersicum esculentum), radish (Raphanus sativus), and parrot feather (Myriophyllum aquaticum) from three studies where exposure to explosives at nontoxic levels occurred showedmore » that extensive transformation of the explosive contaminant occurred, variations were noted in uptake and transformation between terrestrial and aquatic plants, the products had significantly higher polarity and water solubility than the parent compounds, and the molecular sizes of the transformation products were significantly greater than those of the parent compounds.« less
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6. PLANT 2 POWERHOUSE AND TRANSFORMER BUILDING. NOTE ABSENCE OF ...
6. PLANT 2 POWERHOUSE AND TRANSFORMER BUILDING. NOTE ABSENCE OF DIAMOND-SHAPED WINDOWS VISIBLE ON TRANSFORMER BUILDING IN PRE-1970 PHOTOGRAPHS. VIEW TO WEST. - Bishop Creek Hydroelectric System, Plant 2, Bishop Creek, Bishop, Inyo County, CA
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Jaganath, Balusamy; Subramanyam, Kondeti; Mayavan, Subramanian; Karthik, Sivabalan; Elayaraja, Dhandapani; Udayakumar, Rajangam; Manickavasagam, Markandan; Ganapathi, Andy
2014-05-01
An efficient and reproducible Agrobacterium-mediated in planta transformation was developed in Jatropha curcas. The various factors affecting J. curcas in planta transformation were optimized, including decapitation, Agrobacterium strain, pin-pricking, vacuum infiltration duration and vacuum pressure. Simple vegetative in vivo cleft grafting method was adopted in the multiplication of transformants without the aid of tissue culture. Among the various parameters evaluated, decapitated plants on pin-pricking and vacuum infiltrated at 250 mmHg for 3 min with the Agrobacterium strain EHA 105 harbouring the binary vector pGA 492 was proved to be efficient in all terms with a transformation efficiency of 62.66%. Transgene integration was evinced by the GUS histochemical analysis, and the GUS positive plants were subjected to grafting. Putatively transformed J. curcas served as "Scion" and the wild type J. curcas plant severed as "Stock". There was no occurrence of graft rejection and the plants were then confirmed by GUS histochemical analysis, polymerase chain reaction (PCR) and Southern hybridization. Genetic stability of the grafted plants was evaluated by using randomly amplified polymorphic DNA (RAPD), marker which showed 100% genetic stability between mother and grafted plants. Thus, an efficient in planta transformation and grafting based multiplication of J. curcas was established.
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Plant transformation via pollen tube-mediated gene transfer
USDA-ARS?s Scientific Manuscript database
Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...
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Zhu, Hong; Reynolds, L Bruce; Menassa, Rima
2017-06-19
Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.
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Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon
2016-08-01
Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.
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Keadtidumrongkul, Pornthep; Suttangkakul, Anongpat; Pinmanee, Phitsanu; Pattana, Kanokwan; Kittiwongwattana, Chokchai; Apisitwanich, Somsak; Vuttipongchaikij, Supachai
2017-08-01
The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.
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Plant phosphomannose isomerase as a selectable marker for rice transformation
Hu, Lei; Li, Hao; Qin, Ruiying; Xu, Rongfang; Li, Juan; Li, Li; Wei, Pengcheng; Yang, Jianbo
2016-01-01
The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering. PMID:27174847
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Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita
2011-05-01
We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.
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Xu, Yang; Zheng, Xinxin; Song, Yunzhi; Zhu, Lifei; Yu, Zipeng; Gan, Liming; Zhou, Shumei; Liu, Hongmei; Wen, Fujiang; Zhu, Changxiang
2018-06-11
Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na + levels. Salt-responsive genes, including Na + /H + exchangers (NHX1) and high-affinity K + transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na + . Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.
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Vieira, Pabline Marinho; Santos, Mirella Pupo; Andrade, Cristiana Moura; Souza-Neto, Otacílio Antônio; Ulhoa, Cirano José; Aragão, Francisco José Lima
2017-12-01
Aquaporins (AQPs) and aquaglyceroporins (AQGPs) are integral membrane proteins that mediate the transport of water and solutes, such as glycerol and urea, across membranes. AQP and AQGP genes represent a valuable tool for biotechnological improvement of plant tolerance to environmental stresses. We previously isolated a gene encoding for an aquaglyceroporin (ThAQGP), which was up-regulated in Trichoderma harzianum during interaction with the plant pathogen Fusarium solani. This gene was introduced into Nicotiana tabacum and plants were physiologically characterized. Under favorable growth conditions, transgenic progenies did not had differences in both germination and growth rates when compared to wild type. However, physiological responses under drought stress revealed that transgenic plants presented significantly higher transpiration rate, stomatal conductance, photosynthetic efficiency and faster turgor recovery than wild type. Quantitative RT-PCR analysis demonstrated the presence of ThAQGP transcripts in transgenic lines, showing the cause-effect relationship between the observed phenotype and the expression of the transgene. Our results underscore the high potential of T. harzianum as a source of genes with promising applications in transgenic plants tolerant to drought stress. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé; Faber, Anne-Marie; Morsomme, Pierre; Boutry, Marc
2011-02-01
Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth
Wilson, Robert H.; Alonso, Hernan; Whitney, Spencer M.
2016-01-01
In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted “trial and error” protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth. PMID:26926260
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Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth.
Wilson, Robert H; Alonso, Hernan; Whitney, Spencer M
2016-03-01
In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted "trial and error" protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth.
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Nontransgenic Genome Modification in Plant Cells1[W][OA
Marton, Ira; Zuker, Amir; Shklarman, Elena; Zeevi, Vardit; Tovkach, Andrey; Roffe, Suzy; Ovadis, Marianna; Tzfira, Tzvi; Vainstein, Alexander
2010-01-01
Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. PMID:20876340
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Fedoreyeva, L I; Dilovarova, T A; Ashapkin, V V; Martirosyan, Yu Ts; Khavinson, V Kh; Kharchenko, P N; Vanyushin, B F
2017-04-01
Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10 -7 -10 -9 M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.
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Rice, J Hollis; Mundell, Richard E; Millwood, Reginald J; Chambers, Orlando D; Stewart, C Neal; Davies, H Maelor
2013-08-06
The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes. Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field. N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.
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Bobik, Krzysztof; Duby, Geoffrey; Nizet, Yannick; Vandermeeren, Caroline; Stiernet, Patrick; Kanczewska, Justyna; Boutry, Marc
2010-04-01
The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.
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Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan
2011-01-01
Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and
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Novel and potential application of cryopreservation to plant genetic transformation.
Wang, Biao; Zhang, Zhibo; Yin, Zhenfang; Feng, Chaohong; Wang, Qiaochun
2012-01-01
The world population now is 6.7 billion and is predicted to reach 9 billion by 2050. Such a rapid growing population has tremendously increased the challenge for food security. Obviously, it is impossible for traditional agriculture to ensure the food security, while plant biotechnology offers considerable potential to realize this goal. Over the last 15 years, great benefits have been brought to sustainable agriculture by commercial cultivation of genetically modified (GM) crops. Further development of new GM crops will with no doubt contribute to meeting the requirements for food by the increasing population. The present article provides updated comprehensive information on novel and potential application of cryopreservation to genetic transformation. The major progresses that have been achieved in this subject include (1), long-term storage of a large number of valuable plant genes, which offers a good potential for further development of novel cultivars by genetic transformation; (2), retention of regenerative capacity of embryogenic tissues and protoplasts, which ensures efficient plant regeneration system for genetic transformation; (3), improvement of transformation efficiency and plant regeneration of transformed cells; (4), long-term preservation of transgenic materials with stable expression of transgenes and productive ability of recombinant proteins, which allows transgenic materials to be stored in a safe manner before being analyzed and evaluated, and allows establishment of stable seed stocks for commercial production of homologous proteins. Data provided in this article clearly demonstrate that cryo-technique has an important role to play in the whole chain of genetic transformation. Further studies coupling cryotechnique and genetic transformation are expected to significantly improve development of new GM crops. Copyright © 2011 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Pramono, Andri; Fauzantoro, Ahmad; Rizki Hidayati, Irma; Hygea, Arina; Puspita, Oktaviani Sandra; Muktamiroh, Hikmah; Simanjuntak, Kristina; Gozan, Misri
2018-03-01
Tobacco plays an important role in international trade as one of the export commodities. Indonesia is one of the good quality export contributors of tobacco leaves in the world. Nevertheless, tobacco is used only as a raw material for the cigarette industries, and the rise on anti-cigarette regulations prompted the exploration of alternative product from tobacco plants. The content of alkaloids, flavonoids, terpenoids and steroids in tobacco leaves were reported in literatures as antibacterial. Therefore, this study proposed in vitro assay of the ethanolic heat reflux extract (EHRE) of Nicotiana tabacum var. Virginia against nosocomial bacteria pathogen ((Pseudomonas aeruginosa (ATCC 27853), Eschericia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212)). Kirby-bauer diffusion method was used for this assay. The concentration of the EHRE for Kirby-bauer assay were 20; 40; 60; 80; and 100%. The presence of clear zones on Kirby-bauer test, against the growth of each nosocomial bacteria pathogen show that tobacco extract has antibacterial effect. Statistical analysis result showed that each extract concentration had significant difference value (p <0,05). This study indicated that the content (alkaloids, flavonoids, terpenoids and steroids) of tobacco leaf extracts (N. tabacum) has potential as antibacterial against nosocomial bacteria pathogen. Nevertheless, optimization of tobacco leaf extract to obtain maximum active ingredient still needs to be done. This study is important for further development of the tobacco leaf extract as antibacterial
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Centritto, Mauro; Haworth, Matthew; Marino, Giovanni; Pallozzi, Emanuele; Tsonev, Tsonko; Velikova, Violeta; Nogues, Isabel; Loreto, Francesco
2014-09-01
Isoprene emission by terrestrial plants is believed to play a role in mitigating the effects of abiotic stress on photosynthesis. Ultraviolet-B light (UV-B) induces damage to the photosynthetic apparatus of plants, but the role of isoprene in UV-B tolerance is poorly understood. To investigate this putative protective role, we exposed non-emitting (NE) control and transgenic isoprene emitting (IE) Nicotiana tabacum (tobacco) plants to high intensity UV-B exposure. Methanol emissions increased with UV-B intensity, indicating oxidative damage. However, isoprene emission was unaffected during exposure to UV-B radiation, but declined in the 48 h following UV-B treatment at the highest UV-B intensities of 9 and 15 Wm(-2). Photosynthesis and the performance of photosystem II (PSII) declined to similar extents in IE and NE plants following UV-B exposure, suggesting that isoprene emission did not ameliorate the immediate impact of UV-B on photosynthesis. However, after the stress, photosynthesis and PSII recovered in IE plants, which maintained isoprene formation, but not in NE plants. Recovery of IE plants was also associated with elevated antioxidant levels and cycling; suggesting that both isoprene formation and antioxidant systems contributed to reinstating the integrity and functionality of cellular membranes and photosynthesis following exposure to excessive levels of UV-B radiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju
2017-01-01
Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.
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Mzid, Rim; Zorrig, Walid; Ben Ayed, Rayda; Ben Hamed, Karim; Ayadi, Mariem; Damak, Yosra; Lauvergeat, Virginie; Hanana, Mohsen
2018-06-01
Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H 2 O 2 , and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY 2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.
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Manganese toxicity to chlorophyll synthesis in tobacco callus. [Nicotiana tabacum
DOE Office of Scientific and Technical Information (OSTI.GOV)
Clairmont, K.B.; Hagar, W.G.; Davis, E.A.
1986-01-01
Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectivity inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant.
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Melis, A; Thielen, A P
1980-02-08
In the present study we used three types of Nicotiana tabacum, cv John William's Broad Leaf (the wild type and two mutants, the yellow-green Su/su and the yellow Su/su var. Aurea) in order to correlat functional properties of Photosystem II and Photosystem I with the structural organization of their chloroplasts. The effective absorption cross-section of Photosystem II and Photosystem I centers was measured by means of the rate constant of their photoconversion under light-limiting conditions. In agreement with earlier results (Okabe, K., Schmid, G.H. and Straub, J. (1977) Plant Physiol. 60, 150--156) the photosynthetic unit size for both System II and System I in the two mutants was considerably smaller as compared to the wild type. We observed biphasic kinetics in the photoconversion of System II in all three types of N. tabacum. However, the photoconversion of System I occurred with monophasic and exponential kinetics. Under our experimental conditions, the effective cross-section of Photosystem I was comparable to that of the fast System II component (alpha centers). The relative amplitude of the slow System II component (beta centers) varied between 30% in the wild type to 70% in the Su/su var. Aurea mutant. The increased fraction of beta centers is correlated with the decreased fraction of appressed photosynthetic membranes in the chloroplasts of the two mutants. As a working hypothesis, it is suggested that beta centers are located on photosynthetic membranes directly exposed to the stroma medium.
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Hua, Jianfeng; Lin, Xiangui; Yin, Rui; Jiang, Qian; Shao, Yufang
2009-01-01
A pot experiment was conducted to study the effects of arbuscular mycorrhizal (AM) fungi (from contaminated or uncontaminated soils) on arsenic (As) uptake of tobacco (Nicotiana tabacum L.) in As-contaminated soil. Mycorrhizal colonization rate, dry weight, As and P uptake by plants, concentrations of water-extractable As and As fractions were determined. A low mycorrhizal colonization rate (< 25%) was detected. Our research indicated that AM fungi isolated from polluted soils were no more effective than those from unpolluted soils when grown in symbiosis with tobacco. No significant differences were observed in roots and stalks dry weights among all treatments. Leaves and total plant dry weights were much higher in Glomus versiforme treatment than that in control treatment. As contents in roots and stalks from mycorrhizal treatments were much lower than that from control treatment. Total plant As content exhibited the same trend. P concentrations in tobacco were not affected by colonization, nor were stalks, leaves and total plant P contents. Roots P contents were remarkably lower in HN treatments than in other treatments. Meanwhile, decreased soil pH and lower water-extractable As concentrations and higher levels of As fraction bound to well-crystallized hydrous oxides of Fe and Al were found in mycorrhizal treatments than in controls. The protective effect of mycorrhiza against plant As uptake may be associated with changes in As solubility mediated by changing soil pH. These results indicated that under As stress, proper mechanisms employed by AM fungi can protect tobacco against As uptake. Results confirmed that AM fungi can play an important role in food quality and safety.
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Application of Carbon Nanotubes for Plant Genetic Transformation
NASA Astrophysics Data System (ADS)
Burlaka, Olga M.; Pirko, Yaroslav V.; Yemets, Alla I.; Blume, Yaroslav B.
In this chapter, the current state of using carbon nanotubes (CNTs; single- and multi-walled) that have attracted great interdisciplinary interest in recent decades due to their peculiar properties for genetic transformation of prokaryotic and eukaryotic cells will be enlightened. The covalent and non-covalent surface chemistry for the CNT functionalization with focus on the potential applications of surface modifications in design of biocompatible CNTs will be discussed. The properties of CNTs that are favorable for biotechnological use and current status of technical approaches that allow the increase in biocompatibility and lower nanotoxicity of engineered CNTs will be described. Decisions proposed by non-covalent surface modification of CNTs will be discussed. Existing data concerning mechanisms of CNT cell entry and factors governing toxicity, cellular uptake, intracellular traffic, and biodegradation of CNTs along with bioavailability of molecular cargoes of loaded CNTs will be discussed. Eco-friendly production of water dispersions of biologically functionalized multi-walled and single-walled CNTs for use as nano-vehicles for the DNA delivery in plant genetic transformation of plants will be described. The background, advantages, and problems of using CNTs in developing of novel methods of genetic transformation, including plant genetic transformation, will be highlighted. Special attention will be paid to the limitations of conventional gene transfer techniques and promising features of CNT-based strategies having improved efficacy, reproducibility, and accuracy along with less time consumption. Issues impeding manipulation of CNTs such as entangled bundle formation, low water solubility, inert properties of pristine CNTs, etc., and ways to solve arising tasks will be overviewed.
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Plant Products for Pharmacology: Application of Enzymes in Their Transformations
Zarevúcka, Marie; Wimmer, Zdeněk
2008-01-01
Different plant products have been subjected to detailed investigations due to their increasing importance for improving human health. Plants are sources of many groups of natural products, of which large number of new compounds has already displayed their high impact in human medicine. This review deals with the natural products which may be found dissolved in lipid phase (phytosterols, vitamins etc.). Often subsequent convenient transformation of natural products may further improve the pharmacological properties of new potential medicaments based on natural products. To respect basic principles of sustainable and green procedures, enzymes are often employed as efficient natural catalysts in such plant product transformations. Transformations of lipids and other natural products under the conditions of enzyme catalysis show increasing importance in environmentally safe and sustainable production of pharmacologically important compounds. In this review, attention is focused on lipases, efficient and convenient biocatalysts for the enantio- and regioselective formation / hydrolysis of ester bond in a wide variety of both natural and unnatural substrates, including plant products, eg. plant oils and other natural lipid phase compounds. The application of enzymes for preparation of acylglycerols and transformation of other natural products provides big advantage in comparison with employing of conventional chemical methods: Increased selectivity, higher product purity and quality, energy conservation, elimination of heavy metal catalysts, and sustainability of the employed processes, which are catalyzed by enzymes. Two general procedures are used in the transformation of lipid-like natural products: (a) Hydrolysis/alcoholysis of triacylglycerols and (b) esterification of glycerol. The reactions can be performed under conventional conditions or in supercritical fluids/ionic liquids. Enzyme-catalyzed reactions in supercritical fluids combine the advantages of
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Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J Paul; De Lorenzo, Giulia; Cervone, Felice
2004-07-01
Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.
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Cell-to-cell movement of plastids in plants.
Thyssen, Gregory; Svab, Zora; Maliga, Pal
2012-02-14
Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.
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Mhlongo, M I; Tugizimana, F; Piater, L A; Steenkamp, P A; Madala, N E; Dubery, I A
2017-01-22
To counteract biotic stress factors, plants employ multilayered defense mechanisms responsive to pathogen-derived elicitor molecules, and regulated by different phytohormones and signaling molecules. Here, lipopolysaccharide (LPS), a microbe-associated molecular pattern (MAMP) molecule, was used to induce defense responses in Nicotiana tabacum cell suspensions. Intracellular metabolites were extracted with methanol and analyzed using a liquid chromatography-mass spectrometry (UHPLC-qTOF-MS/MS) platform. The generated data were processed and examined with multivariate and univariate statistical tools. The results show time-dependent dynamic changes and accumulation of glycosylated signaling molecules, specifically those of azelaic acid, salicylic acid and methyl-salicylate as contributors to the altered metabolomic state in LPS-treated cells. Copyright © 2016 Elsevier Inc. All rights reserved.
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Kalayou, Shewit; Haileselassie, Mekonnen; Gebre-egziabher, Gebremedhin; Tiku'e, Tsegay; Sahle, Samson; Taddele, Habtamu; Ghezu, Mussie
2012-01-01
Objective To screen the antibacterial activity of nine ethnoveterinary plants traditionally used for the treatment of mastitis, wound and gastrointestinal complications. Methods Hydroalcoholic exctracts of medicinal plants namely, Achyranthes aspera (A. aspera) L. (Family Asparagaceae), Ficus caria (F. caria) (Family Moraceae), Malvi parviflora (M. parviflora) (Family Malvaceae), Vernonia species (V. species) (local name Alakit, Family Asteraceae), Solanum hastifolium (S. hastifolium) (Family Solanaceae), Calpurinia aurea (C. aurea) (Ait) Benth (Family Fabaceae), Nicotiana tabacum (N. tabacum) L. (Family Solanaceae), Ziziphus spina-christi (Z. spina-christi) (Family Rhamnaceae), Croton macrostachys (C. macrostachys) (Family Euphorbiaceae), were screened against clinical bacterial isolates of veterinary importance from October 2007 to April 2009. The antibacterial activity was tested using disc diffusion at two concentrations (200 mg/mL and 100 mg/mL) and broth dilution methods using 70% methanol macerated leaf extracts. Results With the exception of S. hastifolium all plant extracts exhibited antibacterial activity. Among the medicinal plants tested C. aurea, C. macrostachyus, A. aspera, N. tabacum and vernonia species (Alakit) showed the most promising antimicrobial properties. Conclusions It can be concluded that many of the tested plants have antibacterial activity and supports the traditional usage of the plants for mastitis, wound and gastrointestinal complications treatment. Further studies into their toxicity and phytochemistry is advocated. PMID:23569962
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Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J. Paul; De Lorenzo, Giulia; Cervone, Felice
2004-01-01
Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. PMID:15247378
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Dan, Yinghui; Zhang, Song; Zhong, Heng; Yi, Hochul; Sainz, Manuel B
2015-02-01
Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these
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Profiling of Altered Metabolomic States in Nicotiana tabacum Cells Induced by Priming Agents
Mhlongo, Msizi I.; Steenkamp, Paul A.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Dubery, Ian A.
2016-01-01
Metabolomics has developed into a valuable tool for advancing our understanding of plant metabolism. Plant innate immune defenses can be activated and enhanced so that, subsequent to being pre-sensitized, plants are able to launch a stronger and faster defense response upon exposure to pathogenic microorganisms, a phenomenon known as priming. Here, three contrasting chemical activators, namely acibenzolar-S-methyl, azelaic acid and riboflavin, were used to induce a primed state in Nicotiana tabacum cells. Identified biomarkers were then compared to responses induced by three phytohormones—abscisic acid, methyljasmonate, and salicylic acid. Altered metabolomes were studied using a metabolite fingerprinting approach based on liquid chromatography and mass spectrometry. Multivariate data models indicated that these inducers cause time-dependent metabolic perturbations in the cultured cells and revealed biomarkers of which the levels are affected by these agents. A total of 34 metabolites were annotated from the mass spectral data and online databases. Venn diagrams were used to identify common biomarkers as well as those unique to a specific agent. Results implicate 20 cinnamic acid derivatives conjugated to (i) quinic acid (chlorogenic acids), (ii) tyramine, (iii) polyamines, or (iv) glucose as discriminatory biomarkers of priming in tobacco cells. Functional roles for most of these metabolites in plant defense responses could thus be proposed. Metabolites induced by the activators belong to the early phenylpropanoid pathway, which indicates that different stimuli can activate similar pathways but with different metabolite fingerprints. Possible linkages to phytohormone-dependent pathways at a metabolomic level were indicated in the case of cells treated with salicylic acid and methyljasmonate. The results contribute to a better understanding of the priming phenomenon and advance our knowledge of cinnamic acid derivatives as versatile defense metabolites. PMID
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Wang, Jia; Rajakulendran, Nirusan; Amirsadeghi, Sasan; Vanlerberghe, Greg C
2011-08-01
The plant mitochondrial electron transport chain (ETC) includes a non-energy conserving alternative oxidase (AOX) thought to dampen reactive oxygen species (ROS) generation by the ETC and/or facilitate carbon metabolism by uncoupling it from ATP turnover. When wild-type (WT) Nicotiana tabacum grown at 28°C/22°C (light/dark) were transferred to 12°C/5°C, they showed a large induction of leaf Aox1a mRNA and AOX protein within 24 h. Transfer to cold also resulted in a large accumulation of monosaccharides, an increase in transcript level of genes encoding important ROS-scavenging enzymes and a moderate increase in lipid peroxidation. Transgenic plants with suppressed AOX level showed less cold-induced sugar accumulation than WT while transgenic plants with enhanced AOX levels showed enhanced sugar accumulation. This is inconsistent with the hypothesis that AOX acts to burn excess carbohydrate, but rather suggests a role for AOX to aid sugar accumulation, at least during cold stress. At 28°C/22°C, plants with suppressed AOX had elevated levels of lipid peroxidation compared with WT, while plants with enhanced AOX had reduced lipid peroxidation. This is consistent with the hypothesis that AOX dampens ROS generation and oxidative damage. However, this inverse relationship between AOX level and lipid peroxidation did not hold upon shift to cold. Under this stress condition, plants with strong suppression of AOX show enhanced induction of ROS-scavenging enzymes compared with WT and decline in lipid peroxidation. These data suggest that, under stress conditions, the lack of AOX enhances a mitochondrial stress-signaling pathway able to increase the ROS-scavenging capacity of the cell. Copyright © Physiologia Plantarum 2011.
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Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.
Sigeno, Asako; Hayashi, Sugane; Terachi, Toru; Yamagishi, Hiroshi
2009-11-01
Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.
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Reef-coral proteins as visual, non-destructive reporters for plant transformation.
Wenck, A; Pugieux, C; Turner, M; Dunn, M; Stacy, C; Tiozzo, A; Dunder, E; van Grinsven, E; Khan, R; Sigareva, M; Wang, W C; Reed, J; Drayton, P; Oliver, D; Trafford, H; Legris, G; Rushton, H; Tayab, S; Launis, K; Chang, Y-F; Chen, D-F; Melchers, L
2003-11-01
Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.
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Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu
2012-03-01
A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.
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USDA-ARS?s Scientific Manuscript database
The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...
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Ju, Jong Il; Ko, Jung-Moon; Kim, So Hyeon; Baek, Ju Yeoul; Cha, Hyeon-Cheol; Lee, Sang Hoon
2006-08-01
In plant cell culture, the delivery of nutrition and gas (mainly oxygen) to the cells is the most important factor for viability. In this paper, we propose a polydimethylsiloxane (PDMS)-based microculture system that is designed to have good aeration. PDMS is known to have excellent air permeability, and through the experimental method, we investigated the relation between the degree of air delivery and the thickness of the PDMS sheet covering the culture chamber. We determined the proper thickness of the cover sheet, and cultured protoplasts of Nicotiana tabacum in a culture chamber covered with a PDMS sheet having thickness of 400 microm. The cells were successfully divided, and lived well inside the culture chamber for 10 days. In addition, protoplasts were cultured inside the culture chambers covered with the cover glass and the PDMS sheet, respectively, and the microcolonies were formed well inside the PDMS covered chamber after 10 days.
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Evolutionary diversification of type-2 HDAC structure, function and regulation in Nicotiana tabacum.
Nicolas-Francès, Valérie; Grandperret, Vincent; Liegard, Benjamin; Jeandroz, Sylvain; Vasselon, Damien; Aimé, Sébastien; Klinguer, Agnès; Lamotte, Olivier; Julio, Emilie; de Borne, François Dorlhac; Wendehenne, David; Bourque, Stéphane
2018-04-01
Type-2 HDACs (HD2s) are plant-specific histone deacetylases that play diverse roles during development and in responses to biotic and abiotic stresses. In this study we characterized the six tobacco genes encoding HD2s that mainly differ by the presence or the absence of a typical zinc finger in their C-terminal part. Of particular interest, these HD2 genes exhibit a highly conserved intron/exon structure. We then further investigated the phylogenetic relationships among the HD2 gene family, and proposed a model of the genetic events that led to the organization of the HD2 family in Solanaceae. Absolute quantification of HD2 mRNAs in N. tabacum and in its precursors, N. tomentosiformis and N. sylvestris, did not reveal any pseudogenization of any of the HD2 genes, but rather specific regulation of HD2 expression in these three species. Functional complementation approaches in Arabidopsis thaliana demonstrated that the four zinc finger-containing HD2 proteins exhibit the same biological function in response to salt stress, whereas the two HD2 proteins without zinc finger have different biological function. Copyright © 2018 Elsevier B.V. All rights reserved.
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Atmospheric transformation of plant volatiles disrupts host plant finding
NASA Astrophysics Data System (ADS)
Li, Tao; Blande, James D.; Holopainen, Jarmo K.
2016-09-01
Plant-emitted volatile organic compounds (VOCs) play important roles in plant-insect interactions. Atmospheric pollutants such as ozone (O3) can react with VOCs and affect the dynamics and fidelity of these interactions. However, the effects of atmospheric degradation of plant VOCs on plant-insect interactions remains understudied. We used a system comprising Brassica oleracea subsp. capitata (cabbage) and the specialist herbivore Plutella xylostella to test whether O3-triggered VOC degradation disturbs larval host orientation, and to investigate the underlying mechanisms. Larvae oriented towards both constitutive and larva-induced cabbage VOC blends, the latter being the more attractive. Such behaviour was, however, dramatically reduced in O3-polluted environments. Mechanistically, O3 rapidly degraded VOCs with the magnitude of degradation increasing with O3 levels. Furthermore, we used Teflon filters to collect VOCs and their reaction products, which were used as odour sources in behavioural tests. Larvae avoided filters exposed to O3-transformed VOCs and spent less time searching on them compared to filters exposed to original VOCs, which suggests that some degradation products may have repellent properties. Our study clearly demonstrates that oxidizing pollutants in the atmosphere can interfere with insect host location, and highlights the need to address their broader impacts when evaluating the ecological significance of VOC-mediated interactions.
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Hayford, Maria B.; Medford, June I.; Hoffman, Nancy L.; Rogers, Stephen G.; Klee, Harry J.
1988-01-01
The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666057
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Dalton, Heidi L.; Blomstedt, Cecilia K.; Neale, Alan D.; Gleadow, Ros; DeBoer, Kathleen D.; Hamill, John D.
2016-01-01
Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana. PMID
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Ismaili, Ahmad; Jalali-Javaran, Mokhtar; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Forouzandeh-Moghadam, Mehdi; Memari, Hamid Rajabi
2007-05-01
Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CH1s (constant heavy-chain domains). The antigen-specific binding fragments of these heavy-chain antibodies therefore comprise one single domain (the so-called 'VHH') and are of great importance in biotechnological applications. To evaluate the expression and biological activity of sdAbs (single-domain antibodies) in plants, which, on account of their small size and antigen-recognition properties, would have a major impact on antibody-engineering strategies, we constructed a pBI121-VHH gene encoding the recombinant sdAb fragments with specificity for a cancer-associated mucin, MUC1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC1 antigens and immunocytochemistry with cancerous cell lines confirmed that the activity of these molecules compared favourably with that of the parent recombinant antibodies. Protein purification was achieved by using sequential (NH4)2SO4 precipitation, gel filtration and immunoaffinity chromatography. Analysis of the purified VHH by ELISA indicated that the purified antibody fragments were able to react successfully with a MUC1-related peptide. These results reaffirm that the tobacco plant is a suitable host for the production of correctly folded VHH antibody fragments with diagnostic and therapeutic applications.
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Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate
Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping
2011-01-01
Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121
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Atmospheric transformation of plant volatiles disrupts host plant finding
Li, Tao; Blande, James D.; Holopainen, Jarmo K.
2016-01-01
Plant-emitted volatile organic compounds (VOCs) play important roles in plant-insect interactions. Atmospheric pollutants such as ozone (O3) can react with VOCs and affect the dynamics and fidelity of these interactions. However, the effects of atmospheric degradation of plant VOCs on plant-insect interactions remains understudied. We used a system comprising Brassica oleracea subsp. capitata (cabbage) and the specialist herbivore Plutella xylostella to test whether O3-triggered VOC degradation disturbs larval host orientation, and to investigate the underlying mechanisms. Larvae oriented towards both constitutive and larva-induced cabbage VOC blends, the latter being the more attractive. Such behaviour was, however, dramatically reduced in O3-polluted environments. Mechanistically, O3 rapidly degraded VOCs with the magnitude of degradation increasing with O3 levels. Furthermore, we used Teflon filters to collect VOCs and their reaction products, which were used as odour sources in behavioural tests. Larvae avoided filters exposed to O3-transformed VOCs and spent less time searching on them compared to filters exposed to original VOCs, which suggests that some degradation products may have repellent properties. Our study clearly demonstrates that oxidizing pollutants in the atmosphere can interfere with insect host location, and highlights the need to address their broader impacts when evaluating the ecological significance of VOC-mediated interactions. PMID:27651113
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Buschmann, Henrik
2016-01-01
The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.
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Sidorchuk, Yuriy Vladimirovich; Deineko, Elena Victorovna
2017-06-01
Microsporogenesis patterns of the polyploid (2n = 4x = 96) and diploid (2n = 2x = 48) Nicotiana tabacum L. (cv. Havana Petit line SR1) plants have been analyzed and compared. Four types of abnormal positions of the second-division spindles-tripolar, parallel, proximal, and fused-have been observed. Of these abnormalities, only tripolar (2.4%) and parallel (1.4%) spindles are observable in diploid plants. As for polyploids, the increased ploidy is accompanied by an increase in the incidence of tripolar (22.8%) and parallel (8.1%) spindle orientations and emergence of two remaining abnormalities (proximal and fused spindles, 3.3%). As has been shown, the spindle position abnormalities in diploid plants have no effect on the meiotic products, whereas both dyads and triads are detectable among the tetrads in polyploid plants. Analysis of cytoskeletal remodeling has allowed for the insight into the role of interzonal radial microtubule system in spindle positioning during the second division. The reason underlying the change in spindle positioning is disturbed polymerization-depolymerization processes and interdigitation of microtubule plus ends within the interzonal cytoskeleton system in late telophase I-interkinesis and prophase II. As has been demonstrated, fused second-division spindles are formed as a result of fused cytoskeletal structures in prophase-prometaphase II in the case when the nuclei are drawn abnormally close to one another. © 2017 International Federation for Cell Biology.
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Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D
2011-04-01
An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.
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Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P
2013-05-01
Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.
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Viktorovtá, Jitka; Novakova, Martina; Trbolova, Ladislava; Vrchotova, Blanka; Lovecka, Petra; Mackova, Martina; Macek, Tomas
2014-01-01
Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.
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COMBINED EFFECT OF SULFUR DIOXIDE AND OZONE ON BEAN AND TOBACCO PLANTS
Plants of two cultivars of Phaseolus vulgaris and one cultivar of Nicotiana tabacum were exposed to a replicated series of concentrations of sulfur dioxide (SO2), ozone (03), and combinations of these two air pollutants for single four-hour periods. Experiments were performed in ...
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Nagatoshi, Yukari; Ikeda, Miho; Kishi, Hiroyuki; Hiratsu, Keiichiro; Muraguchi, Atsushi; Ohme-Takagi, Masaru
2016-03-01
Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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Kawaoka, Akiyoshi; Matsunaga, Etsuko; Endo, Saori; Kondo, Shinkichi; Yoshida, Kazuya; Shinmyo, Atsuhiko; Ebinuma, Hiroyasu
2003-01-01
We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii × Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance. PMID:12857800
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Kawaoka, Akiyoshi; Matsunaga, Etsuko; Endo, Saori; Kondo, Shinkichi; Yoshida, Kazuya; Shinmyo, Atsuhiko; Ebinuma, Hiroyasu
2003-07-01
We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.
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Mafurah, Joseph Juma; Ma, Huifei; Zhang, Meixiang; Xu, Jing; He, Feng; Ye, Tingyue; Shen, Danyu; Chen, Yanyu; Rajput, Nasir Ahmed; Dou, Daolong
2015-01-01
Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.
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Wang, Z-Y; Bell, J; Lehmann, D
2004-07-01
Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.
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Hotto, Amber M; Huston, Zoe E; Stern, David B
2010-09-29
The roles of non-coding RNAs in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation. Evidence for accumulation of natural antisense RNAs (asRNAs) in chloroplasts comes from the expressed sequence tag database and cDNA libraries, while functional data have been largely obtained from artificial asRNAs. In this study, we used Nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asRNA, AS5, which is complementary to the region which encodes the 5S rRNA and tRNAArg. AS5-overexpressing (AS5ox) plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. Analysis of AS5 transcripts revealed four distinct species in wild-type (WT) and AS5ox plants, and additional AS5ox-specific products. Of the corresponding sense strand transcripts, tRNAArg overaccumulated several-fold in transgenic plants whereas 5S rRNA was unaffected. However, run-on transcription showed that the 5S-trnR region was transcribed four-fold more in the AS5ox plants compared to WT, indicating that overexpression of AS5 was associated with decreased stability of 5S rRNA. In addition, polysome analysis of the transformants showed less 5S rRNA and rbcL mRNA associated with ribosomes. Our results suggest that AS5 can modulate 5S rRNA levels, giving it the potential to affect Chloroplast translation and plant growth. More globally, overexpression of asRNAs via chloroplast transformation may be a useful strategy for defining their functions.
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Hsiao, Paoyuan; Sanjaya; Su, Ruey-Chih; Teixeira da Silva, Jaime A; Chan, Ming-Tsair
2007-03-01
Gene transformation is an integral tool for plant genetic engineering. All antibiotic resistant genes currently employed are of bacterial origin and their presence in the field is undesirable. Therefore, we developed a novel and efficient plant native non-antibiotic selection system for the selection of transgenic plants in the model system Arabidopsis. This new system is based on the enhanced expression of Arabidopsis tryptophan synthase beta 1 (AtTSB1) and the use of 5-methyl-tryptophan (5MT, a tryptophan [Trp] analog) and/or CdCl2 as selection agent(s). We successfully integrated an expression cassette containing an AtT-SB1 cDNA driven by a cauliflower mosaic virus 35S promoter into Arabidopsis by floral dip transformation. Transgenic plants were efficiently selected on MS medium supplemented with 75 microM 5MT or 300 microM CdCl2 devoid of antibiotics. TSB1 selection was as efficient as the conventional hygromycin selection system. Northern blot analysis of transgenic plants selected by 5MT and CdCl2 revealed increased TSB1 mRNA transcript whereas uneven transcript levels of hygromycin phosphotransferase II (hpt) (control) was observed. Gas chromatography-mass spectrometry revealed 10-15 fold greater free Trp content in AtT-SB1 transgenic plants than in wild-type plants grown with or without 5MT or CdCl2. Taken together, the TSB1 system provides a novel selection system distinct from conventional antibiotic selection systems.
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Iida, Asako; Yamashita, Toshiya; Yamada, Yasuyuki; Morikawa, Hiromichi
1991-01-01
Plasmid DNA pB1221 harboring β-glucuronidase gene was delivered to synchronized cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells of different cell cycle stages by a pneumatic particle gun. The cells bombarded at M and G2 phases gave 4 to 6 times higher transformation efficiency than those bombarded at the S and G1 phases. ImagesFigure 2 PMID:16668589
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Li, Renyi; Islam, Saif Ul; Wu, Zujian; Ye, Xiujuan
2016-01-01
Bensulfuron-methyl (BSM) is widely used in paddy soil for weed control. BSM residue in the soil has been known to inhibit the growth of sensitive crop plants. However, it is unknown whether BSM residue can affect the agrosystem in general. In this study, we have found significant effects of BSM on the infestation of Bemisia tabaci, Myzus persicae, and Tobacco mosaic virus (TMV) in Nicotiana tabacum. The soil was treated with BSM before the pest inoculation. The herbicide-treated tobaccos showed resistance to B. tabaci, but this resistance could not be detected until 15-day post-infestation when smaller number of adults B. tabaci appeared. In M. persicae assay, the longevity of all development stages of insects, and the fecundity of insects were not significantly affected when feeding on BSM-treated plants. In TMV assay, the BSM treatment also reduced virus-induced lesions in early infection time. However, the titer of TMV in BSM treated plants increased greatly over time and was over 40-fold higher than the mock-infected control plants after 20 days. Further studies showed that BSM treatment increased both jasmonic acid (JA) and salicylic acid (SA) levels in tobacco, as well as the expression of target genes in the JA and SA signaling pathways, such as NtWIPK, NtPR1a, and NtPAL. NtPR1a and NtPAL were initially suppressed after virus-inoculation, while NtRDR1 and NtRDR6, which play a key role in fighting virus infection, only showed up- or were down-regulated 20 days post virus-inoculation. Taken together, our results suggested that BSM residue in the soil may affect the metabolism of important phytohormones such as JA and SA in sensitive plants and consequently affect the plant immune response against infections such as whitefly, aphids, and viruses. PMID:28083007
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Response morphology and anatomy of tobacco (Nicotiana tabacum L.) plant on waterlogging
NASA Astrophysics Data System (ADS)
Nurhidayati, Tutik; Wardhani, Selfrina Puri; Purnobasuki, Hery; Hariyanto, Sucipto; Jadid, Nurul; Nurcahyani, Desy Dwi
2017-11-01
This study has conducted research on morphological and anatomical responses of some varieties of tobacco plants to waterlogging stress. Parameters measured were morphology, anatomy, and plants sensitivity index. Results were analyzed using two-way ANOVA followed by the Tukey test. The results show that waterlogging stress can reduce the growth of tobacco plants, including a decrease in plant height with the lowest value of 15.6 cm, root length reduction to the lowest value of 4.6 cm and plant dry weight reduction to the lowest value of 0.26 gr. But waterlogging stress can increase the number of adventitious roots with the highest value of 18.33. In addition, waterlogging stress can lead to the formation of aerenchyma tissue. The sensitivity index showed that plant varieties that are resistant to waterlogging stress are the varieties Kemloko 3 (index value of 0.03), varieties of Paiton 2 (index value of 0.18), and the varieties Kemloko 2 (index value of 0.42).
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Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity
NASA Astrophysics Data System (ADS)
Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana
To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed
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“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta
Hansen, Geneviève; Chilton, Mary-Dell
1996-01-01
We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts. PMID:8962167
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Nahar, Noor; Rahman, Aminur; Nawani, Neelu N; Ghosh, Sibdas; Mandal, Abul
2017-11-01
We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200μM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100μM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28μg/g d wt.) than that found in the shoots of non-transgenic controls (40μg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400μg/g d. wt.) than that (2100μg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid. Copyright © 2017 Elsevier GmbH. All rights reserved.
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Burklew, Caitlin E; Ashlock, Jordan; Winfrey, William B; Zhang, Baohong
2012-01-01
Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al(2)O(3) nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al(2)O(3) nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al(2)O(3) nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al(2)O(3) nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al(2)O(3) nanoparticles in the environment.
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Burklew, Caitlin E.; Ashlock, Jordan; Winfrey, William B.; Zhang, Baohong
2012-01-01
Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al2O3 nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al2O3 nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al2O3 nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al2O3 nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al2O3 nanoparticles in the environment. PMID:22606225
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An orange fluorescent protein tagging system for real-time pollen tracking.
Rice, J Hollis; Millwood, Reginald J; Mundell, Richard E; Chambers, Orlando D; Abercrombie, Laura L; Davies, H Maelor; Stewart, C Neal
2013-09-27
Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.
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Alteration of gene expression by restriction enzymes electroporated into plant cells.
Ashraf, M; Altschuler, M; Galasinski, S; Griffiths, T D
1993-06-01
The alteration in the expression of a beta-glucuronidase (GUS) reporter gene was used to monitor the effect of restriction endonucleases electroporated into the tobacco (Nicotiana tabacum L.) protoplasts. Restriction enzyme (RE) Hind III which does not have a recognition site within the gene cassette, had little effect on enzyme activity. In contrast restriction endonucleases Hae III and Sau3A1 which possess 8 and 16 recognition sites in the GUS cassette, were found to reduce the enzyme activity by 89% and 94% respectively when compared to control electroporations. Restriction-site mutation analysis (RSM) and Southern blot analysis indicated the enzymatic degradation of GUS coding sequence by the REs Hae III and Sau3A1. Results of this study suggest that on electroporation, REs can enter into plant cells and alter the expression of the GUS gene. The alteration of gene expression is thus correlated with the digestion of GUS template DNA. Future applications of this technique could include addressing fundamental questions with regard to DNA repair, site-specific recombination, identifying mutations, insertional mutagenesis, enhancement of stable transformation and gene tagging in plants.
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Fasakin, E A; Aberejo, B A
2002-11-01
The effectiveness of pulverised plant materials; Tithonium diversifolia, Afromomum melegueta, Nicotiana tabacum, Monodora myristica and Piper guineense as ovicidal, larvicidal and adult deterents of fish beetle (Dermestes maculatus) in smoked catfish (Clarias gariepinus) during storage were evaluated. Leaves of T. diversifolia, N. tabacum and seeds of A. melegueta, M. myristica and P. guineese were dried and pulverised into powder. Adults and larvae of third generation (F3) of D. maculatus were introduced into Kilner jars containing disinfested fish samples. Pulverised plant materials were applied to the surface of the fish samples at 10% (w/w) and monitored for 40 days, while egg hatchability of the insects was monitored for seven days. The result showed that all the plant materials had varying degree of insecticidal activities. Pulverised powder of P. guineense and A. melegueta were the most effective and significantly (P < 0.05) inhibited egg hatchability and adult emergence of D. maculatus in smoked catfish. N. tabacum gave the lowest insecticidal effect on adults, larvae and eggs of D. maculatus. However, the larvae of D. maculatus were not significantly (P > 0.05) affected by the plant materials. The percentage weight loss in fish treated with P. guineense and A. melegueta were minimal compared with the untreated fish sample. The result of this study showed that pulverised plant materials obtained from P. guineense and A. melegueta could be used to deter egg hatchability and adult emergence of D. maculatus in smoked catfish during storage. This could also reduce percentage losses due to insect infestation on smoked fish during storage.
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Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru
2014-01-01
Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars. PMID:25003197
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Studies of genetic transformation of higher plants using irradiated pollen
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chyi, Y.S.
Pandey has reported extensively on an unusual genetic phenomenon he called egg transformation. When compatible pollen was treated wth genetically lethal dosage of ..gamma..-radiation (100,000 rad), and used as mentor pollen to overcome selfincompatibility of several Nicotiana species, some genetic characters were found to be transferred from the radiation killed pollen to nonhybrid progeny. Observed transformants were fertile, cytogenetically normal, and had maternal phenotypes except for those specific traits transferred from the donors. Heavily irradiated pollen was believed to discharge its radiation-fragmented DNA (chromatin) into the embryo sac and bring about the transformation of the egg. The frequency of genemore » transfer was reported to be over 50%, and happened for all three characters Pandey studied - self incompatible specificities, flower color, and pollen color. Plant species studied were tomato, pea, apple, rapeseed, and Nicotiana species, including various stocks from Dr. Pandey. Treatments included pollinations with soley irradiated donor pollen, with a mixture of irradiated donor and normal self pollen, with a mixture of normal donor and self pollen, and double pollinations with irradiated donor pollen and normal self pollen, using different time intervals to separate the two pollinations. A total of 6210 pollinations were made, and 17,522 seedlings representing 87,750 potential transformational events were screened. In no case was an unambiguous transformant recovered. This research was unable to confirm or expand upon the findings of Dr. Pandey, or elucidate the mechanisms underlying such phenomena. Alternative explanations for Pandey's data were postulated. This approach to gene transfer by using irradiated pollen appears to be of little practical use to plant breeders.« less
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Ruhlman, Tracey A; Rajasekaran, Kanniah; Cary, Jeffrey W
2014-11-01
The chloroperoxidase (cpo) gene from Pseudomonas pyrrocinia was transformed into the plastid genome (plastome) of Nicotiana tabacum var. Petit Havana and transplastomic lines were compared with a nuclear transformant for the same gene. Southern analysis confirmed integration in the plastome and western blotting confirmed the presence of the chloroperoxidase protein (CPO) in higher abundance in transplastomic plants than in cpo nuclear transformants. Northern analysis of primary plastome transformants for cpo showed 15-fold higher transcript abundance than in the nuclear transformant, yet this extent of enhancement was not observed in western blot, enzyme or bioassay, indicating a bottleneck at the post-transcriptional level. Representative plants from the two transplastomic lines showed resistance to fungal pathogens in vitro (Aspergillus flavus, Fusarium verticillioides, and Verticillium dahliae) and in planta (Alternaria alternata). Published by Elsevier Ireland Ltd.
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Potency of Nicotiana tabacum as anti - microfouling
NASA Astrophysics Data System (ADS)
Aunurohim, Nurilma, Dian Ahmada; Kuswytasari, Nengah Dwianita
2017-06-01
In general, the attachment and growth of organisms on the surface of the object or material immersed in the sea are called Biofouling. Biofouling microscopic called microfouling, where it acts as a precursor of the next engaging organisms that are generally larger in size (called macrofouling). In recent time, biofouling control over the use of chemicals in antifouling paints. Usage tributyltin polishing copolymer paints (TBT - SPC paint) containing TBT has adverse effects on non-target marine organisms. This study uses tobacco dust as an anti-microfouling, which is the waste generated during the process of tobacco leaves. Five of bacterial isolates have been founded at Surabaya coastal in a preliminary test with iron plates. Then those isolates tested to detect and visualized of biofilm. The activity of anti-bacteria had been done and the result is known that more high of tobacco extract given, a diameter of zone inhibit high too. The extract of tobacco dust can be applied most effectively to be anti-microfouling is a concentration of 40%, isolates 3 and 4 are a type of bacteria that is most inhibited growth. So, in this result, Nicotiana tabacum is potential as an anti-microfouling.
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Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki
2008-07-01
Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.
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Jian, Bo; Hou, Wensheng; Wu, Cunxiang; Liu, Bin; Liu, Wei; Song, Shikui; Bi, Yurong; Han, Tianfu
2009-06-25
Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. Superroot of Lotus corniculatus is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic L. corniculatus plants from Superroot-derived leaves through A. tumefaciens-mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification. In the present study, we developed an A. rhizogenes-mediated transformation of Superroot-derived L. corniculatus for gene function investigation, combining the efficient A. rhizogenes-mediated transformation and the rapid regeneration system of Superroot. The transformation system using A. rhizogenes K599 harbouring pGFPGUSPlus was improved by validating some parameters which may influence the transformation frequency. Using stem sections with one node as explants, a 2-day pre-culture of explants, infection with K599 at OD(600) = 0.6, and co-cultivation on medium (pH 5.4) at 22 degrees C for 2 days enhanced the transformation frequency significantly. As proof of concept, Superroot-derived L. corniculatus was transformed with a gene from wheat encoding an Na+/H+ antiporter (TaNHX2) using the described system. Transgenic Superroot plants were obtained and had increased salt tolerance, as expected from the expression of TaNHX2. A rapid and efficient tool for gene function investigation in L. corniculatus was developed, combining the simplicity and high efficiency of the Superroot regeneration system and the availability of A. rhizogenes-mediated transformation. This system was improved by validating some parameters influencing the transformation frequency, which could reach 92% based on GUS detection. The combination
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Cross-Resistance to Short Residual Sulfonylurea Herbicides in Transgenic Tobacco Plants 1
Gabard, Jerome M.; Charest, Pierre J.; Iyer, V. N.; Miki, Brian L.
1989-01-01
Transgenic Nicotiana tabacum plants, produced by Agrobacterium tumefaciens-mediated transformation with a mutant gene (csr1-1) coding for acetohydroxyacid synthase (AHAS) from a chlorsulfuron resistant Arabidopsis thaliana line GH50 (GW Haughn et al. [1988] Mol Gen Genet 211: 266-271; GW Haughn, C Somerville [1986] Mol Gen Genet 204: 430-434), were selected directly on 80 micrograms per liter (225 nanomolar) chlorsulfuron. The expression of csr-1 in two separate transgenic lines CHL-1 and CHL-2 was confirmed by biochemical and genetic analyses. The AHAS activity of GH50 and the equivalent component of AHAS activity in CHL-2 was resistant to three short residual sulfonylurea herbicides, DPX-M6316, DPX-A7881, and DPX-L5300, in addition to chlorsulfuron but not to the sulfonylurea CGA 131′036. Cross-resistance to the imidazolinones AC 263, 499, AC 252, 214, and AC 243,997 was not observed. Parallel observations were made on the inhibition of seedling growth in soil or on culture medium. The relevance of these findings for the application of transgenic plants in agriculture is discussed. Images Figure 1 PMID:16667071
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Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P.; Snell, Kristi D.
2011-01-01
An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated. PMID:21325565
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Shikata, Masahito; Ezura, Hiroshi
2016-01-01
Tomato is a model plant for fruit development, a unique feature that classical model plants such as Arabidopsis and rice do not have. The tomato genome was sequenced in 2012 and tomato is becoming very popular as an alternative system for plant research. Among many varieties of tomato, Micro-Tom has been recognized as a model cultivar for tomato research because it shares some key advantages with Arabidopsis including its small size, short life cycle, and capacity to grow under fluorescent lights at a high density. Mutants and transgenic plants are essential materials for functional genomics research, and therefore, the availability of mutant resources and methods for genetic transformation are key tools to facilitate tomato research. Here, we introduce the Micro-Tom mutant database "TOMATOMA" and an efficient transformation protocol for Micro-Tom.
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Genetic transformation of the plant pathogens Phytophthora capsici and Phytophthora parasitica.
Bailey, A M; Mena, G L; Herrera-Estrella, L
1991-01-01
Phytophthora capsici and P.parasitica were transformed to hygromycin B resistance using plasmids pCM54 and pHL1, which contain the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Ustilago maydis heat shock hsp70 gene. Enzymes Driselase and Novozyme 234 were used to generate protoplasts which were then transformed following exposure to plasmid DNA and polyethylene glycol 6000. Transformation frequencies of over 500 transformants per micrograms of DNA per 1 x 10(6) protoplasts were obtained. Plasmid pCM54 appears to be transmitted in Phytophthora spp. as an extra-chromosomal element through replication, as shown by Southern blot hybridization and by the loss of plasmid methylation. In addition, transformed strains retained their capacity of infecting Serrano pepper seedlings and Mc. Intosh apple fruits, the host plants for P.capsici and P.parasitica, respectively. Images PMID:1651483
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Dek, Mohd Sabri Pak; Padmanabhan, Priya; Sherif, Sherif; Subramanian, Jayasankar; Paliyath, And Gopinadhan
2017-07-15
Phosphatidylinositol 3-kinase (PI3K) is a key enzyme that phosphorylates phosphatidylinositol at 3'-hydroxyl position of the inositol head group initiating the generation of several phosphorylated phosphatidylinositols, collectively referred to as phosphoinositides. The function of PI3K in plant senescence and ethylene signal transduction process was studied by expression of Solanum lycopersicum PI3K in transgenic Nicotiana tabacum , and delineating its effect on flower senescence. Detached flowers of transgenic tobacco plants with overexpressed Sl - PI3K (OX) displayed accelerated senescence and reduced longevity, when compared to the flowers of wild type plants. Flowers from PI3K-overexpressing plants showed enhanced ethylene production and upregulated expression of 1-aminocyclopropane-1-carboxylic acid oxidase 1 ( ACO1 ). Real time polymerase chain reaction (PCR) analysis showed that PI3K was expressed at a higher level in OX flowers than in the control. Seedlings of OX-lines also demonstrated a triple response phenotype with characteristic exaggerated apical hook, shorter hypocotyls and increased sensitivity to 1-aminocyclopropane-1-carboxylate than the control wild type seedlings. In floral tissue from OX-lines, Solanum lycopersicum phosphatidylinositol 3-kinase green fluorescent protein (PI3K-GFP) chimera protein was localized primarily in stomata, potentially in cytoplasm and membrane adjacent to stomatal pores in the guard cells. Immunoblot analysis of PI3K expression in OX lines demonstrated increased protein level compared to the control. Results of the present study suggest that PI3K plays a crucial role in senescence by enhancing ethylene biosynthesis and signaling.
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Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants.
Walters, D A; Vetsch, C S; Potts, D E; Lundquist, R C
1992-01-01
Embryogenic maize (Zea mays L.) callus cultures were transformed by microprojectile bombardment with a chimeric hygromycin phosphotransferase (HPT) gene and three transformed lines were obtained by selecting for hygromycin resistance. All lines contained one or a few copies of the intact HPT coding sequence. Fertile, transgenic plants were regenerated and the transmission of the chimeric gene was demonstrated through two complete generations. One line inherited the gene in the manner expected for a single, dominant locus, whereas two did not.
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Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E
2010-08-09
Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system
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Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas
2010-09-01
A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.
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Evaluation of toxicity of trichloroethylene for plants
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ryu, S.B.; Davis, L.C.; Dana, J.
1996-12-31
Trichloroethylene (TCE) exposure of several species of plants was studied. Although earlier studies indicated that the root systems of plants could tolerate an aqueous phase concentration of 1 mM for a day, toxicity to whole plants was observed at somewhat lower levels in the gas phase in this study. The tested species included pumpkin (Cucurbita maxima), tomato (Lycopersicon esculentum), sweet potato (Dioscoria batata), tobacco (Nicotiana tabacum), soybean (Glycine max L. Merr), and alfalfa (Medicago sativa). Damage was observable as wilting or failure of the gravitropic response of shoots at levels above about 0.2 mM in the gas phase, which correspondsmore » to 0.5 mM in the aqueous phase. Plants were usually killed quickly at gas phase concentrations above 0.4 mM.« less
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Wang, Meng; Xu, Zongchang; Ding, Anming; Kong, Yingzhen
2018-05-24
Xyloglucan endotransglucosylase/hydrolase genes ( XTHs ) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum , Nicotiana sylvestrisis , Nicotiana tomentosiformis , Arabidopsis , and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.
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Mhlongo, Msizi I.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Steenkamp, Paul A.; Dubery, Ian A.
2016-01-01
Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role. PMID:26978774
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Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. [Nicotiana tabacum
DOE Office of Scientific and Technical Information (OSTI.GOV)
Constantin, M.J.; Henke, R.R.; Mansur, M.A.
1977-01-01
Incorporating activated charcoal (AC) in culture media has been shown to affect growth and development of various organisms. Since AC stimulates the development of tobacco haploid plantlets from cultured anthers, research was conducted to determine the effect of activated charcoal on pith-derived callus growth and shoot development in Nicotiana tabacum cv. Wisconsin 38. Our results indicate that the hormones required for callus growth and shoot development in Wisconsin-38 tobacco are adsorbed by AC, thereby inhibiting callus growth and prohibiting shoot development. This effect was observed even when AC was removed from the medium by filtration prior to culturing the callus.
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Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu
2014-01-01
The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121
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Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu
2014-01-01
The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.
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Cis- and trans-zeatin differentially modulate plant immunity.
Großkinsky, Dominik K; Edelsbrunner, Kerstin; Pfeifhofer, Hartwig; van der Graaff, Eric; Roitsch, Thomas
2013-07-01
Phytohormones are essential regulators of various processes in plant growth and development. Several phytohormones are also known to regulate plant responses to environmental stress and pathogens. Only recently, cytokinins have been demonstrated to play an important role in plant immunity. Increased levels of cytokinins such as trans-zeatin, which are considered highly active, induced resistance against mainly (hemi)biotrophic pathogens in different plant species. In contrast, cis-zeatin is commonly regarded as a cytokinin exhibiting low or no activity. Here we comparatively study the impact of both zeatin isomers on the infection of Nicotiana tabacum by the (hemi)biotrophic microbial pathogen Pseudomonas syringae. We demonstrate a biological effect of cis-zeatin and a differential effect of the two zeatin isomers on symptom development, defense responses and bacterial multiplication.
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Described are methods to measure the polychlorinated biphenyl (PCB) emissions from the stacks of municipal waste, industrial waste, and sewage sludge incinerators and from capacitor and transformer filling plants. The PCB emissions from the incineration plants are collected by im...
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Tri-trophic insecticidal effects of African plants against cabbage pests.
Amoabeng, Blankson W; Gurr, Geoff M; Gitau, Catherine W; Nicol, Helen I; Munyakazi, Louis; Stevenson, Phil C
2013-01-01
Botanical insecticides are increasingly attracting research attention as they offer novel modes of action that may provide effective control of pests that have already developed resistance to conventional insecticides. They potentially offer cost-effective pest control to smallholder farmers in developing countries if highly active extracts can be prepared simply from readily available plants. Field cage and open field experiments were conducted to evaluate the insecticidal potential of nine common Ghanaian plants: goat weed, Ageratum conyzoides (Asteraceae), Siam weed, Chromolaena odorata (Asteraceae), Cinderella weed, Synedrella nodiflora (Asteraceae), chili pepper, Capsicum frutescens (Solanaceae), tobacco, Nicotiana tabacum (Solanaceae) cassia, Cassia sophera (Leguminosae), physic nut, Jatropha curcas (Euphorbiaceae), castor oil plant, Ricinus communis (Euphorbiaceae) and basil, Ocimum gratissimum (Lamiaceae). In field cage experiments, simple detergent and water extracts of all botanical treatments gave control of cabbage aphid, Brevicoryne brassicae and diamondback moth, Plutella xylostella, equivalent to the synthetic insecticide Attack® (emamectin benzoate) and superior to water or detergent solution. In open field experiments in the major and minor rainy seasons using a sub-set of plant extracts (A. conyzoides, C. odorata, S. nodiflora, N. tabacum and R. communis), all controlled B. brassicae and P. xylostella more effectively than water control and comparably with or better than Attack®. Botanical and water control treatments were more benign to third trophic level predators than Attack®. Effects cascaded to the first trophic level with all botanical treatments giving cabbage head weights, comparable to Attack® in the minor season. In the major season, R. communis and A conyzoides treatment gave lower head yields than Attack® but the remaining botanicals were equivalent or superior to this synthetic insecticide. Simply-prepared extracts from readily
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Tri-Trophic Insecticidal Effects of African Plants against Cabbage Pests
Amoabeng, Blankson W.; Gurr, Geoff M.; Gitau, Catherine W.; Nicol, Helen I.; Stevenson, Phil C.
2013-01-01
Botanical insecticides are increasingly attracting research attention as they offer novel modes of action that may provide effective control of pests that have already developed resistance to conventional insecticides. They potentially offer cost-effective pest control to smallholder farmers in developing countries if highly active extracts can be prepared simply from readily available plants. Field cage and open field experiments were conducted to evaluate the insecticidal potential of nine common Ghanaian plants: goat weed, Ageratum conyzoides (Asteraceae), Siam weed, Chromolaena odorata (Asteraceae), Cinderella weed, Synedrella nodiflora (Asteraceae), chili pepper, Capsicum frutescens (Solanaceae), tobacco, Nicotiana tabacum (Solanaceae) cassia, Cassia sophera (Leguminosae), physic nut, Jatropha curcas (Euphorbiaceae), castor oil plant, Ricinus communis (Euphorbiaceae) and basil, Ocimum gratissimum (Lamiaceae). In field cage experiments, simple detergent and water extracts of all botanical treatments gave control of cabbage aphid, Brevicoryne brassicae and diamondback moth, Plutella xylostella, equivalent to the synthetic insecticide Attack® (emamectin benzoate) and superior to water or detergent solution. In open field experiments in the major and minor rainy seasons using a sub-set of plant extracts (A. conyzoides, C. odorata, S. nodiflora, N. tabacum and R. communis), all controlled B. brassicae and P. xylostella more effectively than water control and comparably with or better than Attack®. Botanical and water control treatments were more benign to third trophic level predators than Attack®. Effects cascaded to the first trophic level with all botanical treatments giving cabbage head weights, comparable to Attack® in the minor season. In the major season, R. communis and A conyzoides treatment gave lower head yields than Attack® but the remaining botanicals were equivalent or superior to this synthetic insecticide. Simply-prepared extracts from readily
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Pinçon, Gaelle; Chabannes, Matthieu; Lapierre, Catherine; Pollet, Brigitte; Ruel, Katia; Joseleau, Jean-Paul; Boudet, Alain M.; Legrand, Michel
2001-01-01
Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078
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Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood
2014-04-01
Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.
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Gordon-Kamm, William; Dilkes, Brian P.; Lowe, Keith; Hoerster, George; Sun, Xifan; Ross, Margit; Church, Laura; Bunde, Chris; Farrell, Jeff; Hill, Patrea; Maddock, Sheila; Snyder, Jane; Sykes, Louisa; Li, Zhongsen; Woo, Young-min; Bidney, Dennis; Larkins, Brian A.
2002-01-01
The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation. PMID:12185243
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Chi-Ham, Cecilia L; Boettiger, Sara; Figueroa-Balderas, Rosa; Bird, Sara; Geoola, Josef N; Zamora, Pablo; Alandete-Saez, Monica; Bennett, Alan B
2012-06-01
The Public Intellectual Property Resource for Agriculture (PIPRA) was founded in 2004 by the Rockefeller Foundation in response to concerns that public investments in agricultural biotechnology benefiting developing countries were facing delays, high transaction costs and lack of access to important technologies due to intellectual property right (IPR) issues. From its inception, PIPRA has worked broadly to support a wide range of research in the public sector, in specialty and minor acreage crops as well as crops important to food security in developing countries. In this paper, we review PIPRA's work, discussing the failures, successes, and lessons learned during its years of operation. To address public sector's limited freedom-to-operate, or legal access to third-party rights, in the area of plant transformation, we describe PIPRA's patent 'pool' approach to develop open-access technologies for plant transformation which consolidate patent and tangible property rights in marker-free vector systems. The plant transformation system has been licensed and deployed for both commercial and humanitarian applications in the United States (US) and Africa, respectively. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
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Invasive plants transform the three-dimensional structure of rain forests
Asner, Gregory P.; Hughes, R. Flint; Vitousek, Peter M.; Knapp, David E.; Kennedy-Bowdoin, Ty; Boardman, Joseph; Martin, Roberta E.; Eastwood, Michael; Green, Robert O.
2008-01-01
Biological invasions contribute to global environmental change, but the dynamics and consequences of most invasions are difficult to assess at regional scales. We deployed an airborne remote sensing system that mapped the location and impacts of five highly invasive plant species across 221,875 ha of Hawaiian ecosystems, identifying four distinct ways that these species transform the three-dimensional (3D) structure of native rain forests. In lowland to montane forests, three invasive tree species replace native midcanopy and understory plants, whereas one understory invader excludes native species at the ground level. A fifth invasive nitrogen-fixing tree, in combination with a midcanopy alien tree, replaces native plants at all canopy levels in lowland forests. We conclude that this diverse array of alien plant species, each representing a different growth form or functional type, is changing the fundamental 3D structure of native Hawaiian rain forests. Our work also demonstrates how an airborne mapping strategy can identify and track the spread of certain invasive plant species, determine ecological consequences of their proliferation, and provide detailed geographic information to conservation and management efforts. PMID:18316720
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Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; ...
2017-01-18
Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Cai, Yingqi; McClinchie, Elizabeth; Price, Ann
Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less
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Transformation of pecan and regeneration of transgenic plants.
McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E
1993-09-01
A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.
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Fresquet-Corrales, Sandra; Roque, Edelín; Sarrión-Perdigones, Alejandro; Rochina, Maricruz; López-Gresa, María P.; Díaz-Mula, Huertas M.; Bellés, José M.; Tomás-Barberán, Francisco; Beltrán, José P.
2017-01-01
Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce “bloat-safe” plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed. PMID:28902886
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Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin
2016-01-01
Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.
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Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong
2013-01-01
The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764
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Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong
2013-08-01
The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.
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Engineering metabolic pathways in plants by multigene transformation.
Zorrilla-López, Uxue; Masip, Gemma; Arjó, Gemma; Bai, Chao; Banakar, Raviraj; Bassie, Ludovic; Berman, Judit; Farré, Gemma; Miralpeix, Bruna; Pérez-Massot, Eduard; Sabalza, Maite; Sanahuja, Georgina; Vamvaka, Evangelia; Twyman, Richard M; Christou, Paul; Zhu, Changfu; Capell, Teresa
2013-01-01
Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.
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Hoyerová, Klára; Perry, Lucie; Hand, Paul; Laňková, Martina; Kocábek, Tomáš; May, Sean; Kottová, Jana; Pačes, Jan; Napier, Richard; Zažímalová, Eva
2008-01-01
We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants. PMID:18184737
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Kuluev, B R; Safiullina, M G; Kniazev, A V; Chemeris, A V
2013-01-01
We obtained transgenic tobacco plants demonstrating overexpression of NtEXPA5 gene that encodes alpha-expansin of Nicotiana tabacum. The transgenic plants were characterized by increased size of leaves and stems. However, size of flowers remained almost unchanged. The increase of organ sizes was induced by cell stretching only. Moreover, the number of cell divisions was even decreased. The obtained data suggest tight interaction between cell stretching regulation and cell division, which together provide the basic mechanism aimed at the controlling of plant organ sizes.
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Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D
2017-07-01
Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
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Ncube, Efficient N; Mhlongo, Msizi I; Piater, Lizelle A; Steenkamp, Paul A; Dubery, Ian A; Madala, Ntakadzeni E
2014-01-01
Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures. Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof. Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.
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Ontogeny of plants under various gravity condition
NASA Astrophysics Data System (ADS)
Laurinavičius, R.; Švegždienṡ, D.; Raklevičienė, D.; Kenstavičienė, P.
2001-01-01
The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.
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Ashraf, Muhammad Aleem; Shahid, Ahmad Ali; Rao, Abdul Qayyum; Bajwa, Kamran Shehzad; Husnain, Tayyab
2014-01-01
The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant. PMID:24424501
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mauzey-Amato, Jacqueline M.; Dai, Ziyu
2000-11-01
Optimization of the transgene expression system is one of the critical steps for the high level production of heterologous proteins in plants, where the promoter is a key component regulating transgene expression. In this study, the activity of the rpL34 promoter was analyzed in transgenic tobacco (Nicotiana tabacum) NTI calli. A DNA fragment containing the rpL34 promoter and the reporter gene B-D-glucuronidase (GUS) were cloned into binary vector pZD427 to generate the transgene expression vector pZD428. The insertion was verified by enzyme restriction digestion and agarose gel electrophoresis analyses. The DNA fragment containing the rpL34 promoter and GUS reporter genemore » was then integrated into the tobacco genomes via Agrobacterium funiefaciens-mediated NT suspension cell transformation. The transformed CaNi were induced on Murashige and Skoog (MS) plates containing proper amounts of 2,4-D, cefotoxime, and kanamycin. Two hundred and sixty transformed calli were harvested for GUS activity and protein concentration measurements. GUS activity analyses revealed the specific activity up to 278,358 units per milligram total soluble protein. The GUS activity under the control of the rpL34 promoter is much higher than that under the control of the cauliflower mosaic virus 35S promoter, a commonly used promoter in plant biology. These results suggest that the rpL34 promoter is one of the most active promoters that can be used for heterologous protein production in calli and suspension cells.« less
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Scholthof, Karen-Beth G
2017-02-01
One of the seminal events in plant pathology was the discovery by Francis O. Holmes that necrotic local lesions induced on certain species of Nicotiana following rub-inoculation of Tobacco mosaic virus (TMV) was due to a specific interaction involving a dominant host gene (N). From this, Holmes had an idea that if the N gene from N. glutinosa was introgressed into susceptible tobacco, the greatly reduced titer of TMV would, by extension, prevent subsequent infection of tomato and pepper plants by field workers whose hands were contaminated with TMV from their use of chewing and smoking tobacco. The ultimate outcome has many surprising twists and turns, including Holmes' failure to obtain fertile crosses of N. glutinosa × N. tabacum after 3 years of intensive work. Progress was made with N. digluta, a rare amphidiploid that was readily crossed with N. tabacum. And, importantly, the first demonstration by Holmes of the utility of interspecies hybridization for virus resistance was made with Capsicum (pepper) species with the identification of the L gene in Tabasco pepper, that he introgressed into commercial bell pepper varieties. Holmes' findings are important as they predate Flor's gene-for-gene hypothesis, show the use of interspecies hybridization for control of plant pathogens, and the use of the local lesion as a bioassay to monitor resistance events in crop plants.
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Bastaki, Nasmah K.; Cullis, Christopher A.
2014-01-01
Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation. PMID:25549243
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Bastaki, Nasmah K; Cullis, Christopher A
2014-12-19
Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.
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Geric Stare, Barbara; Fouville, Didier; Širca, Saša; Gallot, Aurore; Urek, Gregor; Grenier, Eric
2011-02-01
While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties.
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Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing; Zhou, Xue-Rong; Shrestha, Pushkar; Divi, Uday K; Ral, Jean-Philippe; Mansour, Maged P; Nichols, Peter D; James, Christopher N; Horn, Patrick J; Chapman, Kent D; Beaudoin, Frederic; Ruiz-López, Noemi; Larkin, Philip J; de Feyter, Robert C; Singh, Surinder P; Petrie, James R
2014-02-01
High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. © 2013 CSIRO. Plant Biotechnology Journal published by Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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USDA-ARS?s Scientific Manuscript database
Plant Transformation Technologies is a comprehensive, authoritative book focusing on cutting-edge plant biotechnologies, offering in-depth, forward-looking information on methods for controlled and accurate genetic engineering. In response to ever-increasing pressure for precise and efficient integr...
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Sequencing and phylogenetic analysis of tobacco virus 2, a polerovirus from Nicotiana tabacum.
Zhou, Benguo; Wang, Fang; Zhang, Xuesong; Zhang, Lina; Lin, Huafeng
2017-07-01
The complete genome sequence of a new virus, provisionally named tobacco virus 2 (TV2), was determined and identified from leaves of tobacco (Nicotiana tabacum) exhibiting leaf mosaic, yellowing, and deformity, in Anhui Province, China. The genome sequence of TV2 comprises 5,979 nucleotides, with 87% nucleotide sequence identity to potato leafroll virus (PLRV). Its genome organization is similar to that of PLRV, containing six open reading frames (ORFs) that potentially encode proteins with putative functions in cell-to-cell movement and suppression of RNA silencing. Phylogenetic analysis of the nucleotide sequence placed TV2 alongside members of the genus Polerovirus in the family Luteoviridae. To the best our knowledge, this study is the first report of a complete genome sequence of a new polerovirus identified in tobacco.
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Pan, Lili; Sun, Jianteng; Wu, Xiaodan; Wei, Zi; Zhu, Lizhong
2016-11-01
The occurrence and fate of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) have received significant attention. However, there is limited information on the metabolism relationship between OH-pentaBDEs and MeO-pentaBDEs that were frequently detected with relatively high concentrations in the environment. In this study, the biotransformation between OH-BDE-99 and MeO-BDE-99 was investigated in rice, wheat, and soybean plants. All the three plants can metabolize OH-BDE-99 to corresponding homologous methoxylated metabolites, while the transformation from MeO-BDE-99 to OH-BDE-99 could only be found in soybean. The conversion of parent compounds was the highest in soybean, followed by wheat and rice. Transformation products were found mainly in the roots, with few metabolites being translocated to the shoots and solution after exposure. The results of this study provide valuable information for a better understanding of the accumulation and transformation of OH-PBDEs and MeO-PBDEs in different plants. Copyright © 2016. Published by Elsevier B.V.
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Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F
2014-02-21
Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.
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Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong
2015-01-01
The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production.
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Restivo, Francesco Maria; Laccone, Maria Concetta; Buschini, Annamaria; Rossi, Carlo; Poli, Paola
2002-03-01
Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.
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Kirst, Henning; Shen, Yanxin; Vamvaka, Evangelia; Betterle, Nico; Xu, Dongmei; Warek, Ujwala; Strickland, James A; Melis, Anastasios
2018-04-06
Downregulation in the expression of the signal recognition particle 43 (SRP43) gene in tobacco conferred a truncated photosynthetic light-harvesting antenna (TLA property), and resulted in plants with a greater leaf-to-stem ratio, improved photosynthetic productivity and canopy biomass accumulation under high-density cultivation conditions. Evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a survival advantage for the organism in the wild, where sunlight is often the growth-limiting factor. In crop monocultures, however, this property is strongly counterproductive, when growth takes place under direct and excess sunlight. The large arrays of light-harvesting antennae in crop plants cause the surface of the canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis and forcing them to engage in wasteful dissipation of the excess energy. Evidence in this work showed that downregulation by RNA-interference approaches of the Nicotiana tabacum signal recognition particle 43 (SRP43), a nuclear gene encoding a chloroplast-localized component of the photosynthetic light-harvesting assembly pathway, caused a decrease in the light-harvesting antenna size of the photosystems, a corresponding increase in the photosynthetic productivity of chlorophyll in the leaves, and improved tobacco plant canopy biomass accumulation under high-density cultivation conditions. Importantly, the resulting TLA transgenic plants had a substantially greater leaf-to-stem biomass ratio, compared to those of the wild type, grown under identical agronomic conditions. The results are discussed in terms of the potential benefit that could accrue to agriculture upon application of the TLA-technology to crop plants, entailing higher density planting with plants having a greater biomass and leaf-to-stem ratio, translating into greater crop yields per plant with canopies in a novel agronomic configuration.
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Fan, Wei; Li, Rong; Li, Sifan; Ping, Wenli; Li, Shujun; Naumova, Alexandra; Peelen, Tamara; Yuan, Zheng; Zhang, Dabing
2016-01-01
Reliable methods are needed to detect the presence of tobacco components in tobacco products to effectively control smuggling and classify tariff and excise in tobacco industry to control illegal tobacco trade. In this study, two sensitive and specific DNA based methods, one quantitative real-time PCR (qPCR) assay and the other loop-mediated isothermal amplification (LAMP) assay, were developed for the reliable and efficient detection of the presence of tobacco (Nicotiana tabacum) in various tobacco samples and commodities. Both assays targeted the same sequence of the uridine 5′-monophosphate synthase (UMPS), and their specificities and sensitivities were determined with various plant materials. Both qPCR and LAMP methods were reliable and accurate in the rapid detection of tobacco components in various practical samples, including customs samples, reconstituted tobacco samples, and locally purchased cigarettes, showing high potential for their application in tobacco identification, particularly in the special cases where the morphology or chemical compositions of tobacco have been disrupted. Therefore, combining both methods would facilitate not only the detection of tobacco smuggling control, but also the detection of tariff classification and of excise. PMID:27635142
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Maodzeka, Antony; Hussain, Nazim; Wei, Liquan; Zvobgo, Gerald; Mapodzeke, James Mutemachani; Adil, Muhammad Faheem; Jabeen, Salma; Wang, Feng; Jiang, Lixi; Shamsi, Imran Haider
2017-01-01
In the present study, the effects of lead (Pb) uptake and toxicity were investigated in a hydroponic culture using 7 tobacco (Nicotiana tabacum L.) genotypes (Bina 1 [B1], Kutsaga Mammoth 10 [KM10], Nanjing 3 [N3], Kutsaga 35 [K35], Kutsaga E1 [KE1], Cocker 176 [C176], and Kutsaga RK6 [KRK6]) that differed in Pb tolerance. Lead was applied as a solution of Pb nitrate at concentrations of 0 μM, 10 μM, 250 μM, and 500 μM. After 4 wk of Pb treatment, tissue biomass and photosynthetic parameters were measured and elemental analysis was performed. The results showed decreases in growth and photosynthetic parameters with increases in Pb concentration compared with the control. The least reduction in the recorded physiological parameters was noted in K35, whereas the greatest reduction was observed in N3, which is an obvious indication of genotypic differences. Activities of peroxidase, catalase, and malondialdehyde increased significantly with increases in Pb concentration, with genotypes K35 and N3 showing the least and the greatest reduction, respectively. The results demonstrate the phototoxic nature of Pb on plants, and it can be concluded that in Pb-prone areas genotypes K35 and B1 can be used for cultivation because they can grow efficiently in the presence of high Pb concentrations while restricting Pb uptake in the aboveground parts, as seen by the higher Pb tolerance index. Environ Toxicol Chem 2017;36:175-181. © 2016 SETAC. © 2016 SETAC.
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Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien
2014-01-01
Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.
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Miele, Marco; Costantini, Susan; Colonna, Giovanni
2011-02-02
Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm in vivo the molecular modelling data, osmotin has been purified from nicotiana tabacum seeds and its nine residues peptide synthesized. We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico.
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Direct transformation and plant regeneration of the haploid liverwort Marchantia polymorpha L.
Takenaka, M; Yamaoka, S; Hanajiri, T; Shimizu-Ueda, Y; Yamato, K T; Fukuzawa, H; Ohyama, K
2000-06-01
Thalli of the haploid liverwort Marchantial polymorpha were successfully used for direct particle bombardment with plasmid pMT, which carries a hygromycin phosphotransferase gene (hpt) controlled by the CaMV 35S promoter and the NOS polyadenylation region. Hygromycin-resistant cell masses arose from the thallus surface and developed directly into hygromycin-resistant thalli. Southern blot analyses indicated that these thalli carried at least 1-4 copies of the hpt gene, which were stably transmitted to their asexual thallus progenies via gemma propagation for three generations. This transformation and direct plant regeneration protocol is expected to be a valuable tool for the molecular analysis of this lower land plant.
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Shi, Kai; Li, Xin; Zhang, Guan-Qun; Xia, Xiao-Jian; Chen, Zhi-Xiang; Yu, Jing-Quan
2013-01-01
Plant RNA-dependent RNA Polymerase 1 (RDR1) is an important element of the RNA silencing pathway in the plant defense against viruses. RDR1 expression can be elicited by viral infection and salicylic acid (SA), but the mechanisms of signaling during this process remains undefined. The involvement of hydrogen peroxide (H2O2) and nitric oxide (NO) in RDR1 induction in the compatible interactions between Tobacco mosaic tobamovirus (TMV) and Nicotiana tabacum, Nicotiana benthamiana, and Arabidopsis thaliana was examined. TMV inoculation onto the lower leaves of N. tabacum induced the rapid accumulation of H2O2 and NO followed by the increased accumulation of RDR1 transcripts in the non-inoculated upper leaves. Pretreatment with exogenous H2O2 and NO on upper leaf led to increased RDR1 expression and systemic TMV resistance. Conversely, dimethylthiourea (an H2O2 scavenger) and 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (an NO scavenger) partly blocked TMV- and SA-induced RDR1 expression and increased TMV susceptibility, whereas pretreatment with exogenous H2O2 and NO failed to diminish TMV infection in N. benthamiana plants with naturally occurring RDR1 loss-of-function. Furthermore, in N. tabacum and A. thaliana, TMV-induced H2O2 accumulation was NO-dependent, whereas NO generation was not affected by H2O2. These results suggest that, in response to TMV infection, H2O2 acts downstream of NO to mediate induction of RDR1, which plays a critical role in strengthening RNA silencing to restrict systemic viral infection. PMID:24098767
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Diagnostics of the power oil-filled transformer equipment of thermal power plants
NASA Astrophysics Data System (ADS)
Eltyshev, D. K.; Khoroshev, N. I.
2016-08-01
Problems concerning improvement of the diagnostics efficiency of the electrical facilities and functioning of the generation and distribution systems through the examples of the power oil-filled transformers, as the responsible elements referring to the electrical part of thermal power plants (TPP), were considered. Research activity is based on the fuzzy logic system allowing working both with statistical and expert information presented in the form of knowledge accumulated during operation of the power oil-filled transformer facilities. The diagnostic algorithm for various types of transformers, with the use of the intellectual estimation model of its thermal state on the basis of the key diagnostic parameters and fuzzy inference hierarchy, was developed. Criteria for taking measures allowing preventing emergencies in the electric power systems were developed. The fuzzy hierarchical model for the state assessment of the power oil-filled transformers of 110 kV, possessing high degree of credibility and setting quite strict requirements to the limits of variables of the equipment diagnostic parameters, was developed. The most frequent defects of the transformer standard elements, related with the disturbance of the isolation properties and instrumentation operation, were revealed after model testing on the real object. Presented results may be used both for the express diagnostics of the transformers state without disconnection from the power line and for more detailed analysis of the defects causes on the basis of the advanced list of the diagnostic parameters; information on those parameters may be received only after complete or partial disconnection.
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Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing; Zhou, Xue-Rong; Shrestha, Pushkar; Divi, Uday K; Ral, Jean-Philippe; Mansour, Maged P; Nichols, Peter D; James, Christopher N; Horn, Patrick J; Chapman, Kent D; Beaudoin, Frederic; Ruiz-López, Noemi; Larkin, Philip J; de Feyter, Robert C; Singh, Surinder P; Petrie, James R
2014-01-01
High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. PMID:24151938
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USDA-ARS?s Scientific Manuscript database
Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce ...
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Persson, S; Wyatt, S E; Love, J; Thompson, W F; Robertson, D; Boss, W F
2001-07-01
To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.
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NASA Technical Reports Server (NTRS)
Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)
2001-01-01
To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.
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Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan
2013-01-01
A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.
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Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan
2013-01-01
A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926
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Ghanta, Srijani; Bhattacharyya, Dipto; Sinha, Ragini; Banerjee, Anindita; Chattopadhyay, Sharmila
2011-05-01
The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.
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Persson, Staffan; Wyatt, Sarah E.; Love, John; Thompson, William F.; Robertson, Dominique; Boss, Wendy F.
2001-01-01
To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+ uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+ accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of 45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium. PMID:11457960
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Cotton transformation via pollen tube pathway.
Wang, Min; Zhang, Baohong; Wang, Qinglian
2013-01-01
Although many gene transfer methods have been employed for successfully obtaining transgenic cotton, the major constraint in cotton improvement is the limitation of genotype because the majority of transgenic methods require plant regeneration from a single transformed cell which is limited by cotton tissue culture. Comparing with other plant species, it is difficult to induce plant regeneration from cotton; currently, only a limited number of cotton cultivars can be cultured for obtaining regenerated plants. Thus, development of a simple and genotype-independent genetic transformation method is particularly important for cotton community. In this chapter, we present a simple, cost-efficient, and genotype-independent cotton transformation method-pollen tube pathway-mediated transformation. This method uses pollen tube pathway to deliver transgene into cotton embryo sacs and then insert foreign genes into cotton genome. There are three major steps for pollen tube pathway-mediated genetic transformation, which include injection of -foreign genes into pollen tube, integration of foreign genes into plant genome, and selection of transgenic plants.
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An Examination of the Plastid DNA of Hypohaploid Nicotiana plumbaginifolia Plants
Cannon, Gordon C.; Van, K. Tran Thanh; Heinhorst, Sabine; Trinh, T. H.; Weissbach, Arthur
1989-01-01
DNA was extracted from different morphological types of hypohaploid Nicotiana plumbaginifolia plants. The cellular levels of chloroplast DNA (expressed as percent of total DNA) were found to be approximately two- to threefold higher in two albino hypohaploids than in a green hypohaploid. The level of chloroplast DNA in the green hypohaploid was not significantly different from either in vitro or in vivo grown haploid N. plumbaginifolia plants. Molecular hybridization with DNA probes for the large subunit of ribulose bisphosphate carboxylase from spinach and with Pvull fragments representing the entire Nicotiana tabacum chloroplast genome revealed no gross qualitative differences in the chloroplast DNAs of hypohaploid plants. Based on these observations we have concluded that the lack of chloroplast function observed in the albino forms of hypohaploid N. plumbaginifolia plants is not due to changes in the chloroplast genome. Images Figure 1 Figure 2 PMID:16666781
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Vanessa, Álvarez-López; Ángeles, Prieto-Fernández; Sergio, Roiloa; Beatriz, Rodríguez-Garrido; Rolf, Herzig; Markus, Puschenreiter; Susan, Kidd Petra
2017-03-01
We evaluated the effect of compost amendment and/or bacterial inoculants on the growth and metal accumulation of Salix caprea (clone BOKU 01 AT-004) and Nicotiana tabacum (in vitro-bred clone NBCu10-8). Soil was collected from an abandoned Pb/Zn mine and rhizobacterial inoculants were previously isolated from plants growing at the same site. Plants were grown in untreated or compost-amended (5% w/w) soil and were inoculated with five rhizobacterial strains. Non-inoculated plants were also established as a control. Compost addition increased the shoot DW yield of N. tabacum but not S. caprea, while it decreased soil metal availability and lowered shoot Cd/Zn concentrations in tobacco plants. Compost amendment enhanced the shoot Cd/Zn removal due to the growth promotion of N. tabacum or to the increase in metal concentration in S. caprea leaves. Bacterial inoculants increased photosynthetic efficiency (particularly in N. tabacum) and sometimes modified soil metal availability, but this did not lead to a significant increase in Cd/Zn removal. Compost amendment was more effective in improving the Cd and Zn phytoextraction efficiency than bioaugmentation.
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"Chitin-specific" peroxidases in plants.
Maksimov, I V; Cherepanova, E A; Khairullin, R M
2003-01-01
The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega(Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.
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Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong
2015-01-01
The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153
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Advancing Crop Transformation in the Era of Genome Editing.
Altpeter, Fredy; Springer, Nathan M; Bartley, Laura E; Blechl, Ann E; Brutnell, Thomas P; Citovsky, Vitaly; Conrad, Liza J; Gelvin, Stanton B; Jackson, David P; Kausch, Albert P; Lemaux, Peggy G; Medford, June I; Orozco-Cárdenas, Martha L; Tricoli, David M; Van Eck, Joyce; Voytas, Daniel F; Walbot, Virginia; Wang, Kan; Zhang, Zhanyuan J; Stewart, C Neal
2016-07-01
Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. © 2016 American Society of Plant Biologists. All rights reserved.
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Jeong, Yu Jeong; An, Chul Han; Woo, Su Gyeong; Park, Ji Hye; Lee, Ki-Won; Lee, Sang-Hoon; Rim, Yeonggil; Jeong, Hyung Jae; Ryu, Young Bae; Kim, Cha Young
2016-09-01
The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4'-O-β-D-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104-240 µg/g fresh weight in SM (F2). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.
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Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter
2015-01-01
Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.
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Decolorization of RBBR by plant cells and correlation with the transformation of PCBs.
Chroma, Ludmila; Macek, Tomas; Demnerova, Katerina; Macková, Martina
2002-11-01
An extracellular H2O2-requiring Remazol Brilliant Blue R (RBBR) decolorizing enzyme activity was detected after cultivation of cells of various plant species both in liquid medium and when growing on agar plates containing RBBR. Level of the enzyme activity was compared with the ability to metabolize polychlorinated biphenyls (PCBs). The ability to decolorize RBBR was tested in the presence and absence of PCBs. The cultures with high PCB-transforming activity proved to exhibit RBBR oxidase much more resistant towards the influence of PCBs. In addition low activities of lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) were detected in medium and in plant cells. No correlation of MnP and LiP activities with PCB degradation could be found. The RBBR decolorization could be used as a rough screening method for plant cultures able to metabolize PCBs.
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Largia, Muthiah Joe Virgin; Satish, Lakkakula; Johnsi, Rajaiah; Shilpha, Jayabalan; Ramesh, Manikandan
2016-08-01
Agrobacterium rhizogenes mediated transformation has been experimented in leaf explants of the memory herb Bacopa monnieri in order to assess the regeneration potential of hairy roots (HR) followed by the elicitation of transformed plants for increased Bacoside A production. Out of the four strains tested, A4 and MTCC 532 derived HR exhibited regrowth in MS basal medium while MTCC 2364 derived HR showed regeneration in MS medium supplemented with suitable phyto hormones. R1000 derived HR possessed no regeneration potential. Comparable to A4, MTCC 532 derived HR displayed maximum regrowth frequency of about 85.71 ± 1.84 % with an increase in biomass to threefold. Therefore, five HR plant lines (MTCC 532 derived) were generated and maintained in MS basal liquid medium in which HR3 topped the others in producing a huge biomass of about 67.09 ± 0.66 g FW. PCR amplification and southern hybridization analysis of rol A gene (280 bp) has been performed in order to confirm the transformation process. Moreover, HR3 plant line has accumulated highest total phenolic content of about 165.68 ± 0.82 mg GAE/g DW and highest total flavonoid content of about 497.78 ± 0.57 mg QRE/g DW when compared to other lines and untransformed controls. In addition, HR3 plant extract showed 85.58 ± 0.14 % of DPPH (2, 2-diphenyl-1-picryl hydrazyl) inhibition displaying its reliable anti oxidant potential. Further on elicitation with 10 mg/L chitosan for 2 weeks, HR3 has produced 5.83 % of Bacoside A which is fivefold and threefold increased production when compared to untransformed and transformed unelicited controls respectively. This is the first report on eliciting HR plants for increased metabolite accumulation in B. monnieri.
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Li, Le; Miao, Weiguo; Liu, Wenbo; Zhang, Shujian
2017-01-01
Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1–45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall. PMID:28141855
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Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue
NASA Technical Reports Server (NTRS)
Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.
1989-01-01
The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.
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Lücker, Joost; Schwab, Wilfried; van Hautum, Bianca; Blaas, Jan; van der Plas, Linus H. W.; Bouwmeester, Harro J.; Verhoeven, Harrie A.
2004-01-01
Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting β-pinene, limonene, and γ-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes. PMID:14718674
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Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A
2016-05-01
Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.
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Quiapim, Andréa C.; Brito, Michael S.; Bernardes, Luciano A.S.; daSilva, Idalete; Malavazi, Iran; DePaoli, Henrique C.; Molfetta-Machado, Jeanne B.; Giuliatti, Silvana; Goldman, Gustavo H.; Goldman, Maria Helena S.
2009-01-01
The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process. PMID:19052150
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Applications of optical manipulation in plant biology
NASA Astrophysics Data System (ADS)
Buer, Charles S.
. The spring tension of these strands was measured in normal and cold-hardened G. biloba and N. tabacum callus cells. There was little change in flexibility between the groups of cultured cells in either species studied. Microspheres were attached to Hechtian strands in normal cultured Nicotiana tabacum and the cells were deplasmolyzed and replasmolyzed to determine the fate of Hechtian strands. The microspheres either moved to the plasma membrane and adhered or moved to the cell wall and adhered. The attached microspheres occasionally moved independently on the same strand. Inserted microspheres provided a visual probe to follow physiological events within a plant cell.
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Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun
2014-01-01
Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631
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Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun
2014-08-22
Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.
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Plants having modified response to ethylene
Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.
1997-01-01
The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.
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Plants having modified response to ethylene
Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.
1998-10-20
The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 67 figs.
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Plants having modified response to ethylene
Meyerowitz, Elliot M.; Chang, Caren; Bleecker, Anthony B.
1998-01-01
The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.
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Plants having modified response to ethylene
Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.
1997-11-18
The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 31 figs.
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Advancing Crop Transformation in the Era of Genome Editing[OPEN
Blechl, Ann E.; Brutnell, Thomas P.; Conrad, Liza J.; Gelvin, Stanton B.; Jackson, David P.; Kausch, Albert P.; Lemaux, Peggy G.; Medford, June I.; Orozco-Cárdenas, Martha L.; Tricoli, David M.; Van Eck, Joyce; Voytas, Daniel F.
2016-01-01
Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. PMID:27335450
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Ritala, Anneli; Dong, Lemeng; Imseng, Nicole; Seppänen-Laakso, Tuulikki; Vasilev, Nikolay; van der Krol, Sander; Rischer, Heiko; Maaheimo, Hannu; Virkki, Arho; Brändli, Johanna; Schillberg, Stefan; Eibl, Regine; Bouwmeester, Harro; Oksman-Caldentey, Kirsi-Marja
2014-04-20
The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC-MS screening tool was developed for the rapid evaluation of production clones. The GC-MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 μg/g dry weight (DW) and a maximum of 31.3 μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag. Copyright © 2014 Elsevier B.V. All rights reserved.
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Ruiz-Vera, Ursula M; De Souza, Amanda P; Long, Stephen P; Ort, Donald R
2017-01-01
Down-regulation of photosynthesis is among the most common responses observed in C 3 plants grown under elevated atmospheric CO 2 concentration ([CO 2 ]). Down-regulation is often attributed to an insufficient capacity of sink organs to use or store the increased carbohydrate production that results from the stimulation of photosynthesis by elevated [CO 2 ]. Down-regulation can be accentuated by inadequate nitrogen (N) supply, which may limit sink development. While there is strong evidence for down-regulation of photosynthesis at elevated [CO 2 ] in enclosure studies most often involving potted plants, there is little evidence for this when [CO 2 ] is elevated fully under open-air field treatment conditions. To assess the importance of sink strength on the down-regulation of photosynthesis and on the potential of N to mitigate this down-regulation under agriculturally relevant field conditions, two tobacco cultivars ( Nicotiana tabacum L. cv. Petit Havana; cv. Mammoth) of strongly contrasting ability to produce the major sink of this crop, leaves, were grown under ambient and elevated [CO 2 ] and with two different N additions in a free air [CO 2 ] (FACE) facility. Photosynthetic down-regulation at elevated [CO 2 ] reached only 9% in cv. Mammoth late in the season likely reflecting sustained sink strength of the rapidly growing plant whereas down-regulation in cv. Petit Havana reached 25%. Increased N supply partially mitigated down-regulation of photosynthesis in cv. Petit Havana and this mitigation was dependent on plant developmental stage. Overall, these field results were consistent with the hypothesis that sustained sink strength, that is the ability to utilize photosynthate, and adequate N supply will allow C 3 crops in the field to maintain enhanced photosynthesis and therefore productivity as [CO 2 ] continues to rise.
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Schäfer, Holger; Ramamoorthy, Siva; Wink, Michael
2017-01-01
Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells. Two versions of rhEPO clones, with or without the N-terminal calreticulin (cal) fusion sequence, were produced by cloning the amplified construct into gateway binary vector pK7WG2D. Following Agrobacterium rhizogenes mediated transformation of tobacco explants; integration and expression of constructs in hairy roots were confirmed by several tests at DNA, RNA and protein levels. The amount of intracellular rhEPO from hairy root cultures with cal signal peptide was measured up to 66.75 ng g-1 of total soluble protein. The presence of the ER signal peptide (cal) was essential for the secretion of rhEPO into the spent medium; no protein was detected from hairy root cultures without ER signal peptide. The addition of polyvinylpyrrolidone enhanced the stabilization of secreted rhEPO leading to a 5.6 fold increase to a maximum concentration of 185.48 pg rhEPOHR g-1 FW hairy root cultures. The rhizo-secreted rhEPO was separated by HPLC and its biological activity was confirmed by testing distinct parameters for proliferation and survival in retinal pigment epithelial cells (ARPE). In addition, the rhEPO was detected to an amount 14.8 ng g-1 of total soluble leaf protein in transgenic T0 generation plantlets regenerated from hairy root cultures with cal signal peptide. PMID:28800637
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Biolistics Transformation of Wheat
NASA Astrophysics Data System (ADS)
Sparks, Caroline A.; Jones, Huw D.
We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).
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Kim, Ji Seong; Lee, Jeong Eun; Nie, Hualin; Lee, Yong Jae; Kim, Sun Tae; Kim, Sun-Hyung
2018-02-01
In this study, the effects of the plant growth-promoting rhizobacterium (PGPR), Bacillus sp. JS on the growth of tobacco (Nicotiana tabacum 'Xanthi') and lettuce (Lactuca sativa 'Crispa'), were evaluated by comparing various growth parameters between plants treated with the bacterium and those exposed to water or nutrient broth as control. In both tobacco and lettuce, fresh weight and length of shoots were increased upon exposure to Bacillus sp. JS. To explain the overall de novo expression of plant proteins by bacterial volatiles, two-dimensional gel electrophoresis was performed on samples from PGPR-treated tobacco plants. Our results showed that chlorophyll a/b binding proteins were significantly up-regulated, and total chlorophyll content was also increased. Our findings indicate the potential benefits of using Bacillus sp. JS as a growth-promoting factor in agricultural practice, and highlight the need for further research to explore these benefits.
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Pandey, Sonika; Patel, Manish Kumar; Jha, Bhavanath
2016-01-01
Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas. PMID:27411057
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Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath
2016-01-01
Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.
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Marchev, Andrey; Yordanova, Zhenya; Alipieva, Kalina; Zahmanov, Georgi; Rusinova-Videva, Snezhana; Kapchina-Toteva, Veneta; Simova, Svetlana; Popova, Milena; Georgiev, Milen I
2016-09-01
To develop a protocol to transform Verbascum eriophorum and to study the metabolic differences between mother plants and hairy root culture by applying NMR and processing the datasets with chemometric tools. Verbascum eriophorum is a rare species with restricted distribution, which is poorly studied. Agrobacterium rhizogenes-mediated genetic transformation of V. eriophorum and hairy root culture induction are reported for the first time. To determine metabolic alterations, V. eriophorum mother plants and relevant hairy root culture were subjected to comprehensive metabolomic analyses, using NMR (1D and 2D). Metabolomics data, processed using chemometric tools (and principal component analysis in particular) allowed exploration of V. eriophorum metabolome and have enabled identification of verbascoside (by means of 2D-TOCSY NMR) as the most abundant compound in hairy root culture. Metabolomics data contribute to the elucidation of metabolic alterations after T-DNA transfer to the host V. eriophorum genome and the development of hairy root culture for sustainable bioproduction of high value verbascoside.
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Lactoferrin derived resistance against plant pathogen in transgenic plants
USDA-ARS?s Scientific Manuscript database
Lactoferrin (LF) is a ubiquitous cationic iron-binding milk glycoprotein and it is known to exert a broad-spectrum primary defense activity against bacteria, fungi, protozoa and viruses in mammals. The Bovine lactoferrin gene was introduced to tobacco (Nicotiana tabacum var Xanthi), Arabidopsis (A. ...
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Ye, Shanwen; Cai, Changyang; Ren, Huibo; Wang, Wenjia; Xiang, Mengqi; Tang, Xiaoshan; Zhu, Caiping; Yin, Tengfei; Zhang, Li; Zhu, Qiang
2017-01-01
Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established. PMID:28798758
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Rott, Markus; Martins, Nádia F.; Thiele, Wolfram; Lein, Wolfgang; Bock, Ralph; Kramer, David M.; Schöttler, Mark A.
2011-01-01
Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b6f complex to the metabolic demand for ATP and NADPH. While the cytochrome b6f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential β-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b6f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO2 fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed. PMID:21278125
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USDA-ARS?s Scientific Manuscript database
A high-throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S), that contain selective marke...
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60. VIEW OF THE CURRENT TRANSFORMER VAULT. THIS CURRENT TRANSFORMER ...
60. VIEW OF THE CURRENT TRANSFORMER VAULT. THIS CURRENT TRANSFORMER WAS USED TO SENSE HIGH CURRENT BEING GENERATED ON GENERATOR NUMBER 3 AND REDUCE IT TO A LOWER, EXACT ANALOG VALUE THAT COULD BE SAFELY HANDLED AND MONITORED WITH THE CONTROL CIRCUITRY. THE CURRENT TRANSFORMER IS LOCATED IN THE CENTER OF THE PHOTOGRAPH. THE CONNECTING BUS ABOVE THE TRANSFORMER WAS REMOVED FOR SALVAGE. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT
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Ecker, Joseph R [Carlsbad, CA; Nehring, Ramlah [La Jolla, CA; McGrath, Robert B [Philadelphia, PA
2007-05-22
Nucleic acid and polypeptide sequences are described which relate to an EIN6 gene, a gene involved in the plant ethylene response. Plant transformation vectors and transgenic plants are described which display an altered ethylene-dependent phenotype due to altered expression of EIN6 in transformed plants.
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Genetic transformation protocols using zygotic embryos as explants: an overview.
Tahir, Muhammad; Waraich, Ejaz A; Stasolla, Claudio
2011-01-01
Genetic transformation of plants is an innovative research tool which has practical significance for the development of new and improved genotypes or cultivars. However, stable introduction of genes of interest into nuclear genomes depends on several factors such as the choice of target tissue, the method of DNA delivery in the target tissue, and the appropriate method to select the transformed plants. Mature or immature zygotic embryos have been a popular choice as explant or target tissue for genetic transformation in both angiosperms and gymnosperms. As a result, considerable protocols have emerged in the literature which have been optimized for various plant species in terms of transformation methods and selection procedures for transformed plants. This article summarizes the recent advances in plant transformation using zygotic embryos as explants.
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Smigocki, Ann C; Wilson, Dennis
2004-12-01
The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.
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Lubovská, Zuzana; Dobrá, Jana; Storchová, Helena; Wilhelmová, Naďa; Vanková, Radomíra
2014-11-01
Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations. Copyright © 2014 Elsevier GmbH. All rights reserved.
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Fukushige, Hirotada; Hildebrand, David F
2005-03-23
Fatty acid hydroperoxide lyase (HL) is the key enzyme for the production of the "green note"compounds, leaf aldehyde [(2E)-hexenal] and leaf alcohol [(3Z)-hexenol], in plant tissues. A cDNA encoding HL was cloned from leaves of watermelon (Citrullus lanatus) and expressed in Nicotiana tabacum. The enzyme is 3 times more active with 13-hydroperoxylinolenic acid than with 13-hydroperoxylinoleic acid. The activity against 9-hydroperoxides of polyunsaturated fatty acids is minimal. Enzyme activity of the watermelon HL in the transgenic leaves was approximately 50 times higher than endogenous HL activity in the wild-type N. tabacum plants. When compared with Arabidopsis HL also expressed in N. tabacum, the highest HL activity is 10 times higher in watermelon HL overexpressing leaves than in Arabidopsis HL overexpressers.
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Yassi, Annalee; Tate, Robert B; Routledge, Michael
2003-07-01
This study is an extension of a previously published analysis of cancer mortality in a transformer manufacturing plant where there had been extensive use of mineral oil transformer fluid. The objectives of the present study were to update the mortality analysis and include deaths for the past 6 years as well as to do an analysis of cancer incidence of the cohort. A cohort of 2,222 males working at a transformer manufacturing plant between 1946 and 1975 was constructed. Using a classical historical cohort study design, cancer incidence and mortality were determined through record linkage with Canadian provincial and national registries. The rates of cancer incidence and mortality experienced by this cohort were compared to that of the Canadian male population. A statistically significant increased risk of developing and dying of pancreatic cancer was found but not an increase in overall cancer mortality. This was consistent with the previous report from this group. Interestingly, the cohort demonstrated a statistically significant risk of overall cancer incidence and specific increased incidence of gallbladder cancer. This study contributes further evidence to the growing body of literature indicating the carcinogenic properties of mineral oils used in occupational settings, in particular those used prior to 1970s. Copyright 2003 Wiley-Liss, Inc.
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Evangelou, Michael W H; Bauer, Uwe; Ebel, Mathias; Schaeffer, Andreas
2007-06-01
Phytoextraction, the use of plants to extract contaminants from soils and groundwater, is a promising approach for cleaning up soils contaminated with heavy metals. In order to enhance phytoextraction the use of chelating agents has been proposed. This study aims to assess whether ethylene diamine disuccinate (EDDS), a biodegradable chelator, can be used for enhanced phytoextraction purposed, as an alternative to ethylene diamine tetraacetate (EDTA). EDDS revealed a higher toxicity to tobacco (Nicotiana tabacum) in comparison to EDTA, but no toxicity to microorganisms. The uptake of Cu was increased by the addition of EDTA and EDDS, while no increase was observed in the uptake of Cd. Both chelating agents showed a very low root to shoot translocation capability and the translocation factor was lower than the one of the control. Heavy metals where significantly more phytoavailable than in the control, even after harvesting, resulting in a high heavy metal leaching possibility, probably owing to a low biodegradation rate of EDDS. New seedlings which were transplanted into the EDDS treated pots 7d after the phytoextraction experiment, showed signs of necrosis and chlorosis, which resulted in a significantly lower biomass in comparison to the control. The seedlings on the EDTA treated pots showed no toxicity signs. Contrary to previous opinions the results of this study revealed the chelating agents EDTA and EDDS as unsuitable for enhanced phytoextraction using tobacco.
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Localization of arginine decarboxylase in tobacco plants.
Bortolotti, Cristina; Cordeiro, Alexandra; Alcázar, Rubén; Borrell, Antoni; Culiañez-Macià, Francisco A.; Tiburcio, Antonio F.; Altabella, Teresa
2004-01-01
The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli, and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.
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High Efficiency Transformation of Cultured Tobacco Cells 1
An, Gynheung
1985-01-01
Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants. Images Fig. 1 PMID:16664453
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Zhao, Yujun; Zhang, Yifeng; Su, Ping; Yang, Jian; Huang, Luqi; Gao, Wei
2017-01-01
Tripterygium wilfordii is a perennial woody liana medicinal plant with several crucial biological activities. Although studies on tissue culture have previously been conducted, research on genetic transformation is much more challenging and therefore results in slower progress. In the present study, a highly efficient transformation system involving the particle bombardment of T. wilfordii with the reporter egfp gene using the PDS-1000/He system was established. A total of seven parameters affecting the genetic transformation were investigated using an L 18 (6 × 3 6 )-type orthogonal array. The result indicated that DNA delivery conditions of 3-cm target distance, 1100 psi helium pressure, 28 mmHg chamber vacuum pressure, three times number of bombardment, CaCl 2 as precipitation agent, 2 μg plasmid DNA concentration and 48 h post-bombardment incubation time were optimal for T. wilfordii cell suspensions transformation. The average transformation efficiency was 19.17%. Based on this transformation system, the overexpression of two T. wilfordii farnesyl pyrophosphate synthase genes ( TwFPSs ) was performed in cell suspensions. Integration of the TwFPSs in the genome was verified by PCR analysis and also by Southern blotting using hygromycin gene as a probe. Real-time quantitative PCR analysis showed that the expression of TwFPS1&2 was highly up regulated in transgenic cell suspensions compared with control cells. The detection of metabolites showed that TwFPS1 & 2 could highly increase the celastrol content (973.60 μg/g) in transgenic cells. These results indicated that this transformation system is an effective protocol for characterizing the function of genes in the terpenoid biosynthetic pathway.
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Block, Anna; Guo, Ming; Li, Guangyong; Elowsky, Christian; Clemente, Thomas E.; Alfano, James R.
2009-01-01
Summary The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N-terminal two-thirds was sufficient for mitochondrial localization. A HopG1-GFP fusion lacking HopG1’s N-terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N-terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1’s target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions. PMID:19863557
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Sai, Liman; Liu, Siqi; Qian, Xuexue; Yu, Yahui; Xu, Xiaofeng
2018-05-21
In this study, water-soluble fluorescent carbon nanodots (CNDs) were directly injected into the leaf of nicotiana tabacum. With the help of UV-to-blue light conversion nanomaterial, the photosynthetic rate of the leaf was improved 18% upon additional 6 W UV irradiation. The photostability and toxicity of different kinds of CNDs were discussed. The results showed that CNDs functionalized with NH 2 -groups on their surfaces could maintain good fluorescence in plant leaf, and CNDs with complex surface groups tended to have high toxicity to the plant. The NH 2 -functionalized CNDs with non-toxicity and good photostability were used as in vivo light conversion material for direct utilization of UV light in the solar energy. Copyright © 2018 Elsevier B.V. All rights reserved.
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Genetic transformation of carnation (Dianthus caryophylus L.).
Nontaswatsri, Chalermsri; Fukai, Seiichi
2010-01-01
This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).
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Coursol, Sylvie; Fromentin, Jérôme; Noirot, Elodie; Brière, Christian; Robert, Franck; Morel, Johanne; Liang, Yun-Kuan; Lherminier, Jeannine; Simon-Plas, Françoise
2015-02-01
The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells. © 2014 INRA New Phytologist © 2014 New Phytologist Trust.
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Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.
Morozov, Sergey Y; Milyutina, Irina A; Bobrova, Vera K; Ryazantsev, Dmitry Y; Erokhina, Tatiana N; Zavriev, Sergey K; Agranovsky, Alexey A; Solovyev, Andrey G; Troitsky, Alexey V
2015-12-01
The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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Abanda-Nkpwatt, Daniel; Müsch, Martina; Tschiersch, Jochen; Boettner, Mewes; Schwab, Wilfried
2006-01-01
Four Methylobacterium extorquens strains were isolated from strawberry (Fragaria x ananassa cv. Elsanta) leaves, and one strain, called ME4, was tested for its ability to promote the growth of various plant seedlings. Seedling weight and shoot length of Nicotiana tabacum, Lycopersicon esculentum, Sinapis alba, and Fragaria vesca increased significantly in the presence of the pink-pigmented facultative methylotroph (PPFM), but the germination behaviour of seeds from six other plants was not affected. The cell-free supernatant of the bacterial culture stimulated germination, suggesting the production of a growth-promoting agent by the methylotroph. Methanol emitted from N. tabacum seedlings, as determined by proton-transfer-reaction mass spectrometry (PTR-MS), ranged from 0.4 to 0.7 ppbv (parts per billion by volume), while significantly lower levels (0.005 to 0.01 ppbv) of the volatile alcohol were measured when the seedlings were co-cultivated with M. extorquens ME4, demonstrating the consumption of the gaseous methanol by the bacteria. Additionally, by using cells of the methylotrophic yeast Pichia pastoris transformed with the pPICHS/GFP vector harbouring a methanol-sensitive promoter in combination with the green fluorescence protein (GFP) reporter gene, stomata were identified as the main source of the methanol emission on tobacco cotyledons. Methylobacterium extorquens strains can nourish themselves using the methanol released by the stomata and release an agent promoting the growth of the seedlings of some crop plants.
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Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima
2012-07-01
Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.
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Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y
2015-11-26
This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.
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[The application of genome editing in identification of plant gene function and crop breeding].
Zhou, Xiang-chun; Xing, Yong-zhong
2016-03-01
Plant genome can be modified via current biotechnology with high specificity and excellent efficiency. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system are the key engineered nucleases used in the genome editing. Genome editing techniques enable gene targeted mutagenesis, gene knock-out, gene insertion or replacement at the target sites during the endogenous DNA repair process, including non-homologous end joining (NHEJ) and homologous recombination (HR), triggered by the induction of DNA double-strand break (DSB). Genome editing has been successfully applied in the genome modification of diverse plant species, such as Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. In this review, we summarize the application of genome editing in identification of plant gene function and crop breeding. Moreover, we also discuss the improving points of genome editing in crop precision genetic improvement for further study.
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Lombari, P; Ercolano, E; El Alaoui, H; Chiurazzi, M
2003-04-01
We describe herein a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants. In this new procedure, dedifferentiated root explants, used as starting material, are the source of a large number of cells that are competent for the regeneration procedure, with a high susceptibility to Agrobacterium infection. The application of this protocol resulted in a tenfold increase in the number of transformants produced by a single plant in comparison to the widely used hypocotyl transformation procedure. Furthermore, our procedure allowed the use of intact plants stored for a long time at 4 degrees C, thus providing a potential continuous supply of explants for transformation experiments. The overall time of incubation under tissue culture conditions required to obtain a plant transferable into soil is 4 months. The transgenic nature of the transformants was demonstrated by the detection of beta-glucuronidase (GUS) activity in the primary transformants and by molecular analysis. Stable transformation was indicated by Mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of the transgenic plants.
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A dynamic model for plant growth: validation study under changing temperatures
NASA Technical Reports Server (NTRS)
Wann, M.; Raper, C. D. Jr; Raper CD, J. r. (Principal Investigator)
1984-01-01
A dynamic simulation model to describe vegetative growth of plants, for which some functions and parameter values have been estimated previously by optimization search techniques and numerical experimentation based on data from constant temperature experiments, is validated under conditions of changing temperatures. To test the predictive capacity of the model, dry matter accumulation in the leaves, stems, and roots of tobacco plants (Nicotiana tabacum L.) was measured at 2- or 3-day intervals during a 5-week period when temperatures in controlled-environment rooms were programmed for changes at weekly and daily intervals and in ascending or descending sequences within a range of 14 to 34 degrees C. Simulations of dry matter accumulation and distribution were carried out using the programmed changes for experimental temperatures and compared with the measured values. The agreement between measured and predicted values was close and indicates that the temperature-dependent functional forms derived from constant-temperature experiments are adequate for modelling plant growth responses to conditions of changing temperatures with switching intervals as short as 1 day.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lagrimini, L.M.
Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionicmore » peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H{sub 2}O{sub 2} via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.« less
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He, Zhu-Mei; Jiang, Xiao-Ling; Qi, Yu; Luo, Di-Qing
2008-06-01
To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.
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Effect of bacterial inoculants on phytomining of metals from waste incineration bottom ash.
Rosenkranz, Theresa; Kidd, Petra; Puschenreiter, Markus
2018-03-01
Waste incineration bottom ash is considered a secondary resource for valuable trace elements (TE), which is currently neglected in most European countries. Phytomining could potentially recover valuable TE from such waste materials but is still at an exploratory stage with many challenges. The use of bioaugmentation to improve plant growth and TE accumulation of metal-tolerant high biomass plants growing on waste incineration bottom ash was evaluated. Bacterial strains that were previously isolated from rhizosphere, roots and contaminated soil were selected according to their plant growth promoting characteristics and tolerance to the bottom ash substrate. Those selected bacterial strains were tested for their beneficial effects on Nicotiana tabacum and Salix smithiana with regards to phytomining. The rhizobacterial strain Rhodococcus erythropolis P30 enhanced the shoot dry weight of N. tabacum by on average 57% compared to the control plants. Several bacterial inoculants enhanced biomass production and the nutritional status of S. smithiana. Moreover, those bacterial strains previously described to enhance biomass production of N. tabacum and members of the Salicaceae on TE-contaminated soils, also enhanced biomass production of these species on bottom ash. However, bacterial inoculants could not enhance trace element accumulation in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Alinia, F; Ghareyazie, B; Rubia, L; Bennett, J; Cohen, M B
2000-04-01
The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous rice pests--the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young larvae of S. incertulas, C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C. suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.
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Advancing Crop Transformation in the Era of Genome Editing
USDA-ARS?s Scientific Manuscript database
Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than thirty years of technological advances. Genome editing provides new opportunities to...
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NASA Astrophysics Data System (ADS)
Karaliūnas, Mindaugas; Jakštas, Vytautas; Nasser, Kinan E.; Venckevičius, Rimvydas; Urbanowicz, Andrzej; Kašalynas, Irmantas; Valušis, Gintaras
2016-09-01
In this work, a comparative research of biologically active organic molecules in its natural environment using the terahertz (THz) time domain spectroscopy (TDS) and Fourier transform spectroscopy (FTS) systems is carried out. Absorption coefficient and refractive index of Nicotiana tabacum L. leaves containing nicotine, Cannabis sativa L. leaves containing tetrahydrocannabinol, and Humulu lupulus L. leaves containing α-acids, active organic molecules that obtain in natural environment, were measured in broad frequency range from 0.1 to 13 THz at room temperature. In the spectra of absorption coefficient the features were found to be unique for N. tabacum, C. sativa and H. lupulus. Moreover, those features can be exploited for identification of C. sativa sex and N. tabacum origin. The refractive index can be also used to characterize different species.
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Liu, Ying; Liu, Guoxuan; Yang, Yali; Niu, Sufang; Yang, Fuguang; Yang, Shaoxia; Tang, Jianian; Chen, Jianping
2017-12-01
An efficient and reproducible protocol is described for shoot-bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of J. curcas. Treating the explants with high concentrations (5-120 mg/L) of TDZ for short durations (5-80 min) before inoculation culture increased significantly the regeneration frequency and improved the quality of the regenerated buds. The highest shoot-buds induction rate (87.35%) was achieved when petiole explants were treated with 20 mg/L TDZ solution for 20 min and inoculated on hormone-free MS medium for 30 days. Regenerated shoots of 0.5 cm or a little longer were isolated and grafted to seedling stocks of the same species, and then the grafted plantlets were planted on half-strength MS medium containing 0.1 mg/L IBA and 2 mg/L sodium nitroprusside (SNP). This grafting strategy was found to be very effective, to obtain that healthy grafted plantlets ready for acclimatization within 20 days. By the above mentioned protocol and with general Agrobacterium - mediated genetic transformation methods only 65 days were needed to obtain intact transgenic plants.
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Chen, Dan; Ren, Yujun; Deng, Yingtian; Zhao, Jie
2010-01-01
Auxin is an important plant growth regulator, and plays a key role in apical–basal axis formation and embryo differentiation, but the mechanism remains unclear. The level of indole-3-acetic acid (IAA) during zygote and embryo development of Nicotiana tabacum L. is investigated here using the techniques of GC-SIM-MS analysis, immunolocalization, and the GUS activity assay of DR5::GUS transgenic plants. The distribution of ABP1 and PM H+-ATPase was also detected by immunolocalization, and this is the first time that integral information has been obtained about their distribution in the zygote and in embryo development. The results showed an increase in IAA content in ovules and the polar distribution of IAA, ABP1, and PM H+-ATPase in the zygote and embryo, specifically in the top and basal parts of the embryo proper (EP) during proembryo development. For information about the regulation mechanism of auxin, an auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid) and exogenous IAA were, respectively, added to the medium for the culture of ovules at the zygote and early proembryo stages. Treatment with a suitable IAA concentration promoted zygote division and embryo differentiation, while TIBA treatment obviously suppressed these processes and caused the formation of abnormal embryos. The distribution patterns of IAA, ABP1, and PM H+-ATPase were also disturbed in the abnormal embryos. These results indicate that the polar distribution and transport of IAA begins at the zygote stage, and affects zygote division and embryo differentiation in tobacco. Moreover, ABP1 and PM H+-ATPase may play roles in zygote and embryo development and may also be involved in IAA signalling transduction. PMID:20348352
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Sogutmaz Ozdemir, Bahar; Budak, Hikmet
2018-01-01
Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.
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Yang, Li Yun; Yang, Shuang Long; Li, Jun Ying; Ma, Jun Hong; Pang, Tao; Zou, Cong Ming; He, Bin; Gong, Ming
2018-02-05
Temperature remarkably affects the growth and metabolism of plants. Tobacco is an important cash crop, and the long-term effects of different growth temperatures (18.5, 23.5 and 28.5 °C, daily average) on growth, development and plastid pigments metabolism of tobacco plants were investigated in this study. Compared with tobacco plants grown under 23.5 °C, treatments with 18.5 and 28.5 °C inhibited the expansion of leaves. The contents of superoxide anion (O 2 ·- ), hydrogen peroxide (H 2 O 2 ) and malonaldehyde (MDA) in the leaves were significantly increased under 28.5 °C from 0 to 60 days, which in turn accelerated the flowering and senescence of tobacco plants. By contrast, the treatment with 18.5 °C remarkably decreased O 2 .- , H 2 O 2 and MDA, and delayed the flowering and senescence. Furthermore, treatment with 18.5 °C significantly up-regulated the expression of glutamyl-tRNA reductase (Glu-TR) and magnesium chelatase (MgCH), and down-regulated the ferri chelatase (FeCH), protochlorophyllide oxidoreductase, chlorophyllase (CHLase), phaeophorbide a monooxygenase (PaO) and phytoene synthase (PSY), which further promoted the accumulation of chlorophyll (Chls) and reduced the carotenoids (Cars) in leaves. On the contrary, exposing to 28.5 °C remarkably down-regulated the Glu-TR and MgCH, and up-regulated the FeCH, CHLase, PaO and PSY, which in turn decreased the Chls and increased the Cars in tobacco leaves. As compared with the plants grown under 23.5 °C, lower (18.5 °C) and higher (28.5 °C) growth temperature inhibited the growth of tobacco plants. In general, treatment with 28.5 °C accelerated the flowering and senescence of tobacco plants by enhancing the accumulation of O 2 .- and H 2 O 2 in leaves, while exposing to 18.5 °C had the opposite effects. Treatment with 18.5 °C increased the content of Chls and reduced the Cars in leaves. In contrast, Treatment with 28.5 °C decreased the Chls and increased the Cars. Moreover, both
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Swain, S S; Sahu, L; Pal, A; Barik, D P; Pradhan, C; Chand, P K
2012-02-01
Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD(660) ≅ 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.
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Antibody degradation in tobacco plants: a predominantly apoplastic process
2011-01-01
Background Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. Results In this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. Conclusions The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions. PMID:22208820
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Somatic mosaicism in plants with special reference to somatic crossing over
Vig, Baldev K.
1978-01-01
Plant systems in use for the detection of environmental mutagens appear capable of detecting all types of genetic effects which can be studied in animals. The study of somatic mosaicism, however, is better developed in plants than in higher animals. A case is presented here which shows the ability of plant systems in analyzing a host of genetic end points, including chromosome aberrations like deletions, somatic crossing over, numerical inequality, gene conversion, paramutations and point mutations. The systems in general use utilize certain varieties of Tradescantia, Glycine max, Nicotiana tabacum, Antirrhinum majus, Petunia hybrida, and Arabidopsis thaliana. Heterozygous plants or their homozygous counterparts with gene markers affecting chlorophyll development or anthocyanin in floral parts are exploited in these studies. Mutagens produce different frequencies of different types of spots typical of the mode of action of the agent. Analysis of these parameters may be used to predict, at least qualitatively, the kind of genetic damage that might be produced in man. Besides, one can test the validity of interpretation by traditional progeny tests of plants raised from tissue culture from sectors as in Nicotiana and/or by precursor analysis as done in Antirrhinum. The study of mosaicism in plants offers quite inexpensive, rapid, and reliable tests of mutagenicity at least as a preliminary eukaryotic test system. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 9. PMID:367771
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Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.
Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly
2013-01-01
Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.
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Progress of cereal transformation technology mediated by Agrobacterium tumefaciens.
Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko
2014-01-01
Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.
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Progress of cereal transformation technology mediated by Agrobacterium tumefaciens
Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko
2014-01-01
Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132
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Genetic Transformation of Switchgrass
NASA Astrophysics Data System (ADS)
Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu
Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.
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Genetic transformation of switchgrass.
Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu
2009-01-01
Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.
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Xiong, Yehui; Zeng, Hongmei; Zhang, Yuliang; Xu, Dawei; Qiu, Dewen
2013-01-01
RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests. PMID:23630449
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DOE Office of Scientific and Technical Information (OSTI.GOV)
P Yu
Unlike traditional 'wet' analytical methods which during processing for analysis often result in destruction or alteration of the intrinsic protein structures, advanced synchrotron radiation-based Fourier transform infrared microspectroscopy has been developed as a rapid and nondestructive and bioanalytical technique. This cutting-edge synchrotron-based bioanalytical technology, taking advantages of synchrotron light brightness (million times brighter than sun), is capable of exploring the molecular chemistry or structure of a biological tissue without destruction inherent structures at ultra-spatial resolutions. In this article, a novel approach is introduced to show the potential of the advanced synchrotron-based analytical technology, which can be used to study plant-basedmore » food or feed protein molecular structure in relation to nutrient utilization and availability. Recent progress was reported on using synchrotron-based bioanalytical technique synchrotron radiation-based Fourier transform infrared microspectroscopy and diffused reflectance infrared Fourier transform spectroscopy to detect the effects of gene-transformation (Application 1), autoclaving (Application 2), and bio-ethanol processing (Application 3) on plant-based food and feed protein structure changes on a molecular basis. The synchrotron-based technology provides a new approach for plant-based protein structure research at ultra-spatial resolutions at cellular and molecular levels.« less
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Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M.; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima
2012-01-01
Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection. PMID:22822208
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Yao, Youli; Danna, Cristian H.; Zemp, Franz J.; Titov, Viktor; Ciftci, Ozan Nazim; Przybylski, Roman; Ausubel, Frederick M.; Kovalchuk, Igor
2011-01-01
We have previously shown that local exposure of plants to stress results in a systemic increase in genome instability. Here, we show that UV-C–irradiated plants produce a volatile signal that triggers an increase in genome instability in neighboring nonirradiated Arabidopsis thaliana plants. This volatile signal is interspecific, as UV-C–irradiated Arabidopsis plants transmit genome destabilization to naive tobacco (Nicotiana tabacum) plants and vice versa. We report that plants exposed to the volatile hormones methyl salicylate (MeSA) or methyl jasmonate (MeJA) exhibit a similar level of genome destabilization as UV-C–irradiated plants. We also found that irradiated Arabidopsis plants produce MeSA and MeJA. The analysis of mutants impaired in the synthesis and/or response to salicylic acid (SA) and/or jasmonic acid showed that at least one other volatile compound besides MeSA and MeJA can communicate interplant genome instability. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (npr1) mutant, defective in SA signaling, is impaired in both the production and the perception of the volatile signals, demonstrating a key role for NPR1 as a central regulator of genome stability. Finally, various forms of stress resulting in the formation of necrotic lesions also generate a volatile signal that leads to genomic instability. PMID:22028460
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Ponndorf, Daniel; Broer, Inge; Nausch, Henrik
2017-08-01
Increasing the arginine (Arg) content in plants used as feed or food is of interest, since the supplementation of food with conditionally essential Arg has been shown to have nutritional benefits. An increase was achieved by the expression of the Arg-rich bacterial storage component, cyanophycin (CGP), in the chloroplast of transgenic plants. CGP is stable in plants and its degradation into β-aspartic acid (Asp)-Arg dipeptides, is solely catalyzed by bacterial cyanophycinases (CGPase). Dipeptides can be absorbed by animals even more efficiently than free amino acids (Matthews and Adibi 1976; Wenzel et al. 2001). The simultaneous production of CGP and CGPase in plants could be a source of β-Asp-Arg dipeptides if CGP degradation can be prevented in planta or if dipeptides are stable in the plants. We have shown for the first time that it is possible to co-express CGP and CGPase in the same plant without substrate degradation in planta by transient expression of the cyanobacterial CGPase CPHB (either in the plastid or cytosol), and the non-cyanobacterial CGPase CPHE (cytosol) in CGP-producing Nicotiana tabacum plants. We compared their ability to degrade CGP in planta and in crude plant extracts. No CGP degradation appeared prior to cell homogenization independent of the CGPase produced. In crude plant extracts, only cytosolic CPHE led to a fast degradation of CGP. CPHE also showed higher stability and in vitro activity compared to both CPHB variants. This work is the next step to increase Arg in forage plants using a stable, Arg-rich storage protein.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
LACKS,S.A.
2003-10-09
Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants wasmore » revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).« less
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Li, Wei-zheng; Teng, Xiao-hui; Zhang, Hong-fei; Liu, Ting; Wang, Qiong; Guo, Xian-ru
2017-01-01
We tested the behavioral responses of ovipositing females and natal larvae of two sibling species, a generalist Helicoverpa armigera (Hübner) and a specialist Helicoverpa assulta (Guenée), to odor sources emitted from different combinations of six plant species (tobacco, Nicotiana tabacum; hot pepper, Capsicum annuum; tomato, Solanum esculentum; cotton, Gossypium hirsutum; peanut, Arachis hypogaea; maize, Zea mays). Under the conditions of plant materials versus corresponding controls, both stages of both species could find their corresponding host plants. However, H. assulta females and larvae exhibited a supersensitive and an insensitive response, respectively. Under the conditions of tobacco paired with each plant species, H. assulta females exhibited more specialized ovipositional response to tobacco than its sibling. When each plant species were combined with tobacco and tested against tobacco reference, peanut played an opposite role in the two species in their ovipositional responses to tobacco, and cotton can enhance the approaching response of H. armigera larvae when combined with tobacco. It seems that two attractive host plants also can act antagonistically with respect to host selection of the generalist via volatile exchange. Tomato should better be excluded from host list of H. assulta. PMID:28182679
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Jiang, X; Ou, Z; Ying, P; Yediler, A; Ketrrup, A
2001-06-01
The transportation and transformation of 14C-phenanthrene in a closed 'plant-lava-nutrient solution-air' chamber system was studied by using radioactivity technology. The results showed that in this closed chamber system, phenanthrene was degraded fast. The radioactivity of 14C left at 23d in the nutrient solution was only 25% of applied. At the end of experiment (46d), the distribution sequence of 14C activity in the components of closed chamber system was root (38.55%) > volatile organic compounds (VOCs, 17.68%) > lava (14.35%) > CO2 (11.42%) > stem (2%). 14C-activities in plant tissue were combined with the tissue, and existed in the forms of lava-bound(root 4.68%; stem and leaves 0.68%) and polar metabolites (root 23.14%; stem 0.78%).
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Cheng, X; Sardana, R; Kaplan, H; Altosaar, I
1998-03-17
Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.
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7. TRANSFORMER BANK. NOTE CONCRETE BARRIER WALL FROM WWII ON ...
7. TRANSFORMER BANK. NOTE CONCRETE BARRIER WALL FROM WWII ON LEFT TO PROTECT TRANSFORMERS FROM JAPANESE BOMBS. - Gene Pump Plant, South of Gene Wash Reservoir, 2 miles west of Whitsett Pump Plant, Parker Dam, San Bernardino County, CA
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Tobacco as platform for a commercial production of cyanophycin.
Nausch, Henrik; Hausmann, Tina; Ponndorf, Daniel; Hühns, Maja; Hoedtke, Sandra; Wolf, Petra; Zeyner, Annette; Broer, Inge
2016-12-25
Cyanophycin (CP) is a proteinogenic polymer that can be substituted for petroleum in the production of plastic compounds and can also serve as a source of valuable dietary supplements. However, because there is no economically feasible system for large-scale industrial production, its application is limited. In order to develop a low-input system, CP-synthesis was established in the two commercial Nicotiana tabacum (N. tabacum) cultivars 'Badischer Geudertheimer' (BG) and 'Virginia Golta' (VG), by introducing the cyanophycin-synthetase gene from Thermosynecchococcus elongatus BP-1 (CphA Te ) either via crossbreeding with transgenic N. tabacum cv. Petit Havana SR1 (PH) T2 individual 51-3-2 or by agrobacterium-mediated transformation. Both in F1 hybrids (max. 9.4% CP/DW) and T0 transformants (max. 8.8% CP/DW), a substantial increase in CP content was achieved in leaf tissue, compared to a maximum of 1.7% CP/DW in PH T0 transformants of Hühns et al. (2008). In BG CP, yields were homogenous and there was no substantial difference in the variation of the CP content between primary transformants (T0), clones of T0 individuals, T1 siblings and F1 siblings of hybrids. Therefore, BG meets the requirements for establishing a master seed bank for continuous and reliable CP-production. In addition, it was shown that the polymer is not only stable in planta but also during silage, which simplifies storage of the harvest prior to isolation of CP. Copyright © 2016 Elsevier B.V. All rights reserved.
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Bultreys, Alain; Trombik, Tomasz; Drozak, Anna; Boutry, Marc
2009-09-01
SUMMARY The behaviour of Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 was investigated in response to fungal and oomycete infections. The importance of NpPDR1 in plant defence was demonstrated for two organs in which NpPDR1 is constitutively expressed: the roots and the petal epidermis. The roots of the plantlets of two lines silenced for NpPDR1 expression were clearly more sensitive than those of controls to the fungal pathogens Botrytis cinerea, Fusarium oxysporum sp., F. oxysporum f. sp. nicotianae, F. oxysporum f. sp. melonis and Rhizoctonia solani, as well as to the oomycete pathogen Phytophthora nicotianae race 0. The Ph gene-linked resistance of N. plumbaginifolia to P. nicotianae race 0 was totally ineffective in NpPDR1-silenced lines. In addition, the petals of the NpPDR1-silenced lines were spotted 15%-20% more rapidly by B. cinerea than were the controls. The rapid induction (after 2-4 days) of NpPDR1 expression in N. plumbaginifolia and N. tabacum mature leaves in response to pathogen presence was demonstrated for the first time with fungi and one oomycete: R. solani, F. oxysporum and P. nicotianae. With B. cinerea, such rapid expression was not observed in healthy mature leaves. NpPDR1 expression was not observed during latent infections of B. cinerea in N. plumbaginifolia and N. tabacum, but was induced when conditions facilitated B. cinerea development in leaves, such as leaf ageing or an initial root infection. This work demonstrates the increased sensitivity of NpPDR1-silenced N. plumbaginifolia plants to all of the fungal and oomycete pathogens investigated.
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Cereal transformation through particle bombardment
NASA Technical Reports Server (NTRS)
Casas, A. M.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.; Mitchell, C. A. (Principal Investigator)
1995-01-01
The review focuses on experiments that lead to stable transformation in cereals using microprojectile bombardment. The discussion of biological factors that affect transformation examines target tissues and vector systems for gene transfer. The vector systems include reporter genes, selectable markers, genes of agronomic interest, and vector constructions. Other topics include physical parameters that affect DNA delivery, selection of stably transformed cells and plant regeneration, and analysis of gene expression and transmission to the progeny.
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A novel histone variant localized in nucleoli of higher plant cells.
Tanaka, I; Akahori, Y; Gomi, K; Suzuki, T; Ueda, K
1999-07-01
Immunofluorescence staining with antisera raised against p35, a basic nuclear protein that accumulates in the pollen nuclei of Lilium longiflorum, specifically stained the nucleoli in interphase nuclei of somatic tissues, including root and leaf, and in pachytene nuclei during meiotic division, whereas antisera raised against histone H1 uniformly stained the entire chromatin domain with the exception of the nucleoli in these nuclei. Further, p35-specific antisera stained the nucleoli in root and leaf nuclei of the monocotyledonous plants Tulipa gesneriana, Allium cepa and Triticum aestivum and of the dicotyledonous plants Vicia faba and Nicotiana tabacum. Thus, these novel antisera stained the nucleoli in cells of all higher plants examined, although the staining patterns within nucleoli were somewhat different among plant species and tissues. The full-length cDNA of p35 was cloned on the basis of the partial amino acid sequence. The deduced amino acid composition and amino acid sequence of p35 indicate that this nucleolar protein is a novel variant of histone Hl. Further, p35 was strongly bound to ribosomal DNA in vitro. The results of immunoblotting of histones extracted from each tissue of the various plant species with the nucleolus-specific antibodies also suggested the conservation of similar epitope(s) in both mono- and dicotyledonous plants. From these results, it is suggested that similar variants of histone Hl are specifically distributed in the nucleoli of all plant species and help to organize the nucleolar chromatin.
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Peng, Cheng; Xu, Chen; Liu, Qinglin; Sun, Lijuan; Luo, Yongming; Shi, Jiyan
2017-05-02
Agricultural soil is gradually becoming a primary sink for metal-based nanoparticles (MNPs). The uptake and accumulation of MNPs by crops may contaminate food chain and pose unexpected risks for human health. Here, we investigated the fate and transformation of CuO nanoparticles (NPs) in the soil-rice system during the rice lifecycle. The results show that at the maturation stage, 1000 mg/kg CuO NPs significantly decreased redox potential by 202.75 mV but enhanced electrical conductivity by 497.07 mS/cm compared to controls. Moreover, the bioavailability of highest CuO NPs in the soil was reduced by 69.84% along with the plant growth but then was significantly increased by 165% after drying-wetting cycles. Meanwhile, CuO and Cu combined with humic acid were transformed to Cu 2 S and Cu associated with goethite by X-ray absorption near edge structure analysis. Additionally, CuO NPs had an acute negative effect on the plant growth than bulk particles, which dramatically reduced the fresh weight of grains to 6.51% of controls. Notably, CuO NPs were found to be translocated from soil to plant especially to the chaff and promoted the Cu accumulation in the aleurone layer of rice using micro X-ray fluorescence technique, but could not reach the polished rice.
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Chandra Rai, Avinash; Singh, Major; Shah, Kavita
2012-12-01
Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessive production alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C(2)H(2) type Zinc Finger Protein (ZFP) reported to control the expression of several stress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86 variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant and consistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increased proline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxide concentrations in the transformed tomato plants did not alter much, suggesting a well regulated formation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformed tomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation of osmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, the ZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regions affected with severe drought stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.
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Lu, You; Hatsugai, Noriyuki; Katagiri, Fumiaki; Ishimaru, Carol A; Glazebrook, Jane
2015-11-01
Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.
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Early events in Agrobacterium-mediated genetic transformation of citrus explants.
Peña, Leandro; Pérez, Rosa M; Cervera, Magdalena; Juárez, José A; Navarro, Luis
2004-07-01
Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.
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Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.
Michelmore, R; Marsh, E; Seely, S; Landry, B
1987-12-01
Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.
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Badhan, Ajay; Wang, Yuxi; McAllister, Tim A
2017-01-01
Fourier transformed mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls (Acebes et al., Front Plant Sci 5:303, 2014; Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Badhan et al., BioMed Res Int 2015: 562952, 2015; Roach et al., Plant Physiol 156:1351-1363, 2011). The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here, we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. This method has been successfully used to study complex enzyme saccharification processes like rumen digestion to identify recalcitrant moieties in low-quality forage which resist rumen digestion (Badhan et al., BioMed Res Int 2015: 562952, 2015), as well as to characterize cell wall mutant lines or transgenic lines expressing exogenous hydrolases (Badhan et al., Biotechnol Biofuels 7:1-15, 2014; Roach et al., Plant Physiol 156:1351-1363, 2011). The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low cost and nondestructive manner.
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Tercé-Laforgue, Thérèse; Bedu, Magali; Dargel-Grafin, Céline; Dubois, Frédéric; Gibon, Yves; Restivo, Francesco M; Hirel, Bertrand
2013-10-01
Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.
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Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines
Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.
2014-01-01
Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433
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Wheat (Triticum aestivum L.) transformation using immature embryos.
Ishida, Yuji; Tsunashima, Masako; Hiei, Yukoh; Komari, Toshihiko
2015-01-01
Wheat may now be transformed very efficiently by Agrobacterium tumefaciens. Under the protocol hereby described, immature embryos of healthy plants of wheat cultivar Fielder grown in a well-conditioned greenhouse were pretreated with centrifuging and cocultivated with A. tumefaciens. Transgenic wheat plants were obtained routinely from between 40 and 90 % of the immature embryos, thus infected in our tests. All regenerants were normal in morphology and fully fertile. About half of the transformed plants carried single copy of the transgene, which are inherited by the progeny in a Mendelian fashion.
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Gene transfer and expression in plants.
Lorence, Argelia; Verpoorte, Robert
2004-01-01
Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).
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Chloride regulates leaf cell size and water relations in tobacco plants
Franco-Navarro, Juan D.; Brumós, Javier; Rosales, Miguel A.; Cubero-Font, Paloma; Talón, Manuel; Colmenero-Flores, José M.
2016-01-01
Chloride (Cl–) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl– when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl–-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5mM Cl–) and no water limitation, Cl– specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1–5mM range, Cl– played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl– also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl–, these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl– responds to adaptive functions improving water homeostasis in higher plants. PMID:26602947
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Metabolism of 2,4-dichlorophenol in tobacco engineered with bacterial degradative genes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Perkins, E.J.; Sekine, M.; Gordon, M.P.
1990-05-01
The potential use of plants in toxic waste remediation has been overlooked. While chlorophenols are relatively slowly metabolized in Nicotiana tabacum var. Xanthi leaf extracts, chlorocatechols are rapidly metabolized, presumably by polyphenol oxidases. Our initial focus has been the fate of 2,4-dichlorophenol (2,4DCP) in var. Xanthi plants which express a bacterial 2,4DCP hydroxylase, which converts 2,4DCP to 3,5-dichlorocatechol. The roots of wild type and 2,4DCP hydroxylase transgenic plants growing in hydroponics were exposed to {sup 14}C-2,4DCP. Approximately 95% of {sup 14}C-2,4DCP metabolites remained in the roots when exposed to 2,4DCP. Upon extraction of root tissue, three major metabolites were foundmore » in untransformed plants and four major metabolites in transformed plants. Upon digestion with beta-D-glucosidase, these metabolites disappeared concomitant with the appearance of free 2,4DCP in wild type plants and 2,4DCP and 3,5-dichlorocatechol in transgenic plants. It is apparent that the chlorophenols are not readily available substrates for polyphenol oxidases in whole plants.« less
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Frugivore-Mediated Selection in A Habitat Transformation Scenario
Fontúrbel, Francisco E.; Medel, Rodrigo
2017-01-01
Plant-animal interactions are strong drivers of phenotypic evolution. However, the extent to which anthropogenic habitat transformation creates new selective scenarios for plant-animal interactions is a little explored subject. We examined the effects of native forest replacement by exotic Eucalyptus trees on the frugivore-mediated phenotypic selection coefficients imposed by the relict marsupial Dromiciops gliroides upon traits involved in frugivore attraction and germination success of the mistletoe Tristerix corymbosus (Loranthaceae). We found significant gradients for seed weight and sugar content along the native - transformed habitat gradient. While selection for larger seed weight was more relevant in native habitats, fruits with intermediate sugar content were promoted in transformed habitats. The spatial habitat structure and microclimate features such as the degree of sunlight received influenced the natural selection processes, as they correlated with the phenotypic traits analysed. The response of this plant-frugivore interaction to human disturbance seemed to be context-dependent, in which extremely transformed habitats would offer new opportunities for natural selection on dispersal-related traits. Even in recent transformation events like this, human disturbance acts as a strong contemporary evolution driver. PMID:28349942
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Ohadi R, Mehrnaz S; Alvari, Amene; Samim, M; Abdin, Malik Z
2013-03-01
Encapsulation of plasmid DNA in nanoparticle is expected to enhance the stability of DNA, reproducibility and frequency of the genetic transformation in plants. Here we report the formulation of HMG Co-A reductase gene loaded calcium phosphate nanoparticles (Cap nanoparticles) and their in-vitro, in-vivo characterization. The developed Cap nanoparticles were characterized by DSC, FT-IR, and XRD. Developed Cap nanoparticles were spherical in shape having the particle size and zeta potential in the range of 10.86±0.09nm to 33.42±0.18nm and -25.5±0.07mV to -31.7±0.07mV (for Cap-I to Cap-IV). DNA releasing in acidic media showed, initially slow release followed by fast release with a maximum release of Cap-I (95.77±1.39%) > Cap-II (87.32±2.07%) > Cap-III (76.54±2.01%) > Cap-IV (72.93±1.75%) over 60min. Cap nanoparticles were quite stable at storage condition of 40±0.5°C/75±5%RH, 25±0.5°C/60±RH, 4±0.5°C/ambient humidity and the integrity of pDNA encapsulated was confirmed by gel electrophoresis. Compared to wild type C. intybus, transformation efficiency and enhanced biosynthesis of esculin with the DNA nanoparticles in C. intybus were about 10% and 71%, respectively. Antioxidant activity capacity of the biotransformed plants was significantly higher than the normal plant due to high accumulation of esculin.
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The use of comet assay in plant toxicology: recent advances
Santos, Conceição L. V.; Pourrut, Bertrand; Ferreira de Oliveira, José M. P.
2015-01-01
The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage. PMID:26175750
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Criqui, Marie Claire; de Almeida Engler, Janice; Camasses, Alain; Capron, Arnaud; Parmentier, Yves; Inzé, Dirk; Genschik, Pascal
2002-01-01
The anaphase promoting complex or cyclosome is the ubiquitin-ligase that targets destruction box-containing proteins for proteolysis during the cell cycle. Anaphase promoting complex or cyclosome and its activator (the fizzy and fizzy-related) proteins work together with ubiquitin-conjugating enzymes (UBCs) (E2s). One class of E2s (called E2-C) seems specifically involved in cyclin B1 degradation. Although it has recently been shown that mammalian E2-C is regulated at the protein level during the cell cycle, not much is known concerning the expression of these genes. Arabidopsis encodes two genes belonging to the E2-C gene family (called UBC19 and UBC20). We found that UBC19 is able to complement fission yeast (Schizosaccharomyces pombe) UbcP4-140 mutant, indicating that the plant protein can functionally replace its yeast ortholog for protein degradation during mitosis. In situ hybridization experiments were performed to study the expression of the E2-C genes in various tissues of plants. Their transcripts were always, but not exclusively, found in tissues active for cell division. Thus, the UBC19/20 E2s may have a key function during cell cycle, but may also be involved in ubiquitylation reactions occurring during differentiation and/or in differentiated cells. Finally, we showed that a translational fusion protein between UBC19 and green fluorescent protein localized both in the cytosol and the nucleus in stable transformed tobacco (Nicotiana tabacum cv Bright Yellow 2) cells. PMID:12427990
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Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation
NASA Astrophysics Data System (ADS)
Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina
This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.
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The Bacterial Pathogen Xylella fastidiosa Affects the Leaf Ionome of Plant Hosts during Infection
De La Fuente, Leonardo; Parker, Jennifer K.; Oliver, Jonathan E.; Granger, Shea; Brannen, Phillip M.; van Santen, Edzard; Cobine, Paul A.
2013-01-01
Xylella fastidiosa is a plant pathogenic bacterium that lives inside the host xylem vessels, where it forms biofilm believed to be responsible for disrupting the passage of water and nutrients. Here, Nicotiana tabacum was infected with X. fastidiosa, and the spatial and temporal changes in the whole-leaf ionome (i.e. the mineral and trace element composition) were measured as the host plant transitioned from healthy to diseased physiological status. The elemental composition of leaves was used as an indicator of the physiological changes in the host at a specific time and relative position during plant development. Bacterial infection was found to cause significant increases in concentrations of calcium prior to the appearance of symptoms and decreases in concentrations of phosphorous after symptoms appeared. Field-collected leaves from multiple varieties of grape, blueberry, and pecan plants grown in different locations over a four-year period in the Southeastern US showed the same alterations in Ca and P. This descriptive ionomics approach characterizes the existence of a mineral element-based response to X. fastidiosa using a model system suitable for further manipulation to uncover additional details of the role of mineral elements during plant-pathogen interactions. This is the first report on the dynamics of changes in the ionome of the host plant throughout the process of infection by a pathogen. PMID:23667547
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Tight regulation of plant immune responses by combining promoter and suicide exon elements
Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; ...
2015-07-02
Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightlymore » regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.« less
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Tight regulation of plant immune responses by combining promoter and suicide exon elements
Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; Staskawicz, Brian J.; Loqué, Dominique; Hammond, Ming C.
2015-01-01
Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes. PMID:26138488
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Tight regulation of plant immune responses by combining promoter and suicide exon elements
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.
Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightlymore » regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.« less
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Large scale screening of commonly used Iranian traditional medicinal plants against urease activity
2012-01-01
Background and purpose of the study H. pylori infection is an important etiologic impetus usually leading to gastric disease and urease enzyme is the most crucial role is to protect the bacteria in the acidic environment of the stomach. Then urease inhibitors would increase sensitivity of the bacteria in acidic medium. Methods 137 Iranian traditional medicinal plants were examined against Jack bean urease activity by Berthelot reaction. Each herb was extracted using 50% aqueous methanol. The more effective extracts were further tested and their IC50 values were determined. Results 37 plants out of the 137 crude extracts revealed strong urease inhibitory activity (more than 70% inhibition against urease activity at 10 mg/ml concentration). Nine of the whole studied plants crude extracts were found as the most effective with IC50 values less than 500 μg/ml including; Rheum ribes, Sambucus ebulus, Pistachia lentiscus, Myrtus communis, Areca catechu, Citrus aurantifolia, Myristica fragrans, Cinnamomum zeylanicum and Nicotiana tabacum. Conclusions The most potent urease inhibitory was observed for Sambucus ebulus and Rheum ribes extracts with IC50 values of 57 and 92 μg/ml, respectively. PMID:23351780
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Biolistic transformation of Scoparia dulcis L.
Srinivas, Kota; Muralikrishna, Narra; Kumar, Kalva Bharath; Raghu, Ellendula; Mahender, Aileni; Kiranmayee, Kasula; Yashodahara, Velivela; Sadanandam, Abbagani
2016-01-01
Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.
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Genetic transformation of Dichanthium annulatum (Forssk)--an apomictic tropical forage grass.
Dalton, S J; Bettany, A J E; Bhat, V; Gupta, M G; Bailey, K; Timms, E; Morris, P
2003-06-01
Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.
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Schep, Leo J; Slaughter, Robin J; Beasley, D Michael G
2009-09-01
A wide range of plants contain nicotinic and nicotinic-like alkaloids. Of this diverse group, those that have been reported to cause human poisoning appear to have similar mechanisms of toxicity and presenting patients therefore have comparable toxidromes. This review describes the taxonomy and principal alkaloids of plants that contain nicotinic and nicotinic-like alkaloids, with particular focus on those that are toxic to humans. The toxicokinetics and mechanisms of toxicity of these alkaloids are reviewed and the clinical features and management of poisoning due to these plants are described. This review was compiled by systematically searching OVID MEDLINE and ISI Web of Science. This identified 9,456 papers, excluding duplicates, all of which were screened. Reviewed plants and their principal alkaloids. Plants containing nicotine and nicotine-like alkaloids that have been reported to be poisonous to humans include Conium maculatum, Nicotiana glauca and Nicotiana tabacum, Laburnum anagyroides, and Caulophyllum thalictroides. They contain the toxic alkaloids nicotine, anabasine, cytisine, n-methylcytisine, coniine, n-methylconiine, and gamma-coniceine. These alkaloids act agonistically at nicotinic-type acetylcholine (cholinergic) receptors (nAChRs). The nicotinic-type acetylcholine receptor can vary both in its subunit composition and in its distribution within the body (the central and autonomic nervous systems, the neuromuscular junctions, and the adrenal medulla). Agonistic interaction at these variable sites may explain why the alkaloids have diverse effects depending on the administered dose and duration of exposure. Nicotine and nicotine-like alkaloids are absorbed readily across all routes of exposure and are rapidly and widely distributed, readily traversing the blood-brain barrier and the placenta, and are freely distributed in breast milk. Metabolism occurs predominantly in the liver followed by rapid renal elimination. Following acute exposure
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Transformation of mercury speciation through the SCR system in power plants.
Yang, Hong-min; Pan, Wei-ping
2007-01-01
Coal-fired utility boilers are now identified as the largest source of mercury in the United States. There is speculation that the installation of selective catalytic reduction (SCR) system for reduction of NOx can also prompt the oxidation and removal of mercury. In this paper, tests at six full-scale power plants with similar type of the SCR systems are conducted to investigate the effect of the SCR on the transformation of mercury speciation. The results show that the SCR system can achieve more than 70%-80% oxidation of elemental mercury and enhance the mercury removal ability in these units. The oxidation of elemental mercury in the SCR system strongly depends on the coal properties and the operation conditions of the SCR systems. The content of chloride in the coal is the key factor for the oxidization process and the maximum oxidation of elemental mercury is found when chloride content changes from 400 to 600 ppm. The sulfur content is no significant impact on oxidation of elemental mercury.
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Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.
Lowe, Keith; Wu, Emily; Wang, Ning; Hoerster, George; Hastings, Craig; Cho, Myeong-Je; Scelonge, Chris; Lenderts, Brian; Chamberlin, Mark; Cushatt, Josh; Wang, Lijuan; Ryan, Larisa; Khan, Tanveer; Chow-Yiu, Julia; Hua, Wei; Yu, Maryanne; Banh, Jenny; Bao, Zhongmeng; Brink, Kent; Igo, Elizabeth; Rudrappa, Bhojaraja; Shamseer, P M; Bruce, Wes; Newman, Lisa; Shen, Bo; Zheng, Peizhong; Bidney, Dennis; Falco, S Carl; RegisterIII, James C; Zhao, Zuo-Yu; Xu, Deping; Jones, Todd J; Gordon-Kamm, William James
2016-09-06
While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously non-transformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated by using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa var. indica) callus. {copyright, serif} 2016 American Society of Plant Biologists. All rights reserved.
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Citrus transformation using juvenile tissue explants.
Orbović, Vladimir; Grosser, Jude W
2015-01-01
The most frequently used method for production of citrus transgenic plants is via Agrobacterium-mediated transformation of tissues found on explants obtained from juvenile seedlings. Within the last decade and especially within the last 5-6 years, this robust method was employed to produce thousands of transgenic plants. With the newly applied screening methods that allow easier and faster detection of transgenic shoots, estimates of transformation rate for some cultivars have gone up making this approach even more attractive. Although adjustments have to be made regarding the (varietal) source of the starting material and Agrobacterium strain used in each experiment preformed, the major steps of this procedure have not changed significantly if at all. Transgenic citrus plants produced this way belong to cultivars of rootstocks, sweet oranges, grapefruits, mandarins, limes, and lemons.
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Yu, Lin-Hui; Wu, Jie; Tang, Hui; Yuan, Yang; Wang, Shi-Mei; Wang, Yu-Ping; Zhu, Qi-Sheng; Li, Shi-Gui; Xiang, Cheng-Bin
2016-06-13
Nitrogen is essential for plant survival and growth. Excessive application of nitrogenous fertilizer has generated serious environment pollution and increased production cost in agriculture. To deal with this problem, tremendous efforts have been invested worldwide to increase the nitrogen use ability of crops. However, only limited success has been achieved to date. Here we report that NLP7 (NIN-LIKE PROTEIN 7) is a potential candidate to improve plant nitrogen use ability. When overexpressed in Arabidopsis, NLP7 increases plant biomass under both nitrogen-poor and -rich conditions with better-developed root system and reduced shoot/root ratio. NLP7-overexpressing plants show a significant increase in key nitrogen metabolites, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signalling. More importantly, overexpression of NLP7 also enhances photosynthesis rate and carbon assimilation, whereas knockout of NLP7 impaired both nitrogen and carbon assimilation. In addition, NLP7 improves plant growth and nitrogen use in transgenic tobacco (Nicotiana tabacum). Our results demonstrate that NLP7 significantly improves plant growth under both nitrogen-poor and -rich conditions by coordinately enhancing nitrogen and carbon assimilation and sheds light on crop improvement.
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Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi
2017-01-01
Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T 1 and T 2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T 2 progenies and PCR-identified Apr gene segregation in T 2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.
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Duarte-Galvan, Carlos; Romero-Troncoso, Rene de J; Torres-Pacheco, Irineo; Guevara-Gonzalez, Ramon G; Fernandez-Jaramillo, Arturo A; Contreras-Medina, Luis M; Carrillo-Serrano, Roberto V; Millan-Almaraz, Jesus R
2014-10-09
Soil drought represents one of the most dangerous stresses for plants. It impacts the yield and quality of crops, and if it remains undetected for a long time, the entire crop could be lost. However, for some plants a certain amount of drought stress improves specific characteristics. In such cases, a device capable of detecting and quantifying the impact of drought stress in plants is desirable. This article focuses on testing if the monitoring of physiological process through a gas exchange methodology provides enough information to detect drought stress conditions in plants. The experiment consists of using a set of smart sensors based on Field Programmable Gate Arrays (FPGAs) to monitor a group of plants under controlled drought conditions. The main objective was to use different digital signal processing techniques such as the Discrete Wavelet Transform (DWT) to explore the response of plant physiological processes to drought. Also, an index-based methodology was utilized to compensate the spatial variation inside the greenhouse. As a result, differences between treatments were determined to be independent of climate variations inside the greenhouse. Finally, after using the DWT as digital filter, results demonstrated that the proposed system is capable to reject high frequency noise and to detect drought conditions.
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Flexer, Victoria; Mano, Nicolas
2010-02-15
We propose here a new method for the direct and continuous measurement of O(2) and glucose generated during photosynthesis. Our system is based on amperometric enzyme biosensors comprising immobilized redox enzymes (glucose oxidase (GOx) and bilirubin oxidase (BOD)) and redox hydrogels "wiring" the enzyme reaction centers to electrodes. We found that these electrodes, implanted into a living plant, responded in real time to visible light as an external stimulus triggering photosynthesis. They proved to be highly selective and fast enough and may be a valuable tool in understanding photosynthesis kinetics. Furthermore, we demonstrate that with our electrodes we could harvest glucose and O(2) produced during photosynthesis to produce energy, transforming sunlight into electricity in a simple, green, renewable, and sustainable way.
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Persistence of Agrobacterium tumefaciens in transformed conifers.
Charity, Julia A; Klimaszewska, Krystyna
2005-01-01
Previous studies have shown that the widely used plant transformation vector Agrobacterium tumefaciens can persist in genetically engineered plants in vitro and in transgenic greenhouse-grown plants, despite the use of counter-selective antibiotics. However, little is known regarding Agrobacterium persistence in tree species. To understand the kinetics of A. tumefaciens decline and persistence in transformation experiments, we assayed for the presence of A. tumefaciens in spruce and pine embryogenic tissue for up to 10 weeks post-transformation. The A. tumefaciens populations declined rapidly in the first five days post-cocultivation but generally declined more slowly in pine, relative to spruce. No bacteria were detected in spruce embryogenic tissue beyond four weeks after cocultivation, however in pine there were -100 colony forming units per g tissue at 10 weeks post-cocultivation. We present evidence that the detection limit for PCR using virD2 primers to detect A. tumefaciens in a background of pine needle DNA was approximately 10(9)-10(10) A. tumefaciens cells per g of tissue. We also assayed for A. tumefaciens in transgenic pine and spruce embryogenic tissue and from needles, branches, stems and roots of transformed plants, up to four years post-inoculation. Occasionally A. tumefaciens was detected in embryogenic tissue up to 12 months post-inoculation. A. tumefaciens was never detected in cultured embryogenic tissue more than twelve months after inoculation, nor in developing somatic embryos or germinating plantlets, nor any of the parts of greenhouse-grown plants. From these data we conclude that if A. tumefaciens persists in transgenic conifers, it does so beneath our ability to detect it.
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Lermontova, Inna; Grimm, Bernhard
2000-01-01
The use of herbicides to control undesirable vegetation has become a universal practice. For the broad application of herbicides the risk of damage to crop plants has to be limited. We introduced a gene into the genome of tobacco (Nicotiana tabacum) plants encoding the plastid-located protoporphyrinogen oxidase of Arabidopsis, the last enzyme of the common tetrapyrrole biosynthetic pathway, under the control of the cauliflower mosaic virus 35S promoter. The transformants were screened for low protoporphyrin IX accumulation upon treatment with the diphenyl ether-type herbicide acifluorfen. Leaf disc incubation and foliar spraying with acifluorfen indicated the lower susceptibility of the transformants against the herbicide. The resistance to acifluorfen is conferred by overexpression of the plastidic isoform of protoporphyrinogen oxidase. The in vitro activity of this enzyme extracted from plastids of selected transgenic lines was at least five times higher than the control activity. Herbicide treatment that is normally inhibitory to protoporphyrinogen IX oxidase did not significantly impair the catalytic reaction in transgenic plants and, therefore, did not cause photodynamic damage in leaves. Therefore, overproduction of protoporphyrinogen oxidase neutralizes the herbicidal action, prevents the accumulation of the substrate protoporphyrinogen IX, and consequently abolishes the light-dependent phytotoxicity of acifluorfen. PMID:10631251
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Successful Gene Tagging in Lettuce Using the Tnt1 Retrotransposon from Tobacco
Mazier, Marianne; Botton, Emmanuel; Flamain, Fabrice; Bouchet, Jean-Paul; Courtial, Béatrice; Chupeau, Marie-Christine; Chupeau, Yves; Maisonneuve, Brigitte; Lucas, Hélène
2007-01-01
The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3β-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome. PMID:17351058
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Rothe, Grit; Hachiya, Akira; Yamada, Yasuyuki; Hashimoto, Takashi; Dräger, Birgit
2003-09-01
Putrescine N-methyltransferase (PMT) is the first alkaloid-specific enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 microM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.
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Rudder, Steven; Doohan, Fiona; Creevey, Christopher J; Wendt, Toni; Mullins, Ewen
2014-04-07
Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).
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Zhou, Xue-mei; Zhao, Peng; Wang, Wei; Zou, Jie; Cheng, Tian-he; Peng, Xiong-bo; Sun, Meng-xiang
2015-01-01
Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses. PMID:26205094
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Transformation of fruit trees. Useful breeding tool or continued future prospect?
Petri, César; Burgos, Lorenzo
2005-02-01
Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genesfrom the transformed plant as a research priority in coming years.
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Engineering a Catabolic Pathway in Plants for the Degradation of 1,2-Dichloroethane1[OA
Mena-Benitez, Gilda L.; Gandia-Herrero, Fernando; Graham, Stuart; Larson, Tony R.; McQueen-Mason, Simon J.; French, Christopher E.; Rylott, Elizabeth L.; Bruce, Neil C.
2008-01-01
Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum ‘Xanthi’) plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater. PMID:18467461
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NASA Astrophysics Data System (ADS)
Musharraf, Syed Ghulam; Ali, Arslan; Choudhary, M. Iqbal; Atta-ur-Rahman
2014-04-01
Natural products continue to serve as an important source of novel drugs since the beginning of human history. High-throughput techniques, such as MALDI-MS, can be techniques of choice for the rapid screening of natural products in plant materials. We present here a fast and reproducible matrix-free approach for the direct detection of UV active metabolites in plant materials without any prior sample preparation. The plant material is mechanically ground to a fine powder and then sieved through different mesh sizes. The collected plant material is dispersed using 1 μL solvent on a target plate is directly exposed to Nd:YAG 335 nm laser. The strategy was optimized for the analysis of plant metabolites after study of the different factors affecting the reproducibility and effectiveness of the analysis, including particle sizes effects, types of solvents used to disperse the sample, and the part of the plant analyzed. Moreover, several plant species, known for different classes of metabolites, were screened to establish the generality of the approach. The developed approach was validated by the characterization of withaferin A and nicotine in the leaves of Withania somnifera and Nicotiana tabacum, respectively, through comparison of its MS/MS data with the standard compound. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques were used for the tissue imaging purposes. This approach can be used to directly probe small molecules in plant materials as well as in herbal and pharmaceutical formulations for fingerprinting development.
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Biolistic- and Agrobacterium-mediated transformation protocols for wheat.
Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László
2012-01-01
After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.
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Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration
Ningxia Du; Paula M. Pijut
2009-01-01
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...
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NASA Astrophysics Data System (ADS)
Wang, Quan; Serban, Andrew J.; Wachter, Rebekka M.; Moerner, W. E.
2018-03-01
Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ˜0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.
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Wang, Quan; Serban, Andrew J; Wachter, Rebekka M; Moerner, W E
2018-03-28
Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s -1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.
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Gondek, Krzysztof; Baran, Agnieszka; Kopeć, Michał
2014-12-01
The aim of the study was to determine the influence of the process of low-temperature transformation and the addition of plant material to sewage sludge diversifying the content of mobile forms of heavy metals and their ecotoxicity. The experimental design included: sewage sludge+rape straw, sewage sludge+wheat straw, sewage sludge+sawdust, sewage sludge+bark and sewage sludge with no addition. The mixtures were subjected to thermal transformation in a chamber furnace, under conditions without air. The procedure consisted of two stages: the first stage (130°C for 40 min) focused on drying the material, whereas in the second stage (200°C for 30 min) proper thermal transformation of materials took place. Thermal transformation of the materials, caused an increase in total contents of heavy metals in comparison to the material before transformation. From among elements, the cadmium content changed the most in materials after thermal transformation. As a result of thermal transformation, the content of water soluble form of the heavy metals decreased significantly in all the prepared mixtures. Low toxicity of the extracts from materials for Vibrio fischeri and Lepidium sativum was found in the research, regardless of transformation process. L. sativum showed higher sensitivity to heavy metals occurring in the studied extracts from materials than V. fischeri, evidence of which are the positive significant correlations between the content of metals and the inhibition of root growth of L. sativum. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Liang, Hong-Ming; Lin, Ting-Hsiang; Chiou, Jeng-Min; Yeh, Kuo-Chen
2009-06-01
Evaluation of the remediation ability of zinc/cadmium in hyper- and non-hyperaccumulator plant species through greenhouse studies is limited. To bridge the gap between greenhouse studies and field applications for phytoextraction, we used published data to examine the partitioning of heavy metals between plants and soil (defined as the bioconcentration factor). We compared the remediation ability of the Zn/Cd hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri and the non-hyperaccumulators Nicotiana tabacum and Brassica juncea using a hierarchical linear model (HLM). A recursive algorithm was then used to evaluate how many harvest cycles were required to clean a contaminated site to meet Taiwan Environmental Protection Agency regulations. Despite the high bioconcentration factor of both hyperaccumulators, metal removal was still limited because of the plants' small biomass. Simulation with N. tabacum and the Cadmium model suggests further study and development of plants with high biomass and improved phytoextraction potential for use in environmental cleanup.
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Xu, Jia-Yi; Han, Yong-He; Chen, Yanshan; Zhu, Ling-Jia; Ma, Lena Q
2016-02-01
The ability of As-resistant endophytic bacteria in As transformation and plant growth promotion was determined. The endophytes were isolated from As-hyperaccumulator Pteris vittata (PV) after growing for 60 d in a soil containing 200 mg kg(-1) arsenate (AsV). They were isolated in presence of 10 mM AsV from PV roots, stems, and leaflets, representing 4 phyla and 17 genera. All endophytes showed at least one plant growth promoting characteristics including IAA synthesis, siderophore production and P solubilization. The root endophytes had higher P solubilization ability than the leaflet (60.0 vs. 18.3 mg L(-1)). In presence of 10 mM AsV, 6 endophytes had greater growth than the control, suggesting As-stimulated growth. Furthermore, root endophytes were more resistant to AsV while the leaflet endophytes were more tolerant to arsenite (AsIII), which corresponded to the dominant As species in PV tissues. Bacterial As resistance was positively correlated to their ability in AsV reduction but not AsIII oxidation. The roles of those endophytes in promoting plant growth and As resistance in P. vittata warrant further investigation. Published by Elsevier Ltd.
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Diaz-Vivancos, Pedro; Faize, Lydia; Nicolás, Emilio; Clemente-Moreno, Maria José; Bru-Martinez, Roque; Burgos, Lorenzo; Hernández, José Antonio
2016-01-01
Background and Aims Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. Methods The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. Key Results Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. Conclusions The transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field
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Transformation of PRT6 RNAi construct into tomato (Solanum lycopersicum) cv. Micro-Tom
NASA Astrophysics Data System (ADS)
Suka, Intan Elya; Chew, Bee Lynn; Goh, Hoe-Han; Isa, Nurulhikma Md
2018-04-01
PROTEOLYSIS 6 plays major role in the N-end rule pathway as N-recognin which functions as E3 ligase enzyme. It mediates ubiquitin processes that lead to degradation of unstable substrate protein. The aim of the current study is to transform the PRT6 gene into tomato (Solanum lycopersicum) from the cultivar Micro-Tom and to investigate its function in regulating ripening in tomato fruits. The PRT6_RNAi construct was successfully transformed into Agrobacterium C58 via heat shock method and transformed into seven days old cotyledon explants. Factors affecting transformation efficiency such as co-cultivation time and type of plant growth regulator combination were evaluated. Results from this study found that pre-cultured cotyledons from seven days old seedlings incubated for 2 days in co-cultivation medium increased shoot regeneration. Plant growth hormones zeatin combine with auxin produced a higher number of callus formation but lower shoot proliferation and transformation frequency compared to treatments of single plant hormone in the selection medium. Polymerase chain reaction (PCR) was performed on the regenerated shoots to confirm the integration of PRT6 fragment into the genome of transgenic plants. Based on PCR analysis, all putative shoots were positive transformants.
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Melatonin redirects carbohydrates metabolism during sugar starvation in plant cells.
Kobylińska, Agnieszka; Borek, Sławomir; Posmyk, Małgorzata M
2018-05-01
Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin efficacy eliminates oxidative stress (direct and indirect antioxidant) and moreover induce plant stress reaction and switch on different defence strategies (preventively and interventively actions). In this report, the impact of exogenous melatonin on carbohydrate metabolism in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells during sugar starvation was examined. We analysed starch concentration, α-amylase and PEPCK activity as well as proteolytic activity in culture media. It has been shown that BY-2 cell treatment with 200 nM of melatonin improved viability of sugar-starved cells. It was correlated with higher starch content and phosphoenolpyruvate carboxykinase (PEPCK) activity. The obtained results revealed that exogenous melatonin under specific conditions (stress) can play regulatory role in sugar metabolism, and it may modulate carbohydrate concentration in etiolated BY-2 cells. Moreover, our results confirmed the hypothesis that if the starch is synthesised even in sugar-starved cells, it is highly probable that melatonin shifts the BY-2 cell metabolism on gluconeogenesis pathway and allows for synthesis of carbohydrates from nonsugar precursors, that is amino acids. These points to another defence strategy that was induced by exogenous melatonin applied in plants to overcome adverse environmental conditions. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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2014-01-01
Background Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. Results The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. Conclusions This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT). PMID:24708309
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Nictaba Homologs from Arabidopsis thaliana Are Involved in Plant Stress Responses
Eggermont, Lore; Stefanowicz, Karolina; Van Damme, Els J. M.
2018-01-01
Plants are constantly exposed to a wide range of environmental stresses, but evolved complicated adaptive and defense mechanisms which allow them to survive in unfavorable conditions. These mechanisms protect and defend plants by using different immune receptors located either at the cell surface or in the cytoplasmic compartment. Lectins or carbohydrate-binding proteins are widespread in the plant kingdom and constitute an important part of these immune receptors. In the past years, lectin research has focused on the stress-inducible lectins. The Nicotiana tabacum agglutinin, abbreviated as Nictaba, served as a model for one family of stress-related lectins. Here we focus on three non-chimeric Nictaba homologs from Arabidopsis thaliana, referred to as AN3, AN4, and AN5. Confocal microscopy of ArathNictaba enhanced green fluorescent protein (EGFP) fusion constructs transiently expressed in N. benthamiana or stably expressed in A. thaliana yielded fluorescence for AN4 and AN5 in the nucleus and the cytoplasm of the plant cell, while fluorescence for AN3 was only detected in the cytoplasm. RT-qPCR analysis revealed low expression for all three ArathNictabas in different tissues throughout plant development. Stress application altered the expression levels, but all three ArathNictabas showed a different expression pattern. Pseudomonas syringae infection experiments with AN4 and AN5 overexpression lines demonstrated a significantly higher tolerance of several transgenic lines to P. syringae compared to wild type plants. Finally, AN4 was shown to interact with two enzymes involved in plant defense, namely TGG1 and BGLU23. Taken together, our data suggest that the ArathNictabas represent stress-regulated proteins with a possible role in plant stress responses. On the long term this research can contribute to the development of more stress-resistant plants. PMID:29375596
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Shih, Po-Yuan; Chou, Shu-Jen; Müller, Caroline; Halkier, Barbara Ann; Deeken, Rosalia; Lai, Erh-Min
2018-03-02
Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation. © 2018 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.
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Didierjean, Luc; Gondet, Laurence; Perkins, Roberta; Lau, Sze-Mei Cindy; Schaller, Hubert; O'Keefe, Daniel P.; Werck-Reichhart, Danièle
2002-01-01
The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. PMID:12226498
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[Studies on the role of silicic acid in the development of higher plants].
Werner, D
1967-03-01
Germanium acid, a specific inhibitor of the silicic acid metabolism in diatoms, inhibits the growth of Sinapis alba, Lemna minor, Wolffia arrhiza, Nicotiana tabacum, Tradescantia spec, Zinnia elegans, and Secale cereale when applied in the same concentrations as those used in the case of diatoms (15-75 μg GeO2/ml medium). The growth of Aspergillus niger, Phycomyces blakesleanus, Escherichia coli K 12, Euglena gracilis and Pandorina morum is not influenced by these and higher concentrations of Germanium acid. By application of high concentrations of silicic acid, the growth inhibition produced by germanium acid in Lemna minor is partially reduced. Plants of Lemna minor which have been inhibited by germanium acid are essentially smaller than plants grown in a normal medium; their chlorophyll content is significantly decreased. The growth of the roots in Lemna is particularly inhibited. Isolated growing roots of Lycopersicon pimpinellifolium MILL. are inhibited by small concentrations of Ge(OH)4 (ca. 1,5×10(-4) M/l). In contrast to the growth of older plants, the germination of Secale cereale and Sinapis alba is not influenced by Ge(OH)4. The effects of germanium acid are discussed in relation to the physiological role of silicic acid. The results suggest that the element silicon, in the form of silicic acid, is generally essential for the normal development of higher plants.
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Kosjek, Tina; Negreira, Noelia; Heath, Ester; López de Alda, Miren; Barceló, Damià
2018-01-01
This study aims to identify (bio)transformation products of vincristine, a plant alkaloid chemotherapy drug. A batch biotransformation experiment was set-up using activated sludge at two concentration levels with and without the addition of a carbon source. Sample analysis was performed on an ultra-high performance liquid chromatograph coupled to a high-resolution hybrid quadrupole-Orbitrap tandem mass spectrometer. To identify molecular ions of vincristine transformation products and to propose molecular and chemical structures, we performed data-dependent acquisition experiments combining full-scan mass spectrometry data with product ion spectra. In addition, the use of non-commercial detection and prediction algorithms such as MZmine 2 and EAWAG-BBD Pathway Prediction System, was proven to be proficient for screening for transformation products in complex wastewater matrix total ion chromatograms. In this study eleven vincristine transformation products were detected, nine of which were tentatively identified. Copyright © 2017 Elsevier B.V. All rights reserved.
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High-frequency transformation of Lobelia erinus L. by Agrobacterium-mediated gene transfer.
Tsugawa, H; Kagami, T; Suzuki, M
2004-05-01
A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a beta-glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3-4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high--45% per inoculated disc. Copyright 2004 Springer-Verlag
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NASA Astrophysics Data System (ADS)
Dwi Sri Hastuti, Liana; Faull, Jane
2018-03-01
A pot experiment was carried out to test the effectiveness of nematode-trapping fungi (NTF) isolated from Sumatera for controlling infection by the root-knot nematode (RKN) on Deli tobacco plant. Wheat bran soil containing 109 conidia of Arthrobotrys. oligospora, Candellabrella musiformis and Dactylella eudermata was added to the soil as a dry inoculum. Carbofuran was also applied as chemical agent and comparison treatment. Seedling tobacco (Nicotiana tabacum L.) cv. Deli 4 was inoculated with root knot (Meloidogyne incognita Chitwood.) seven days after the plant were transplanted to the pots. A. oligospora, C. musiformis and D. eudermata were found to be reliable as biocontrol agents, reducing the number of vermiform nematodes, swollen root, sausage shaped and galls in tobacco plant after 7, 15 and 30 days of infection with M. incognita. Treatment with NTF produced results that were comparable with Carbofuran® as a control agent in the reduction of the number of infections in tobacco plant caused by M. incognita in Nicotiana tabacum var. Deli 4. They also optimize the growth of the tobacco plants especially up to 15 days after infection.
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In planta transformation method for T-DNA transfer in orchids
NASA Astrophysics Data System (ADS)
Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko
2014-03-01
Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.
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O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.
1994-01-01
The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216
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Xing, Xuexia; Li, Xiaohui; Zhang, Mingzhen; Wang, Yuan; Liu, Bingyang; Xi, Qiliang; Zhao, Ke; Wu, Yunjie; Yang, Tiezhao
2017-01-22
The root-knot nematode (RKN) Meloidogyne incognita reproduces on the roots of tobacco (Nicotiana tabacum), damaging crops, reducing crop yield, and causing economic losses annually. The development of resistant genotypes is an alternative strategy to effectively control these losses. However, the molecular mechanism responsible for host pathogenesis and defense responses in tobacco specifically against RKNs remain poorly understood. Here, root transcriptome analysis of resistant (Yuyan12) and susceptible (Changbohuang) tobacco varieties infected with RKNs was performed. Moreover, 2623 and 545 differentially expressed genes (DEGs) in RKN-infected roots were observed in Yuyan12 and Changbohuang, respectively, compared to those in non-infected roots, including 289 DEGs commonly expressed in the two genotypes. Among these DEGs, genes encoding cell wall modifying proteins, auxin-related proteins, the ROS scavenging system, and transcription factors involved in various biological and physiochemical processes were significantly expressed in both the resistant and susceptible genotypes. This work is thus the first report on the relationships in the RKN-tobacco interaction using transcriptome analysis, and the results provide important information on the mechanism of RKN resistance in tobacco. Copyright © 2016 Elsevier Inc. All rights reserved.
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Analysis of osmotin, a PR protein as metabolic modulator in plants
Abdin, Malik Zainul; Kiran, Usha; Alam, Afshar
2011-01-01
Osmotin is an abundant cationic multifunctional protein discovered in cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) adapted to an environment of low osmotic potential. Beside its role as osmoregulator, it provides plants protection from pathogens, hence also placed in the PRP family of proteins. The osmotin induced proline accumulation has been reported to confer tolerance against both biotic and abiotic stresses in plants including transgenic tomato and strawberry overexpressing osmotin gene. The exact mechanism of induction of proline by osmotin is however, not known till date. These observations have led us to hypothesize that osmotin could be regulating these plant responses through its involvement either as transcription factor, cell signal pathway modulator or both in plants. We have therefore, undertaken the present investigation to analyze the osmotin protein as transcription factor using bioinformatics tools. The results of available online DNA binding motif search programs revealed that osmotin does not contain DNAbinding motifs. The alignment results of osmotin protein with the protein sequence from DATF showed the homology in the range of 0-20%, suggesting that it might not contain a DNA binding motif. Further to find unique DNA-binding domain, the superimposition of osmotin 3D structure on modeled Arabidopsis transcription factors using Chimera also suggested absence of the same. However, evidence implicating osmotin in cell signaling were found during the study. With these results, we therefore, concluded that osmotin is not a transcription factor, but regulating plant responses to biotic and abiotic stresses through cell signaling. PMID:21383921
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Analysis of osmotin, a PR protein as metabolic modulator in plants.
Abdin, Malik Zainul; Kiran, Usha; Alam, Afshar
2011-01-22
Osmotin is an abundant cationic multifunctional protein discovered in cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) adapted to an environment of low osmotic potential. Beside its role as osmoregulator, it provides plants protection from pathogens, hence also placed in the PRP family of proteins. The osmotin induced proline accumulation has been reported to confer tolerance against both biotic and abiotic stresses in plants including transgenic tomato and strawberry overexpressing osmotin gene. The exact mechanism of induction of proline by osmotin is however, not known till date. These observations have led us to hypothesize that osmotin could be regulating these plant responses through its involvement either as transcription factor, cell signal pathway modulator or both in plants. We have therefore, undertaken the present investigation to analyze the osmotin protein as transcription factor using bioinformatics tools. The results of available online DNA binding motif search programs revealed that osmotin does not contain DNAbinding motifs. The alignment results of osmotin protein with the protein sequence from DATF showed the homology in the range of 0-20%, suggesting that it might not contain a DNA binding motif. Further to find unique DNA-binding domain, the superimposition of osmotin 3D structure on modeled Arabidopsis transcription factors using Chimera also suggested absence of the same. However, evidence implicating osmotin in cell signaling were found during the study. With these results, we therefore, concluded that osmotin is not a transcription factor, but regulating plant responses to biotic and abiotic stresses through cell signaling.
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Effect of promoter driving selectable marker on corn transformation.
Prakash, N Shiva; Prasad, V; Chidambram, Thillai P; Cherian, Shoba; Jayaprakash, T L; Dasgupta, Santanu; Wang, Qi; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S
2008-08-01
Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the
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Illingworth, Christopher J R; Parkes, Kevin E; Snell, Christopher R; Mullineaux, Philip M; Reynolds, Christopher A
2008-03-01
Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.
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Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD
NASA Technical Reports Server (NTRS)
Granger, C. L.; Cyr, R. J.
2000-01-01
Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.
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Ishikawa, Toshiki; Rennie, Emilie A.; Lao, Jeemeng; Yan, Jingwei; Tsai, Alex Yi-Lin; Baidoo, Edward E.K.; Demura, Taku; Kawai-Yamada, Maki
2016-01-01
Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ∼25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologous expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged. PMID:27895225
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A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots.
Mano, Y; Matsuhashi, M
1995-03-01
Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.
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Barley Transformation Using Agrobacterium-Mediated Techniques
NASA Astrophysics Data System (ADS)
Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.
Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.
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Wheat (Triticum aestivum L.) transformation using mature embryos.
Medvecká, Eva; Harwood, Wendy A
2015-01-01
In most protocols for the Agrobacterium-mediated transformation of wheat, the preferred target tissues are immature embryos. However, transformation methods relying on immature embryos require the growth of plants under controlled conditions to provide a continuous supply of good-quality target tissue. The use of mature embryos as a target tissue has the advantage of only requiring good-quality seed as the starting material. Here we describe a transformation method based on the Agrobacterium-mediated transformation of callus cultures derived from mature wheat embryos of the genotype Bobwhite S56.
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2012-01-01
Background Striga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation. Results We used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots. Conclusions This rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions. PMID:22720750
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Tang, Yuanman; Liu, Qiuping; Liu, Ying; Zhang, Linli; Ding, Wei
2017-01-01
Various classes of plant pathogenesis-related proteins have been identified in the past several decades. PR-Q, a member of the PR3 family encoding chitinases, has played an important role in regulating plant resistance and preventing pathogen infection. In this paper, we functionally characterized NtPR-Q in tobacco plants and found that the overexpression of NtPR-Q in tobacco Yunyan87 resulted in higher resistance to Ralstonia solanacearum inoculation. Surprisingly, overexpression of NtPR-Q led to the activation of many defense-related genes, such as salicylic acid (SA)-responsive genes NtPR1a/c , NtPR2 and NtCHN50 , JA-responsive gene NtPR1b and ET production-associated genes NtACC Oxidase and NtEFE26 . Consistent with the role of NtPR-Q in multiple stress responses, NtPR-Q transcripts were induced by the exogenous hormones SA, ethylene and methyl jasmonate, which could enhance the resistance of tobacco to R. solanacearum . Collectively, our results suggested that NtPR-Q overexpression led to the up-regulation of defense-related genes and enhanced plant resistance to R. solanacearum infection.
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Diaz-Vivancos, Pedro; Faize, Lydia; Nicolás, Emilio; Clemente-Moreno, Maria José; Bru-Martinez, Roque; Burgos, Lorenzo; Hernández, José Antonio
2016-06-01
Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. The transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field conditions will be required, it is proposed that J8
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32. Credit JTL. Exterior transformer bank; note lightning arrestors removed ...
32. Credit JTL. Exterior transformer bank; note lightning arrestors removed from pad and smaller arrestors installed on transformers and in area near air switches. - Dam No. 4 Hydroelectric Plant, Potomac River, Martinsburg, Berkeley County, WV
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Huang, Jun; Hirji, Rozina; Adam, Luc; Rozwadowski, Kevin L.; Hammerlindl, Joe K.; Keller, Wilf A.; Selvaraj, Gopalan
2000-01-01
Glycinebetaine (betaine) affords osmoprotection in bacteria, plants and animals, and protects cell components against harsh conditions in vitro. This and a compelling body of other evidence have encouraged the engineering of betaine production in plants lacking it. We have installed the metabolic step for oxidation of choline, a ubiquitous substance, to betaine in three diverse species, Arabidopsis, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of a bacterial choline oxidase gene. The highest levels of betaine in independent transgenics were 18.6, 12.8, and 13 μmol g−1 dry weight, respectively, values 10- to 20-fold lower than the levels found in natural betaine producers. However, choline-fed transgenic plants synthesized substantially more betaine. Increasing the choline supplementation further enhanced betaine synthesis, up to 613 μmol g−1 dry weight in Arabidopsis, 250 μmol g−1 dry weight in B. napus, and 80 μmol g−1 dry weight in tobacco. These studies demonstrate the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of betaine. A moderate stress tolerance was noted in some but not all betaine-producing transgenic lines based on relative shoot growth. Furthermore, the responses to stresses such as salinity, drought, and freezing were variable among the three species. PMID:10712538
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Toward Stable Genetic Engineering of Human O-Glycosylation in Plants1[C][W][OA
Yang, Zhang; Bennett, Eric P.; Jørgensen, Bodil; Drew, Damian P.; Arigi, Emma; Mandel, Ulla; Ulvskov, Peter; Levery, Steven B.; Clausen, Henrik; Petersen, Bent L.
2012-01-01
Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics. PMID:22791304
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Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.
Weil, M; Rausch, T
1990-12-01
The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.
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Organogenesis from transformed tomato explants.
Frary, Anne; Van Eck, Joyce
2005-01-01
Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.
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[Prospects of molecular breeding in medical plants].
Ma, Xiao-Jun; Mo, Chang-Ming
2017-06-01
The molecular-assisted breeding, transgenic breeding and molecular designing breeding are three development directions of plant molecular breeding. Base on these three development directions, this paper summarizes developing status and new tendency of research field of genetic linkage mapping, QTL mapping, association mapping, molecular-assisted selections, pollen-mediated transformations, agrobacterium-mediated transformations, particle gun-mediated transformations, genome editing technologies, whole-genome sequencing, transcriptome sequencing, proteome sequencing and varietal molecular designing. The objective and existing problem of medical plant molecular breeding were discussed the prospect of these three molecular breeding technologies application on medical plant molecular breeding was outlooked. Copyright© by the Chinese Pharmaceutical Association.
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NASA Astrophysics Data System (ADS)
Schicklberger, M. F.; Huang, J.; Felix, P.; Pettenato, A.; Chakraborty, R.
2013-12-01
Nitrogen (N) is an essential component of DNA and proteins and consequently a key element of life. N often is limited in plants, affecting plant growth and productivity. To alleviate this problem, tremendous amounts of N-fertilizer is used, which comes at a high economic price and heavy energy demand. In addition, N-fertilizer also significantly contributes to rising atmospheric greenhouse gas concentrations. Therefore, the addition of fertilizer to overcome N limitation is highly undesirable. To explore reduction in fertilizer use our research focuses on optimizing the interaction between plants and diazotrophic bacteria, which could provide adequate amounts of N to the host-plant. Therefore we investigated the diversity of microbes associated with Tobacco (Nicotiana tabacum) and Switchgrass (Panicum virgatum), considered as potential energy crop for bioenergy production. Several bacterial isolates with representatives from Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes and Bacilli were obtained from the roots, leaves, rhizoplane and rhizosphere of these plants. Majority of these isolates grew best with simple sugars and small organic acids. As shown by PCR amplification of nifH, several of these isolates are potential N2-fixing bacteria. We investigated diazotrophs for their response to elevated temperature and salinity (two common climate change induced stresses found on marginal lands), their N2-fixing ability, and their response to root exudates (which drive microbial colonization of the plant). Together this understanding is necessary for the development of eco-friendly, economically sustainable energy crops by decreasing their dependency on fertilizer.
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Kathiria, Palak; Sidler, Corinne; Golubov, Andrey; Kalischuk, Melanie; Kawchuk, Lawrence M.; Kovalchuk, Igor
2010-01-01
Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance. PMID:20498336
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Molecular and physiological strategies to increase aluminum resistance in plants.
Inostroza-Blancheteau, Claudio; Rengel, Zed; Alberdi, Miren; de la Luz Mora, María; Aquea, Felipe; Arce-Johnson, Patricio; Reyes-Díaz, Marjorie
2012-03-01
Aluminum (Al) toxicity is a primary limitation to plant growth on acid soils. Root meristems are the first site for toxic Al accumulation, and therefore inhibition of root elongation is the most evident physiological manifestation of Al toxicity. Plants may resist Al toxicity by avoidance (Al exclusion) and/or tolerance mechanisms (detoxification of Al inside the cells). The Al exclusion involves the exudation of organic acid anions from the root apices, whereas tolerance mechanisms comprise internal Al detoxification by organic acid anions and enhanced scavenging of free oxygen radicals. One of the most important advances in understanding the molecular events associated with the Al exclusion mechanism was the identification of the ALMT1 gene (Al-activated malate transporter) in Triticum aestivum root cells, which codes for a plasma membrane anion channel that allows efflux of organic acid anions, such as malate, citrate or oxalate. On the other hand, the scavenging of free radicals is dependent on the expression of genes involved in antioxidant defenses, such as peroxidases (e.g. in Arabidopsis thaliana and Nicotiana tabacum), catalases (e.g. in Capsicum annuum), and the gene WMnSOD1 from T. aestivum. However, other recent findings show that reactive oxygen species (ROS) induced stress may be due to acidic (low pH) conditions rather than to Al stress. In this review, we summarize recent findings regarding molecular and physiological mechanisms of Al toxicity and resistance in higher plants. Advances have been made in understanding some of the underlying strategies that plants use to cope with Al toxicity. Furthermore, we discuss the physiological and molecular responses to Al toxicity, including genes involved in Al resistance that have been identified and characterized in several plant species. The better understanding of these strategies and mechanisms is essential for improving plant performance in acidic, Al-toxic soils.
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Masani, Mat Yunus Abdul; Noll, Gundula A; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk
2014-01-01
Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.
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Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection
Masani, Mat Yunus Abdul; Noll, Gundula A.; Parveez, Ghulam Kadir Ahmad; Sambanthamurthi, Ravigadevi; Prüfer, Dirk
2014-01-01
Background Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. PMID:24821306
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Hypergravity Leads to the Redistribution of Calcium Ions in Plant Cell
NASA Astrophysics Data System (ADS)
Nedukha, Olena M.
2008-06-01
The study of hypergravity influence on calcium ions distribution and on the relative amount of Ca2+ in cells of Nicotiana tabacum callus was carried out using the centrifuge. 15-day-old N. tabacum callus grown in a Murashige and Scoog agar medium was exposed to hypergravity at 6.5 g and 14 g for 15 and 60 min. The control samples and the centrifuged callus were loaded with Fluo-4 and then studied by the confocal laser-scanning microscopy. The visible redistribution of Ca2+ in the investigated cells and the appearance of calcium-microdomains in cytoplasm have been established under influence of hypergravity. Readaptation of Ca2+ distribution in the cells occurred in 2-4 h after hypergravity ending. It is suggested that influence of hypergravity lead to change of ionic transport of plasmalemma and endomembranes, and also to efflux of Ca2+ from apoplast.
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2012-01-01
Background The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. Results In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. Conclusion The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences. PMID:22436504
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Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants.
Gichner, Tomás; Znidar, Irena; Száková, Jirina
2008-04-30
Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.
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NASA Astrophysics Data System (ADS)
Singh, Yogranjan; Singh Marabi, Rakesh; Satpute, Gyanesh Kumar; Mishra, Stuti
2012-07-01
An exorbitant sum of different synthetic molecules of chemicals including dyes and pigments are discharged into the environment, mainly via industrial effluents every year worldwide. The physical-chemical treatments for remediation viz adsorption, precipitation, ion exchange or filtration have proved to be disadvantageous because of high cost, low efficiency and inapplicability to a wide variety of dyes, or the formation of by-products and thereby creating waste disposal problems. Similarly the limited ability of micro-organisms to degrade xenobiotic especially sulphonoaromatic compounds, limits the efficiency and, therefore, the use of conventional wastewater treatment plants. In this context, the development of alternative biological treatments to eliminate these pollutants from industrial effluents is an important requirement. Plant metabolism, is extremely diverse and can be exploited to treat recalcitrant pollutants, not degradable by bacteria or fungi and can act as an important global sink for environmental pollutants. The presence of putative metabolites, in leaves of hydrophytes has been observed, indicating the transformation of several xenobiotics. A diverse range of the enzymes involved in the early stages of the detoxification process are closely associated with the redox biochemistry of the cell. The activities of enzymes such as glutathione transferases, peroxidases and cytochrome P450 monooxygenases and its multigenic family have implications with respect to the maintenance of redox homeostasis. Besides activating xenobiotics, cytochromes P450 is involved vitally in cell signaling for counteracting buoyant balance. Signal transduction cascades, including the role of cytochrome P450 monooxygenases in responding to gravitational cues, appear to be affected by buoyancy as well. Gravitropism is the orientation of growth in response to gravity and involves the perception of the gravitational force in the columella cells of the root cap where the primary
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The pea END1 promoter drives anther-specific gene expression in different plant species.
Gómez, María D; Beltrán, José-Pío; Cañas, Luis A
2004-10-01
END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (beta-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the -330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity.
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de Paiva Rolla, Amanda Alves; de Fátima Corrêa Carvalho, Josirley; Fuganti-Pagliarini, Renata; Engels, Cibelle; do Rio, Alexandre; Marin, Silvana Regina Rockenbach; de Oliveira, Maria Cristina Neves; Beneventi, Magda A; Marcelino-Guimarães, Francismar Corrêa; Farias, José Renato Bouças; Neumaier, Norman; Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Nepomuceno, Alexandre Lima
2014-02-01
The development of drought tolerant plants is a high priority because the area suffering from drought is expected to increase in the future due to global warming. One strategy for the development of drought tolerance is to genetically engineer plants with transcription factors (TFs) that regulate the expression of several genes related to abiotic stress defense responses. This work assessed the performance of soybean plants overexpressing the TF DREB1A under drought conditions in the field and in the greenhouse. Drought was simulated in the greenhouse by progressively drying the soil of pot cultures of the P58 and P1142 lines. In the field, the performance of the P58 line and of 09D-0077, a cross between the cultivars BR16 and P58, was evaluated under four different water regimes: irrigation, natural drought (no irrigation) and water stress created using rain-out shelters in the vegetative or reproductive stages. Although the dehydration-responsive element-binding protein (DREB) plants did not outperform the cultivar BR16 in terms of yield, some yield components were increased when drought was introduced during the vegetative stage, such as the number of seeds, the number of pods with seeds and the total number of pods. The greenhouse data suggest that the higher survival rates of DREB plants are because of lower water use due to lower transpiration rates under well watered conditions. Further studies are needed to better characterize the soil and atmospheric conditions under which these plants may outperform the non-transformed parental plants.
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Pitcher, Lynne H.; Brennan, Eileen; Hurley, Arthur; Dunsmuir, Pamela; Tepperman, James M.; Zilinskas, Barbara A.
1991-01-01
Transgenic tobacco (Nicotiana tabacum cultivar W38) plants that overproduce petunia chloroplastic Cu/Zn superoxide dismutase were exposed to ozone dosages that injure control tobacco plants. Based on foliar injury ratings, there was no consistent protection provided to the transgenic plants. These data indicate that an increase in the chloroplastic Cu/Zn superoxide dismutase alone is not sufficient to reduce ozone toxicity. ImagesFigure 1 PMID:16668407
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Identification and dynamics of two classes of aurora-like kinases in Arabidopsis and other plants.
Demidov, Dmitri; Van Damme, Daniël; Geelen, Danny; Blattner, Frank R; Houben, Andreas
2005-03-01
Aurora-like kinases play key roles in chromosome segregation and cytokinesis in yeast, plant, and animal systems. Here, we characterize three Arabidopsis thaliana protein kinases, designated AtAurora1, AtAurora2, and AtAurora3, which share high amino acid identities with the Ser/Thr kinase domain of yeast Ipl1 and animal Auroras. Structure and expression of AtAurora1 and AtAurora2 suggest that these genes arose by a recent gene duplication, whereas the diversification of plant alpha and beta Aurora kinases predates the origin of land plants. The transcripts and proteins of all three kinases are most abundant in tissues containing dividing cells. Intracellular localization of green fluorescent protein-tagged AtAuroras revealed an AtAurora-type specific association mainly with dynamic mitotic structures, such as microtubule spindles and centromeres, and with the emerging cell plate of dividing tobacco (Nicotiana tabacum) BY-2 cells. Immunolabeling using AtAurora antibodies yielded specific signals at the centromeres that are coincident with histone H3 that is phosphorylated at Ser position10 during mitosis. An in vitro kinase assay demonstrated that AtAurora1 preferentially phosphorylates histone H3 at Ser 10 but not at Ser 28 or Thr 3, 11, and 32. The phylogenetic analysis of available Aurora sequences from different eukaryotic origins suggests that, although a plant Aurora gene has been duplicated early in the evolution of plants, the paralogs nevertheless maintained a role in cell cycle-related signal transduction pathways.
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Ibrahim, Evra Raunie
2014-01-01
Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258
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Optimization of Agrobacterium-Mediated Transformation in Soybean
Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan
2017-01-01
High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide
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Optimization of Agrobacterium-Mediated Transformation in Soybean.
Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan
2017-01-01
High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens -mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium -mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD 650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA 3 ) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA 3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA 3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA 3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR
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Bassil, Elias; Hu, Hening; Brown, Patrick H.
2004-01-01
The only defined physiological role of boron in plants is as a cross-linking molecule involving reversible covalent bonds with cis-diols on either side of borate. Boronic acids, which form the same reversible bonds with cis-diols but cannot cross-link two molecules, were used to selectively disrupt boron function in plants. In cultured tobacco (Nicotiana tabacum cv BY-2) cells, addition of boronic acids caused the disruption of cytoplasmic strands and cell-to-cell wall detachment. The effect of the boronic acids could be relieved by the addition of boron-complexing sugars and was proportional to the boronic acid-binding strength of the sugar. Experiments with germinating petunia (Petunia hybrida) pollen and boronate-affinity chromatography showed that boronic acids and boron compete for the same binding sites. The boronic acids appear to specifically disrupt or prevent borate-dependent cross-links important for the structural integrity of the cell, including the organization of transvacuolar cytoplasmic strands. Boron likely plays a structural role in the plant cytoskeleton. We conclude that boronic acids can be used to rapidly and reversibly induce boron deficiency-like responses and therefore are useful tools for investigating boron function in plants. PMID:15466241
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Solon, K; Flores-Alsina, X; Kazadi Mbamba, C; Ikumi, D; Volcke, E I P; Vaneeckhaute, C; Ekama, G; Vanrolleghem, P A; Batstone, D J; Gernaey, K V; Jeppsson, U
2017-04-15
The objective of this paper is to report the effects that control/operational strategies may have on plant-wide phosphorus (P) transformations in wastewater treatment plants (WWTP). The development of a new set of biological (activated sludge, anaerobic digestion), physico-chemical (aqueous phase, precipitation, mass transfer) process models and model interfaces (between water and sludge line) were required to describe the required tri-phasic (gas, liquid, solid) compound transformations and the close interlinks between the P and the sulfur (S) and iron (Fe) cycles. A modified version of the Benchmark Simulation Model No. 2 (BSM2) (open loop) is used as test platform upon which three different operational alternatives (A 1 , A 2 , A 3 ) are evaluated. Rigorous sensor and actuator models are also included in order to reproduce realistic control actions. Model-based analysis shows that the combination of an ammonium ( [Formula: see text] ) and total suspended solids (X TSS ) control strategy (A 1 ) better adapts the system to influent dynamics, improves phosphate [Formula: see text] accumulation by phosphorus accumulating organisms (X PAO ) (41%), increases nitrification/denitrification efficiency (18%) and reduces aeration energy (E aeration ) (21%). The addition of iron ( [Formula: see text] ) for chemical P removal (A 2 ) promotes the formation of ferric oxides (X HFO-H , X HFO-L ), phosphate adsorption (X HFO-H,P , X HFO-L,P ), co-precipitation (X HFO-H,P,old , X HFO-L,P,old ) and consequently reduces the P levels in the effluent (from 2.8 to 0.9 g P.m -3 ). This also has an impact on the sludge line, with hydrogen sulfide production ( [Formula: see text] ) reduced (36%) due to iron sulfide (X FeS ) precipitation. As a consequence, there is also a slightly higher energy production (E production ) from biogas. Lastly, the inclusion of a stripping and crystallization unit (A 3 ) for P recovery reduces the quantity of P in the anaerobic digester supernatant
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Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.
Kanamoto, Hirosuke; Yamashita, Atsushi; Asao, Hiroshi; Okumura, Satoru; Takase, Hisabumi; Hattori, Masahira; Yokota, Akiho; Tomizawa, Ken-Ichi
2006-04-01
Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.
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Hunter, Lydia J. R.; Brockington, Samuel F.; Murphy, Alex M.; Pate, Adrienne E.; Gruden, Kristina; MacFarlane, Stuart A.; Palukaitis, Peter; Carr, John P.
2016-01-01
Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7. PMID:26979928
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Hunter, Lydia J R; Brockington, Samuel F; Murphy, Alex M; Pate, Adrienne E; Gruden, Kristina; MacFarlane, Stuart A; Palukaitis, Peter; Carr, John P
2016-03-16
Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.
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Gas-Pascual, Elisabet; Berna, Anne; Bach, Thomas J; Schaller, Hubert
2014-01-01
The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.
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Sankara Rao, K; Sreevathsa, Rohini; Sharma, Pinakee D; Keshamma, E; Udaya Kumar, M
2008-10-01
Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gou J. Y.; Liu C.; Miller, L. M.
Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the levelmore » of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.« less
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Transformation of Morinda citrifolia via simple mature seed imbibition method.
Lee, J J; Ahmad, S; Roslan, H A
2013-12-15
Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.
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Transformation of Epichloë typhina by electroporation of conidia
2011-01-01
Background Choke, caused by the endophytic fungus Epichloë typhina, is an important disease affecting orchardgrass (Dactylis glomerata L.) seed production in the Willamette Valley. Little is known concerning the conditions necessary for successful infection of orchardgrass by E. typhina. Detection of E. typhina in plants early in the disease cycle can be difficult due to the sparse distribution of hyphae in the plant. Therefore, a sensitive method to detect fungal infection in plants would provide an invaluable tool for elucidating the conditions for establishment of infection in orchardgrass. Utilization of a marker gene, such as the green fluorescent protein (GFP), transformed into Epichloë will facilitate characterization of the initial stages of infection and establishment of the fungus in plants. Findings We have developed a rapid, efficient, and reproducible transformation method using electroporation of germinating Epichloë conidia isolated from infected plants. Conclusions The GFP labelled E. typhina provides a valuable molecular tool to researchers studying conditions and mechanisms involved in the establishment of choke disease in orchardgrass. PMID:21375770
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Fang, Lin; Ishikawa, Toshiki; Rennie, Emilie A.; ...
2016-11-28
Glycosylinositol phosphorylceramides (GIPCs) are a class of glycosylated sphingolipids found in plants, fungi, and protozoa. These lipids are abundant in the plant plasma membrane, forming ~25% of total plasma membrane lipids. Little is known about the function of the glycosylated headgroup, but two recent studies have indicated that they play a key role in plant signaling and defense. Here, we show that a member of glycosyltransferase family 64, previously named ECTOPICALLY PARTING CELLS1, is likely a Golgi-localized GIPC-specific mannosyl-transferase, which we renamed GIPC MANNOSYL-TRANSFERASE1 (GMT1). Sphingolipid analysis revealed that the Arabidopsis thaliana gmt1 mutant almost completely lacks mannose-carrying GIPCs. Heterologousmore » expression of GMT1 in Saccharomyces cerevisiae and tobacco (Nicotiana tabacum) cv Bright Yellow 2 resulted in the production of non-native mannosylated GIPCs. gmt1 displays a severe dwarfed phenotype and a constitutive hypersensitive response characterized by elevated salicylic acid and hydrogen peroxide levels, similar to that we previously reported for the Golgi-localized, GIPC-specific, GDP-Man transporter GONST1 (Mortimer et al., 2013). Unexpectedly, we show that gmt1 cell walls have a reduction in cellulose content, although other matrix polysaccharides are unchanged.« less
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Identification of a New Class of Lipid Droplet-Associated Proteins in Plants1[C][W][OPEN
Horn, Patrick J.; James, Christopher N.; Gidda, Satinder K.; Kilaru, Aruna; Dyer, John M.; Mullen, Robert T.; Ohlrogge, John B.; Chapman, Kent D.
2013-01-01
Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652
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Research and Application of Lipoic Acid in Plants
NASA Astrophysics Data System (ADS)
Xiao, Renjie; Wang, Xiran; Jiang, Leiyu; Tang, Haoru
2018-01-01
Lipoic acid is a kind of small molecular compound with strong oxidizing properties. It has been widely used in medicine and has achieved good results since its discovery. However, it is less used in plants, and the biosynthetic pathway is not clear. The content in the plant is mainly measured by high-performance liquid chromatography(HPLC). At present, it is mainly used as an additive to the culture medium for plant tissue culture and Agrobacterium-mediated plant genetic transformation, in order to reduce the browning rate of explants, improve Agrobacterium-mediated genetic transformation efficiency.
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A method for accelerated trait conversion in plant breeding.
Lewis, Ramsey S; Kernodle, S P
2009-05-01
Backcrossing is often used in cultivar development to transfer one or a few genes to desired genetic backgrounds. The duration necessary to complete such 'trait conversions' is largely dependent upon generation times. Constitutive overexpression of the Arabidopsis thaliana gene FT (FLOWERING LOCUS T) induces early-flowering in many plants. Here, we used tobacco (Nicotiana tabacum L.) as a model system to propose and examine aspects of a modified backcross procedure where transgenic FT overexpression is used to reduce generation time and accelerate gene transfer. In this method, the breeder would select for an FT transgene insertion and the trait(s) of interest at each backcross generation except the last. In the final generation, selection would be conducted for the trait(s) of interest, but against FT, to generate the backcross-derived trait conversion. We demonstrate here that constitutive FT overexpression functions to dramatically reduce days-to-flower similarly in diverse tobacco genetic backgrounds. FT-containing plants flowered in an average of 39 days, in comparison with 87-138 days for non-FT plants. Two FT transgene insertions were found to segregate independently of several disease resistance genes often the focus of backcrossing in tobacco. In addition, no undesirable epigenetic effects on flowering time were observed once FT was segregated away. The proposed system would reduce the time required to complete a trait conversion in tobacco by nearly one-half. These features suggest the possible value of this modified backcrossing system for tobacco or other crop species where long generation times or photoperiod sensitivity may impede timely trait conversion.
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Asano, Kyouhei; Lee, Jung-Bum; Yamamura, Yoshimi; Kurosaki, Fumiya
2013-12-01
Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.
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Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them
Bohmert-Tatarev, Karen; McAvoy, Susan; Peoples, Oliver P.; Snell, Kristi D.
2015-08-04
Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.
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131. View, looking northeast, into transformer bay no. 1 showing ...
131. View, looking northeast, into transformer bay no. 1 showing ca. 1950s General Electric transformer; this transformer was brought to White River from the Snoqualmie Falls plant. It has been abandoned and is being removed. When operational it stepped down 55,000 volts to 6,600 volts. Photo by Jet Lowe, HAER, 1989. - Puget Sound Power & Light Company, White River Hydroelectric Project, 600 North River Avenue, Dieringer, Pierce County, WA
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Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.
Teixeira da Silva, Jaime A; Jha, Sumita
2016-11-01
This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.
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Fincham, J R
1989-01-01
Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater
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Tsai, F Y; Coruzzi, G
1991-01-01
Asparagine synthetase (AS) mRNA in Pisum sativum accumulates preferentially in plants grown in the dark. Nuclear run-on experiments demonstrate that expression of both the AS1 and AS2 genes is negatively regulated by light at the level of transcription. A decrease in the transcriptional rate of the AS1 gene can be detected as early as 20 min after exposure to light. Time course experiments reveal that the levels of AS mRNA fluctuate dramatically during a "normal" light/dark cycle. This is due to a direct effect of light and not to changes associated with circadian rhythm. A novel finding is that the light-repressed expression of the AS1 gene is as dramatic in nonphotosynthetic organs such as roots as it is in leaves. Experiments demonstrate that the small amount of light which passes through the soil is sufficient to repress AS1 expression in roots, indicating that light has a direct effect on AS1 gene expression in roots. The negative regulation of AS gene expression by light was shown to be a general phenomenon in plants which also occurs in nonlegumes such as Nicotiana plumbaginifolia and Nicotiana tabacum. Thus, the AS genes can serve as a model with which to dissect the molecular basis for light-regulated transcriptional repression in plants. Images PMID:1681424
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Fault diagnosis of power transformer based on fault-tree analysis (FTA)
NASA Astrophysics Data System (ADS)
Wang, Yongliang; Li, Xiaoqiang; Ma, Jianwei; Li, SuoYu
2017-05-01
Power transformers is an important equipment in power plants and substations, power distribution transmission link is made an important hub of power systems. Its performance directly affects the quality and health of the power system reliability and stability. This paper summarizes the five parts according to the fault type power transformers, then from the time dimension divided into three stages of power transformer fault, use DGA routine analysis and infrared diagnostics criterion set power transformer running state, finally, according to the needs of power transformer fault diagnosis, by the general to the section by stepwise refinement of dendritic tree constructed power transformer fault
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Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation[OPEN
Lowe, Keith; Wu, Emily; Cho, Myeong-Je; Lenderts, Brian; Chamberlin, Mark; Cushatt, Josh; Ryan, Larisa; Khan, Tanveer; Chow-Yiu, Julia; Hua, Wei; Banh, Jenny; Bao, Zhongmeng; Brink, Kent; Igo, Elizabeth; Rudrappa, Bhojaraja; Shamseer, PM; Shen, Bo; Zheng, Peizhong; Bidney, Dennis; Falco, Carl; Zhao, Zuo-Yu; Xu, Deping
2016-01-01
While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus. PMID:27600536
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False Broomrape: A Physiological Disorder Caused by Growth-Regulator Imbalance 1
Hamilton, James L.; Lowe, Richie H.; Skoog, Folke
1972-01-01
False broomrape on tobacco (Nicotiana tabacum) can be induced by applying cytokinins to the roots, by causing an increase in the cytokinin-auxin ratio in the roots, by removing the apical and auxillary buds, or by applying extracts from tobacco with false broomrape to the roots of healthy plants. It can be prevented by treating debudded plants with auxin. Images PMID:16658161
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Ismail, Hammad; Dilshad, Erum; Waheed, Mohammad Tahir; Mirza, Bushra
2017-03-01
Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102 % increase in total phenolic contents and 53-65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.
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High throughput selection of antibiotic-resistant transgenic Arabidopsis plants.
Nagashima, Yukihiro; Koiwa, Hisashi
2017-05-15
Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation. Copyright © 2017 Elsevier Inc. All rights reserved.
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Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita
2012-10-01
Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.
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Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo
2011-01-01
DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis. PMID:22021419
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Heendeniya, Ravindra G; Yu, Peiqiang
2017-03-20
Alfalfa ( Medicago sativa L.) genotypes transformed with Lc-bHLH and Lc transcription genes were developed with the intention of stimulating proanthocyanidin synthesis in the aerial parts of the plant. To our knowledge, there are no studies on the effect of single-gene and two-gene transformation on chemical functional groups and molecular structure changes in these plants. The objective of this study was to use advanced molecular spectroscopy with multivariate chemometrics to determine chemical functional group intensity and molecular structure changes in alfalfa plants when co-expressing Lc-bHLH and C1-MYB transcriptive flavanoid regulatory genes in comparison with non-transgenic (NT) and AC Grazeland (ACGL) genotypes. The results showed that compared to NT genotype, the presence of double genes ( Lc and C1 ) increased ratios of both the area and peak height of protein structural Amide I/II and the height ratio of α-helix to β-sheet. In carbohydrate-related spectral analysis, the double gene-transformed alfalfa genotypes exhibited lower peak heights at 1370, 1240, 1153, and 1020 cm -1 compared to the NT genotype. Furthermore, the effect of double gene transformation on carbohydrate molecular structure was clearly revealed in the principal component analysis of the spectra. In conclusion, single or double transformation of Lc and C1 genes resulted in changing functional groups and molecular structure related to proteins and carbohydrates compared to the NT alfalfa genotype. The current study provided molecular structural information on the transgenic alfalfa plants and provided an insight into the impact of transgenes on protein and carbohydrate properties and their molecular structure's changes.
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Tercé-Laforgue, Thérèse; Clément, Gilles; Marchi, Laura; Restivo, Francesco M; Lea, Peter J; Hirel, Bertrand
2015-10-01
NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering.
Wu, Yuyong; You, Lili; Li, Shengchun; Ma, Meiqi; Wu, Mengting; Ma, Lixin; Bock, Ralph; Chang, Ling; Zhang, Jiang
2017-01-01
Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5' untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3'UTR from the E. coli ribosomal RNA operon rrnB . Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.
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High-Throughput Cryopreservation of Plant Cell Cultures for Functional Genomics
Ogawa, Yoichi; Sakurai, Nozomu; Oikawa, Akira; Kai, Kosuke; Morishita, Yoshihiko; Mori, Kumiko; Moriya, Kanami; Fujii, Fumiko; Aoki, Koh; Suzuki, Hideyuki; Ohta, Daisaku; Saito, Kazuki; Shibata, Daisuke
2012-01-01
Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics. PMID:22437846
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Kilby, N J; Davies, G J; Snaith, M R
1995-11-01
FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.
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B1-Phytoprostanes Trigger Plant Defense and Detoxification Responses1[w
Loeffler, Christiane; Berger, Susanne; Guy, Alexandre; Durand, Thierry; Bringmann, Gerhard; Dreyer, Michael; von Rad, Uta; Durner, Jörg; Mueller, Martin J.
2005-01-01
Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B1-phytoprostanes (PPB1) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB1-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB1 regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB1 considerably prevented cell death caused by severe CuSO4 poisoning. Several Arabidopsis genes induced by PPB1, such as those coding for adenylylsulfate reductase, tryptophan synthase β-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB1 treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB1. In three different plant species, PPB1-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB1-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB1 represent stress signals that improve plants capacity to cope better with a variety of stresses. PMID:15618427
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Hisano, Hiroshi; Meints, Brigid; Moscou, Matthew J; Cistue, Luis; Echávarri, Begoña; Sato, Kazuhiro; Hayes, Patrick M
2017-04-01
The genetic substitution of transformation amenability alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars. Barley (Hordeum vulgare) cv. 'Golden Promise' is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F 2 generation of immature embryos, derived from 'Haruna Nijo' × 'Golden Promise,' as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross 'Golden Promise' × 'Full Pint.' Based on SNP genotype, we selected lines having 'Golden Promise' alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to 'Golden Promise.' The results validate that the genetic substitution of TFA alleles from 'Golden Promise' can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars.
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Michael, I; Hapeshi, E; Aceña, J; Perez, S; Petrović, M; Zapata, A; Barceló, D; Malato, S; Fatta-Kassinos, D
2013-09-01
This work investigated the application of a solar driven advanced oxidation process (solar Fenton), for the degradation of the antibiotic ofloxacin (OFX) in various environmental matrices at a pilot-scale. All experiments were carried out in a compound parabolic collector pilot plant in the presence of doses of H2O2 (2.5 mg L(-1)) and at an initial Fe(2+) concentration of 2 mg L(-1). The water matrices used for the solar Fenton experiments were: demineralized water (DW), simulated natural freshwater (SW), simulated effluent from municipal wastewater treatment plant (SWW) and pre-treated real effluent from municipal wastewater treatment plant (RE) to which OFX had been spiked at 10 mg L(-1). Dissolved organic carbon removal was found to be dependent on the chemical composition of the water matrix. OFX mineralization was higher in DW (78.1%) than in SW (58.3%) at 12 mg L(-1) of H2O2 consumption, implying the complexation of iron or the scavenging of hydroxyl radicals by the inorganic ions present in SW. On the other hand, the presence of dissolved organic matter (DOM) in SWW and RE, led to lower mineralization per dose of H2O2 compared to DW and SW. The major transformation products (TPs) formed during the solar Fenton treatment of OFX, were elucidated using liquid chromatography-time of flight-mass spectrometry (LC-ToF-MS). The transformation of OFX proceeded through a defluorination reaction, accompanied by some degree of piperazine and quinolone substituent transformation while a hydroxylation mechanism occurred by attack of the hydroxyl radicals generated during the process leading to the formation of TPs in all the water matrices, seven of which were tentatively identified. The results obtained from the toxicity bioassays indicated that the toxicity originates from the DOM present in RE and its oxidation products formed during the photocatalytic treatment and not from the TPs resulted from the oxidation of OFX. Copyright © 2013 The Authors. Published by
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Genetic transformation of fruit trees: current status and remaining challenges.
Gambino, Giorgio; Gribaudo, Ivana
2012-12-01
Genetic transformation has emerged as a powerful tool for genetic improvement of fruit trees hindered by their reproductive biology and their high levels of heterozygosity. For years, genetic engineering of fruit trees has focussed principally on enhancing disease resistance (against viruses, fungi, and bacteria), although there are few examples of field cultivation and commercial application of these transgenic plants. In addition, over the years much work has been performed to enhance abiotic stress tolerance, to induce modifications of plant growth and habit, to produce marker-free transgenic plants and to improve fruit quality by modification of genes that are crucially important in the production of specific plant components. Recently, with the release of several genome sequences, studies of functional genomics are becoming increasingly important: by modification (overexpression or silencing) of genes involved in the production of specific plant components is possible to uncover regulatory mechanisms associated with the biosynthesis and catabolism of metabolites in plants. This review focuses on the main advances, in recent years, in genetic transformation of the most important species of fruit trees, devoting particular attention to functional genomics approaches and possible future challenges of genetic engineering for these species in the post-genomic era.
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ERIC Educational Resources Information Center
2000
How does a tiny seed sprout and grow into a towering tree? Join the kids from M.A.P.L.E as they learn about some of the incredible transformations that a plant goes through during its lifetime. In All About Plant Structure & Growth, uncover the secrets of roots, stems and leaves - structures that are vital to a plant's role as an energy…
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Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha
2014-09-01
In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.
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Mitsunami, Tomoko; Nishihara, Masahiro; Galis, Ivan; Alamgir, Kabir Md; Hojo, Yuko; Fujita, Kohei; Sasaki, Nobuhiro; Nemoto, Keichiro; Sawasaki, Tatsuya; Arimura, Gen-ichiro
2014-01-01
Anthocyanin pigments and associated flavonoids have demonstrated antioxidant properties and benefits for human health. Consequently, current plant bioengineers have focused on how to modify flavonoid metabolism in plants. Most of that research, however, does not consider the role of natural biotic stresses (e.g., herbivore attack). To understand the influence of herbivore attack on the metabolic engineering of flavonoids, we examined tobacco plants overexpressing the Arabidopsis PAP1 gene (encoding an MYB transcription factor), which accumulated anthocyanin pigments and other flavonoids/phenylpropanoids. In comparison to wild-type and control plants, transgenic plants exhibited greater resistance to Spodoptera litura. Moreover, herbivory suppressed the PAP1-induced increase of transcripts of flavonoid/phenylpropanoid biosynthetic genes (e.g., F3H) and the subsequent accumulation of these genes' metabolites, despite the unaltered PAP1 mRNA levels after herbivory. The instances of down-regulation were independent of the signaling pathways mediated by defense-related jasmonates but were relevant to the levels of PAP1-induced and herbivory-suppressed transcription factors, An1a and An1b. Although initially F3H transcripts were suppressed by herbivory, after the S. litura feeding was interrupted, F3H transcripts increased. We hypothesize that in transgenic plants responding to herbivory, there is a complex mechanism regulating enriched flavonoid/phenylpropanoid compounds, via biotic stress signals. PMID:25268129
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Bertolla, Franck; Pepin, Regis; Passelegue-Robe, Eugenie; Paget, Eric; Simkin, Andrew; Nesme, Xavier; Simonet, Pascal
2000-01-01
The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene. PMID:10966449
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Desfeux, C; Clough, S J; Bent, A F
2000-07-01
The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.
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Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium
Cai, Yongping; Lin, Yi
2013-01-01
In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048
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Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song
2015-01-01
Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. PMID:25941315
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2012-01-01
Background In ethnobotanical research, the investigation into traditional knowledge of medicinal plants in the context of migration has been of increasing interest in recent decades since it is influenced and changed by new environmental and social conditions. It most likely undergoes transformation processes to match the different living circumstances in the new location. This study compares the traditional knowledge of medicinal plants held by Tyroleans – and their descendants – who emigrated to Australia, Brazil and Peru at different time scales. The study’s findings allow a discussion of the complexities and dynamics that influence this knowledge within the context of long-distance migration. Methods Information was obtained from 65 informants by free-listing, semi-structured interviews and non-participatory observation in Tyrol (Austria) and the migrants’ countries: Australia, Brazil and Peru. The collected data was analysed using different quantitative approaches, including statistical tests, and compared between the countries of investigation. Results All respondents in all four investigation areas claimed that they had knowledge and made use of medicinal plants to treat basic ailments in their day-to-day lives. Informants made 1,139 citations of medicinal plants in total in free lists, which correspond to 164 botanical taxa (genus or species level) in Tyrol, 87 in Australia, 84 in Brazil and 134 in Peru. Of all the botanical taxa listed, only five (1.1%) were listed in all four countries under investigation. Agreement among informants within free lists was highest in Tyrol (17%), followed by Peru (12.2%), Australia (11.9%) and Brazil (11.2%). The proportion of agreement differs significantly between informants in Australia and Tyrol (p = 0.001), Brazil and Tyrol (p = 0.001) and Peru and Tyrol (p = 0.001) and is similar between informants in the migrant countries, as indicated by statistical tests. We recorded 1,286 use citations
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Chang, Yi-Hsuan; Yan, Hao-Zhi; Liou, Ruey-Fen
2015-02-01
The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response. © 2014 BSPP AND JOHN WILEY & SONS LTD.
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Gou, Jin-Ying; Miller, Lisa M.; Hou, Guichuan; Yu, Xiao-Hong; Chen, Xiao-Ya; Liu, Chang-Jun
2012-01-01
Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility. PMID:22247250
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Howard, Amanda R.; Heppler, Marty L.; Ju, Ho-Jong
Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereasmore » GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.« less
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Fourier transform of delayed fluorescence as an indicator of herbicide concentration.
Guo, Ya; Tan, Jinglu
2014-12-21
It is well known that delayed fluorescence (DF) from Photosystem II (PSII) of plant leaves can be potentially used to sense herbicide pollution and evaluate the effect of herbicides on plant leaves. The research of using DF as a measure of herbicides in the literature was mainly conducted in time domain and qualitative correlation was often obtained. Fourier transform is often used to analyze signals. Viewing DF signal in frequency domain through Fourier transform may allow separation of signal components and provide a quantitative method for sensing herbicides. However, there is a lack of an attempt to use Fourier transform of DF as an indicator of herbicide. In this work, the relationship between the Fourier transform of DF and herbicide concentration was theoretically modelled and analyzed, which immediately yielded a quantitative method to measure herbicide concentration in frequency domain. Experiments were performed to validate the developed method. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Determining the transgene containment level provided by chloroplast transformation.
Ruf, Stephanie; Karcher, Daniel; Bock, Ralph
2007-04-24
Plastids (chloroplasts) are maternally inherited in most crops. Maternal inheritance excludes plastid genes and transgenes from pollen transmission. Therefore, plastid transformation is considered a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. Here, we have assessed the strictness of maternal inheritance and the extent to which plastid transformation technology confers an increase in transgene confinement. We describe an experimental system facilitating stringent selection for occasional paternal plastid transmission. In a large screen, we detected low-level paternal inheritance of transgenic plastids in tobacco. Whereas the frequency of transmission into the cotyledons of F(1) seedlings was approximately 1.58 x 10(-5) (on 100% cross-fertilization), transmission into the shoot apical meristem was significantly lower (2.86 x 10(-6)). Our data demonstrate that plastid transformation provides an effective tool to increase the biosafety of transgenic plants. However, in cases where pollen transmission must be prevented altogether, stacking with other containment methods will be necessary to eliminate the residual outcrossing risk.
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NASA Astrophysics Data System (ADS)
Kamnev, A. A.; Tarantilis, P. A.; Antonyuk, L. P.; Bespalova, L. A.; Polissiou, M. G.; Colina, M.; Gardiner, P. H. E.; Ignatov, V. V.
2001-05-01
Structural and compositional features of bacterial cell samples and of lipopolysaccharide-protein complex isolated from the cell surface of the plant-growth-promoting rhizobacterium Azospirillum brasilense (wild-type strain Sp7) were characterised using Fourier transform (FT) Raman spectroscopy. The structural spectroscopic information obtained is analysed and considered together with analytical data on the content of metal cations (Co 2+, Cu 2+ and Zn 2+) in the bacterial cells grown in a standard medium as well as in the presence of each of the cations (0.2 mM). The latter, being taken up by bacterial cells from the culture medium in significant amounts, were shown to induce certain metabolic changes in the bacterium revealed in FT-Raman spectra, which is discussed from the viewpoint of bacterial response to environmental stresses.
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Yin, Jun-Lin; Wong, Woon-Seng; Jang, In-Cheol; Chua, Nam-Hai
2017-02-01
Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/β-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA 3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
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In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering
Wu, Yuyong; You, Lili; Li, Shengchun; Ma, Meiqi; Wu, Mengting; Ma, Lixin; Bock, Ralph; Chang, Ling; Zhang, Jiang
2017-01-01
Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids. PMID:28871270
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Ederli, Luisa; Morettini, Roberta; Borgogni, Andrea; Wasternack, Claus; Miersch, Otto; Reale, Lara; Ferranti, Francesco; Tosti, Nicola; Pasqualini, Stefania
2006-01-01
The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O3). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O3 response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O3 fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O3-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression. PMID:16935990
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Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A
2010-01-01
Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.
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2011-01-01
Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium
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Youssefian, Shohab; Nakamura, Michimi; Orudgev, Emin; Kondo, Noriaki
2001-01-01
O-Acetylserine(thiol) lyase (OASTL), a key enzyme of plant sulfur metabolism, catalyzes the formation of Cys from sulfide and O-acetylserine. The biosynthesis of Cys is regarded as the exclusive function of sulfur reduction in plants, and a key limiting step in the production of glutathione (GSH), a thiol implicated in various cellular functions, including sulfur transport, gene expression, scavenging of reactive oxygen species (ROS), and resistance to biotic and abiotic stresses. To examine whether an increased capacity for cysteine (Cys) biosynthesis alters cellular responses to such stresses, we studied the differential changes in thiol levels and ROS scavenging of transgenic tobacco (Nicotiana tabacum) plants expressing the wheat (Triticum aestivum) OASTL gene, cys1, to SO2 and to the ROS generator, methyl viologen. Intracellular Cys and GSH contents were generally higher in cys1 transgenics than in controls under normal growth conditions, but became especially elevated in transgenic plants after SO2 exposure. An examination of differences in the ROS scavenging system of the transgenic plants also demonstrated the specific accumulation of Cu/Zn superoxide dismutase transcripts, known to be induced by Cys or GSH, and elevated cellular superoxide dismutase activities. The transgenic plants accordingly showed dramatic reductions in the extent of both foliar and photooxidative damage in response to acute SO2, as well as reduced levels of chlorosis and membrane damage following methyl viologen treatment. Overall, our results imply that OASTL plays a pivotal role in the synthesis of Cys and GSH that are required for regulation of plant responses to oxidative stress. PMID:11457951
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Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal
2015-12-01
Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
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Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment.
Batista, Dora; Fonseca, Sandra; Serrazina, Susana; Figueiredo, Andreia; Pais, Maria Salomé
2008-07-01
To the best of our knowledge, this is the first accurate and reliable protocol for hop (Humulus lupulus L.) genetic transformation using particle bombardment. Based on the highly productive regeneration system previously developed by us for hop var. Eroica, two efficient transformation protocols were established using petioles and green organogenic nodular clusters (GONCs) bombarded with gusA reporter and hpt selectable genes. A total of 36 hygromycin B-resistant (hyg(r)) plants obtained upon continuous selection were successfully transferred to the greenhouse, and a first generation group of transplanted plants was followed after spending a complete vegetative cycle. PCR analysis showed the presence of one of both transgenes in 25 plants, corresponding to an integration frequency of 69.4% and an overall transformation efficiency of 7.5%. Although all final transformants were GUS negative, the integration frequency of gusA gene was higher than that of hpt gene. Petiole-derived transgenic plants showed a higher co-integration rate of 76.9%. Real-time PCR analysis confirmed co-integration in 86% of the plants tested and its stability until the first generation, and identified positive plants amongst those previously assessed as hpt (+) only by conventional PCR. Our results suggest that the integration frequencies presented here, as well as those of others, may have been underestimated, and that PCR results should be taken with precaution not only for false positives, but also for false negatives. The protocols here described could be very useful for future introduction of metabolic or resistance traits in hop cultivars even if slight modifications for other genotypes are needed.
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Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.
2000-01-01
The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238
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Qin, Junhao; Li, Huashou; Lin, Chuxia
2016-08-01
Batch and greenhouse experiments were conducted to examine the effects of Fenton process on transformation of roxarsone in soils and its resulting impacts on the growth of and As uptake by a rice plant cultivar. The results show that addition of Fenton reagent markedly accelerated the degradation of roxarsone and produced arsenite, which was otherwise absent in the soil without added Fenton reagent. Methylation of arsenate was also enhanced by Fenton process in the earlier part of the experiment due to abundant supply of arsenate from Roxarsone degradation. Overall, addition of Fenton reagent resulted in the predominant presence of arsenate in the soils. Fenton process significantly improved the growth of rice in the maturity stage of the first crop, The concentration of methylated As species in the rice plant tissues among the different growth stages was highly variable. Addition of Fenton reagent into the soils led to reduced uptake of soil-borne As by the rice plants and this had a significant effect on reducing the accumulation of As in rice grains. The findings have implications for understanding As biogeochemistry in paddy rice field receiving rainwater-borne H2O2 and for development of mitigation strategies to reduce accumulation of As in rice grains. Copyright © 2016 Elsevier Inc. All rights reserved.
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Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics1[W][OA
Cui, Yaya; Barampuram, Shyam; Stacey, Minviluz G.; Hancock, C. Nathan; Findley, Seth; Mathieu, Melanie; Zhang, Zhanyuan; Parrott, Wayne A.; Stacey, Gary
2013-01-01
Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean. PMID:23124322
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The Plant Ionome Revisited by the Nutrient Balance Concept
Parent, Serge-Étienne; Parent, Léon Etienne; Egozcue, Juan José; Rozane, Danilo-Eduardo; Hernandes, Amanda; Lapointe, Line; Hébert-Gentile, Valérie; Naess, Kristine; Marchand, Sébastien; Lafond, Jean; Mattos, Dirceu; Barlow, Philip; Natale, William
2013-01-01
Tissue analysis is commonly used in ecology and agronomy to portray plant nutrient signatures. Nutrient concentration data, or ionomes, belong to the compositional data class, i.e., multivariate data that are proportions of some whole, hence carrying important numerical properties. Statistics computed across raw or ordinary log-transformed nutrient data are intrinsically biased, hence possibly leading to wrong inferences. Our objective was to present a sound and robust approach based on a novel nutrient balance concept to classify plant ionomes. We analyzed leaf N, P, K, Ca, and Mg of two wild and six domesticated fruit species from Canada, Brazil, and New Zealand sampled during reproductive stages. Nutrient concentrations were (1) analyzed without transformation, (2) ordinary log-transformed as commonly but incorrectly applied in practice, (3) additive log-ratio (alr) transformed as surrogate to stoichiometric rules, and (4) converted to isometric log-ratios (ilr) arranged as sound nutrient balance variables. Raw concentration and ordinary log transformation both led to biased multivariate analysis due to redundancy between interacting nutrients. The alr- and ilr-transformed data provided unbiased discriminant analyses of plant ionomes, where wild and domesticated species formed distinct groups and the ionomes of species and cultivars were differentiated without numerical bias. The ilr nutrient balance concept is preferable to alr, because the ilr technique projects the most important interactions between nutrients into a convenient Euclidean space. This novel numerical approach allows rectifying historical biases and supervising phenotypic plasticity in plant nutrition studies. PMID:23526060
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FamNet: A Framework to Identify Multiplied Modules Driving Pathway Expansion in Plants1
Tohge, Takayuki; Klie, Sebastian; Fernie, Alisdair R.
2016-01-01
Gene duplications generate new genes that can acquire similar but often diversified functions. Recent studies of gene coexpression networks have indicated that, not only genes, but also pathways can be multiplied and diversified to perform related functions in different parts of an organism. Identification of such diversified pathways, or modules, is needed to expand our knowledge of biological processes in plants and to understand how biological functions evolve. However, systematic explorations of modules remain scarce, and no user-friendly platform to identify them exists. We have established a statistical framework to identify modules and show that approximately one-third of the genes of a plant’s genome participate in hundreds of multiplied modules. Using this framework as a basis, we implemented a platform that can explore and visualize multiplied modules in coexpression networks of eight plant species. To validate the usefulness of the platform, we identified and functionally characterized pollen- and root-specific cell wall modules that multiplied to confer tip growth in pollen tubes and root hairs, respectively. Furthermore, we identified multiplied modules involved in secondary metabolite synthesis and corroborated them by metabolite profiling of tobacco (Nicotiana tabacum) tissues. The interactive platform, referred to as FamNet, is available at http://www.gene2function.de/famnet.html. PMID:26754669
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Geospatial assessment of invasive plants on reclaimed mines in Alabama
D. Lemke; C.J. Schweitzer; W. Tadesse; Y. Wang; J.A. Brown
2013-01-01
Throughout the world, the invasion of nonnative plants is an increasing threat to native biodiversity and ecosystem sustainability. Invasion is especially prevalent in areas affected by land transformation and disturbance. Surface mines are a major land transformation, and thus may promote the establishment and persistence of invasive plant communities. Using the Shale...
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Methods for genetic transformation in Dendrobium.
da Silva, Jaime A Teixeira; Dobránszki, Judit; Cardoso, Jean Carlos; Chandler, Stephen F; Zeng, Songjun
2016-03-01
The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.
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Plant Sterol Diversity in Pollen from Angiosperms.
Villette, Claire; Berna, Anne; Compagnon, Vincent; Schaller, Hubert
2015-08-01
Here we have examined the composition of free sterols and steryl esters of pollen from selected angiosperm species, as a first step towards a comprehensive analysis of sterol biogenesis in the male gametophyte. We detected four major sterol structural groups: cycloartenol derivatives bearing a 9β,19-cyclopropyl group, sterols with a double bond at C-7(8), sterols with a double bond at C-5(6), and stanols. All these groups were unequally distributed among species. However, the distribution of sterols as free sterols or as steryl esters in pollen grains indicated that free sterols were mostly Δ(5)-sterols and that steryl esters were predominantly 9β,19-cyclopropyl sterols. In order to link the sterol composition of a pollen grain at anthesis with the requirement for membrane lipid constituents of the pollen tube, we germinated pollen grains from Nicotiana tabacum, a model plant in reproductive biology. In the presence of radiolabelled mevalonic acid and in a time course series of measurements, we showed that cycloeucalenol was identified as the major neosynthesized sterol. Furthermore, the inhibition of cycloeucalenol neosynthesis by squalestatin was in full agreement with a de novo biogenesis and an apparent truncated pathway in the pollen tube.
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A long antisense RNA in plant chloroplasts.
Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R
2010-05-01
Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.
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Zimmermann, Matthias R.; Will, Torsten; Felle, Hubert H.; Furch, Alexandra C.U.
2016-01-01
In stressed plants, electrophysiological reactions (elRs) are presumed to contribute to long-distance intercellular communication between distant plant parts. Because of the focus on abiotic stress-induced elRs in recent decades, biotic stress-triggered elRs have been widely ignored. It is likely that the challenge to identify the particular elR types (action potential [AP], variation potential, and system potential [SP]) was responsible for this course of action. Thus, this survey focused on insect larva feeding (Spodoptera littoralis and Manduca sexta) that triggers distant APs, variation potentials, and SPs in monocotyledonous and dicotyledonous plant species (Hordeum vulgare, Vicia faba, and Nicotiana tabacum). APs were detected only after feeding on the stem/culm, whereas SPs were observed systemically following damage to both stem/culm and leaves. This was attributed to the unequal vascular innervation of the plant and a selective electrophysiological connectivity of the plant tissue. However, striking variations in voltage patterns were detected for each elR type. Further analyses (also in Brassica napus and Cucurbita maxima) employing complementary electrophysiological approaches in response to different stimuli revealed various reasons for these voltage pattern variations: an intrinsic plasticity of elRs, a plant-specific signature of elRs, a specific influence of the applied (a)biotic trigger, the impact of the technical approach, and/or the experimental setup. As a consequence, voltage pattern variations, which are not irregular but rather common, need to be included in electrophysiological signaling analysis. Due to their widespread occurrence, systemic propagation, and respective triggers, elRs should be considered as candidates for long-distance communication in higher plants. PMID:26872949
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59. VIEW OF FUSES AND A CURRENT TRANSFORMER LOCATED IN ...
59. VIEW OF FUSES AND A CURRENT TRANSFORMER LOCATED IN THE SIGNAL POWER CONDITIONING ROOM. THE CURRENT TRANSFORMER (UPPER RIGHT) IS AN INDUCTION COUPLED SENSOR WHICH IS USED TO REDUCE HIGH CURRENT TO ANALOGOUS LOW VALUES SAFE TO USE IN CONTROL ROOM CIRCUITRY. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT
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Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.
Zhang, Wan-Jun; Wang, Tao
2015-05-01
Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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A simple and reliable multi-gene transformation method for switchgrass.
Ogawa, Yoichi; Shirakawa, Makoto; Koumoto, Yasuko; Honda, Masaho; Asami, Yuki; Kondo, Yasuhiro; Hara-Nishimura, Ikuko
2014-07-01
A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.
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Khairy, Alaaldin Idris H; Oh, Mi Jeong; Lee, Seung Min; Kim, Da Som; Roh, Kwang Soo
2016-06-01
Toxic heavy metals such as cadmium (Cd) and copper (Cu) are global problems that are a growing threat to the environment. Despite some heavy metals are required for plant growth and development, others are considered toxic elements and do not play any known physiological role in plant cells. Elevated doses of Cd or Cu cause toxicity in plants and generate damages due to the stress condition and eventually cause a significant reduction in quantity and quality of crop plants. The nitric oxide (NO) donor sodium nitroprusside (SNP) is reported to alleviate the toxicity of some heavy metals like Cd and Cu. In the current study, the role of NO in alleviating stresses of Cd and Cu was investigated in in vitro -grown tobacco ( Nicotiana tabacum ) Based on plant growth, total chlorophyll contents, contents and activities of rubisco and rubisco activase. According to the results of this study, the growth and total chlorophyll contents of Cd/Cu stressed plants were hugely decreased in the absence of SNP, while the supplementation of SNP resulted in a significant increase of both fresh weight and total chlorophyll contents. Remarkable reductions of Rubisco and rubisco activase contents and activities were observed in Cd and Cu-induced plants. SNP supplementation showed the highest contents and activities of rubisco and rubisco activase compared to the control and Cu/Cd-stressed plants. Taken together, our findings suggest that SNP could play a protective role in regulation of plant responses to abiotic stresses such as Cd and Cu by enhancing Rubisco and Rubisco activase.
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Bally, Julia; Paget, Eric; Droux, Michel; Job, Claudette; Job, Dominique; Dubald, Manuel
2008-01-01
Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli. This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco (Nicotiana tabacum) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.
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Efficient and heritable transformation of Phalaenopsis orchids.
Hsing, Hong-Xian; Lin, Yi-Jyun; Tong, Chii-Gong; Li, Min-Jeng; Chen, Yun-Jin; Ko, Swee-Suak
2016-12-01
Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method. We developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2-5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot. This study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding.
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Alam, Pravej; Khan, Zainul Abdeen; Abdin, Malik Zainul; Khan, Jawaid A; Ahmad, Parvaiz; Elkholy, Shereen F; Sharaf-Eldin, Mahmoud A
2017-05-01
Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L -1 (BAP) and 0.5 mg L -1 (NAA) while 80% shoot percentage obtained with 4.0 mg L -1 (BAP) and 0.05 mg L -1 (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.
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Millwood, Reginald J; Moon, Hong S; Poovaiah, Charleson R; Muthukumar, Balasubramaniam; Rice, John Hollis; Abercrombie, Jason M; Abercrombie, Laura L; Green, William Derek; Stewart, Charles Neal
2016-05-01
Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen-specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible-to-no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand-crossed to both male-sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000-40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male-sterile tobacco, and 900-2100 seeds per male-sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI-driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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Yang, Jyisy; Yen, Hungchen E.
2002-01-01
A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mm NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mm NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis. PMID:12376666
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Macková, Hana; Hronková, Marie; Dobrá, Jana; Turečková, Veronika; Novák, Ondřej; Lubovská, Zuzana; Motyka, Václav; Haisel, Daniel; Hájek, Tomáš; Prášil, Ilja Tom; Gaudinová, Alena; Štorchová, Helena; Ge, Eva; Werner, Tomáš; Schmülling, Thomas; Vanková, Radomíra
2013-01-01
Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms. PMID:23669573
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Ismail, Hammad; Dilshad, Erum; Waheed, Mohammad Tahir; Sajid, Moniba; Kayani, Waqas Khan; Mirza, Bushra
2016-12-01
Lettuce is an important edible crop which possesses various medicinal properties. In this study Lactuca sativa L. (cv Grand Rapids) was transformed by Agrobacterium-mediated transformation with rol C gene. Transgene integration and expression was confirmed through PCR and semiquantitative RT-PCR. The transformed extracts were evaluated for their in vitro antioxidant and in vivo analgesic, anti-inflammatory and antidepressant activities in rats. The transformed plants showed 53-98 % increase in total phenolic and 45-58 % increase in total flavonoid contents compared with untransformed plants. Results of total reducing power and total antioxidant capacity exhibited 90-118 and 61-75 % increase in transformed plants, respectively. In contrast to control, DPPH, lipid peroxidation and DNA protection assay showed up to 37, 20 and 50 % enhancement in transformed plants, respectively. The extracts showed similar but significant enhancement behavior in hot plate analgesic and carrageenan-induced hind paw edema test. The transformed extracts showed 72.1 and 78.5 % increase for analgesic and anti-inflammatory activities, respectively. The transformants of rol C gene exhibited prominent antidepressant activity with 64-73 % increase compared with untransformed plants. In conclusion, the present work suggests that transformation with rol C gene can be used to generate lettuce with enhanced medicinally important properties, such as antioxidant, analgesic, anti-inflammatory and antidepressant potential.
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Iki, Taichiro; Ishikawa, Masayuki; Yoshikawa, Manabu
2017-01-01
Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.
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Karthik, Sivabalan; Pavan, Gadamchetty; Sathish, Selvam; Siva, Ramamoorthy; Kumar, Periyasamy Suresh; Manickavasagam, Markandan
2018-04-01
Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301- bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l -1 BASTA ® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.
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JPRS Report, Science & Technology, USSR: Life Sciences.
1989-02-01
Cytokinins in Transgenic Nicotiana Tabacum Plants [V. M. Zakharyev, A. Sh. Tashpulatov, et al.; DOKLADY AKADEMIINAUK SSSR, Vol 301 No 3, Jul 88] 18...and flowers . Objective evaluation of the quality of plant materials is impossible without the use of modern immu- nodiagnosis methods which...deriving diagnostic antisera. The diagnostics are automated. Pure viral antigens for potatoes and a number of other crops have been developed on the
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Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest1[OPEN
2017-01-01
Multicellular glandular trichomes are epidermal outgrowths characterized by the presence of a head made of cells that have the ability to secrete or store large quantities of specialized metabolites. Our understanding of the transcriptional control of glandular trichome initiation and development is still in its infancy. This review points to some central questions that need to be addressed to better understand how such specialized cell structures arise from the plant protodermis. A key and unique feature of glandular trichomes is their ability to synthesize and secrete large amounts, relative to their size, of a limited number of metabolites. As such, they qualify as true cell factories, making them interesting targets for metabolic engineering. In this review, recent advances regarding terpene metabolic engineering are highlighted, with a special focus on tobacco (Nicotiana tabacum). In particular, the choice of transcriptional promoters to drive transgene expression and the best ways to sink existing pools of terpene precursors are discussed. The bioavailability of existing pools of natural precursor molecules is a key parameter and is controlled by so-called cross talk between different biosynthetic pathways. As highlighted in this review, the exact nature and extent of such cross talk are only partially understood at present. In the future, awareness of, and detailed knowledge on, the biology of plant glandular trichome development and metabolism will generate new leads to tap the largely unexploited potential of glandular trichomes in plant resistance to pests and lead to the improved production of specialized metabolites with high industrial or pharmacological value. PMID:28724619
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Determining the transgene containment level provided by chloroplast transformation
Ruf, Stephanie; Karcher, Daniel; Bock, Ralph
2007-01-01
Plastids (chloroplasts) are maternally inherited in most crops. Maternal inheritance excludes plastid genes and transgenes from pollen transmission. Therefore, plastid transformation is considered a superb tool for ensuring transgene containment and improving the biosafety of transgenic plants. Here, we have assessed the strictness of maternal inheritance and the extent to which plastid transformation technology confers an increase in transgene confinement. We describe an experimental system facilitating stringent selection for occasional paternal plastid transmission. In a large screen, we detected low-level paternal inheritance of transgenic plastids in tobacco. Whereas the frequency of transmission into the cotyledons of F1 seedlings was ≈1.58 × 10−5 (on 100% cross-fertilization), transmission into the shoot apical meristem was significantly lower (2.86 × 10−6). Our data demonstrate that plastid transformation provides an effective tool to increase the biosafety of transgenic plants. However, in cases where pollen transmission must be prevented altogether, stacking with other containment methods will be necessary to eliminate the residual outcrossing risk. PMID:17420459
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Harpaz-Saad, Smadar; Azoulay, Tamar; Arazi, Tzahi; Ben-Yaakov, Eran; Mett, Anahit; Shiboleth, Yoel M; Hörtensteiner, Stefan; Gidoni, David; Gal-On, Amit; Goldschmidt, Eliezer E; Eyal, Yoram
2007-03-01
Chlorophyll is a central player in harvesting light energy for photosynthesis, yet the rate-limiting steps of chlorophyll catabolism and the regulation of the catabolic enzymes remain unresolved. To study the role and regulation of chlorophyllase (Chlase), the first enzyme of the chlorophyll catabolic pathway, we expressed precursor and mature versions of citrus (Citrus sinensis) Chlase in two heterologous plant systems: (1) squash (Cucurbita pepo) plants using a viral vector expression system; and (2) transiently transformed tobacco (Nicotiana tabacum) protoplasts. Expression of full-length citrus Chlase resulted in limited chlorophyll breakdown in protoplasts and no visible leaf phenotype in whole plants, whereas expression of a Chlase version lacking the N-terminal 21 amino acids (ChlaseDeltaN), which corresponds to the mature protein, led to extensive chlorophyll breakdown in both tobacco protoplasts and squash leaves. ChlaseDeltaN-expressing squash leaves displayed a dramatic chlorotic phenotype in plants grown under low-intensity light, whereas under natural light a lesion-mimic phenotype occurred, which was demonstrated to follow the accumulation of chlorophyllide, a photodynamic chlorophyll breakdown product. Full-length and mature citrus Chlase versions were localized to the chloroplast membrane fraction in expressing tobacco protoplasts, where processing of the N-terminal 21 amino acids appears to occur. Results obtained in both plant systems suggest that Chlase functions as a rate-limiting enzyme in chlorophyll catabolism controlled via posttranslational regulation.
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Hybrid proline-rich proteins: novel players in plant cell elongation?
Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas
2012-01-01
Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464
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Basnayake, Shiromani W V; Moyle, Richard; Birch, Robert G
2011-03-01
Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11-15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.
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Joyce, Priya; Kuwahata, Melissa; Turner, Nicole; Lakshmanan, Prakash
2010-02-01
A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced.
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Biolistic transformation of cotton embryogenic cell suspension cultures
USDA-ARS?s Scientific Manuscript database
Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...
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Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation
Zambon, Flavia T.; Erpen, Lígia; Soriano, Leonardo; Grosser, Jude
2018-01-01
The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree. PMID:29293649
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Ex vitro composite plants: an inexpensive, rapid method for root biology.
Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G
2005-08-01
Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.
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Effects of root-zone acidity on utilization of nitrate and ammonium in tobacco plants
NASA Technical Reports Server (NTRS)
Henry, L. T.; Raper, C. D. Jr; Raper CD, J. r. (Principal Investigator)
1989-01-01
Tobacco (Nicotiana tabacum L., cv. 'Coker 319') plants were grown for 28 days in flowing nutrient culture containing either 1.0 mM NO3- or 1.0 mM NH4+ as the nitrogen source in a complete nutrient solution. Acidities of the solutions were controlled at pH 6.0 or 4.0 for each nitrogen source. Plants were sampled at intervals of 6 to 8 days for determination of dry matter and nitrogen accumulation. Specific rates of NO3- or NH4+ uptake (rate of uptake per unit root mass) were calculated from these data. Net photosynthetic rates per unit leaf area were measured on attached leaves by infrared gas analysis. When NO3- [correction of NO-] was the sole nitrogen source, root growth and nitrogen uptake rate were unaffected by pH of the solution, and photosynthetic activity of leaves and accumulation of dry matter and nitrogen in the whole plant were similar. When NH4+ was the nitrogen source, photosynthetic rate of leaves and accumulation of dry matter and nitrogen in the whole plant were not statistically different from NO3(-) -fed plants when acidity of the solution was controlled at pH 6.0. When acidity for NH4(+) -fed plants was increased to pH 4.0, however, specific rate of NH4+ uptake decreased by about 50% within the first 6 days of treatment. The effect of acidity on root function was associated with a decreased rate of accumulation of nitrogen in shoots that was accompanied by a rapid cessation of leaf development between days 6 and 13. The decline in leaf growth rate of NH4(+) -fed plants at pH 4.0 was followed by reductions in photosynthetic rate per unit leaf area. These responses of NH4(+) -fed plants to increased root-zone acidity are characteristic of the sequence of responses that occur during onset of nitrogen stress.
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Wu, Dongliang; Navet, Natasha; Liu, Yingchao; Uchida, Janice; Tian, Miaoying
2016-09-06
As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility. In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion. This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.
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Fox, P; Narayanaswamy, K; Genz, A; Drewes, J E
2001-01-01
Water quality transformations during soil aquifer treatment at the Mesa Northwest Water Reclamation Plant (NWWRP) were evaluated by sampling a network of groundwater monitoring wells located within the reclaimed water plume. The Mesa Northwest Water Reclamation Plant has used soil aquifer treatment (SAT) since it began operation in 1990 and the recovery of reclaimed water from the impacted groundwater has been minimal. Groundwater samples obtained represent travel times from several days to greater than five years. Samples were analyzed for a wide range of organic and inorganic constituents. Sulfate was used as a tracer to estimate travel times and define reclaimed water plume movement. Dissolved organic carbon concentrations were reduced to approximately 1 mg/L after 12 to 24 months of soil aquifer treatment with an applied DOC concentration from the NWWRP of 5 to 7 mg/L. The specific ultraviolet absorbance (SUVA) increased during initial soil aquifer treatment on a time-scale of days and then decreased as longer term soil aquifer treatment removed UV absorbing compounds. The trihalomethane formation potential (THMFP) was a function of the dissolved organic carbon concentration and ranged from 50 to 65 micrograms THMFP/mg DOC. Analysis of trace organics revealed that the majority of trace organics were removed as DOC was removed with the exception of organic iodine. The majority of nitrogen was applied as nitrate-nitrogen and the reclaimed water plume had lower nitrate-nitrogen concentrations as compared to the background groundwater. The average dissolved organic carbon concentrations in the reclaimed water plume were less than 50% of the drinking water dissolved organic concentrations from which the reclaimed water originated.
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Aymerich, I; Acuña, V; Barceló, D; García, M J; Petrovic, M; Poch, M; Rodriguez-Mozaz, S; Rodríguez-Roda, I; Sabater, S; von Schiller, D; Corominas, Ll
2016-09-01
Pharmaceuticals are designed to improve human and animal health, but may also be a threat to freshwater ecosystems, particularly after receiving urban or wastewater treatment plant (WWTP) effluents. Knowledge on the fate and attenuation of pharmaceuticals in engineered and natural ecosystems is rather fragmented, and comparable methods are needed to facilitate the comprehension of those processes amongst systems. In this study the dynamics of 8 pharmaceuticals (acetaminophen, sulfapyridine, sulfamethoxazole, carbamazepine, venlafaxine, ibuprofen, diclofenac, diazepam) and 11 of their transformation products were investigated in a WWTP and the associated receiving river ecosystem. During 3 days, concentrations of these compounds were quantified at the influents, effluents, and wastage of the WWTP, and at different distances downstream the effluent at the river. Attenuation (net balance between removal and release from and to the water column) was estimated in both engineered and natural systems using a comparable model-based approach by considering different uncertainty sources (e.g. chemical analysis, sampling, and flow measurements). Results showed that pharmaceuticals load reduction was higher in the WWTP, but attenuation efficiencies (as half-life times) were higher in the river. In particular, the load of only 5 out of the 19 pharmaceuticals was reduced by more than 90% at the WWTP, while the rest were only partially or non-attenuated (or released) and discharged into the receiving river. At the river, only the load of ibuprofen was reduced by more than 50% (out of the 6 parent compounds present in the river), while partial and non-attenuation (or release) was observed for some of their transformation products. Linkages in the routing of some pharmaceuticals (venlafaxine, carbamazepine, ibuprofen and diclofenac) and their corresponding transformation products were also identified at both WWTP and river. Finally, the followed procedure showed that dynamic
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2013-01-01
Background Cotton, one of the world’s leading crops, is important to the world’s textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. Results We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome
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Transformation of the Fungal Soybean Pathogen Cercospora kikuchii with the Selectable Marker bar
Upchurch, Robert G.; Meade, Maura J.; Hightower, Robin C.; Thomas, Robert S.; Callahan, Terrence M.
1994-01-01
An improved transformation protocol, utilizing selection for resistance to the herbicide bialaphos, has been developed for the plant pathogenic fungus Cercospora kikuchii. Stable, bialaphos-resistant transformants are recovered at frequencies eight times higher than those achieved with the previous system that was based on selection for benomyl resistance. In addition to C. kikuchii, this improved method can also be used to transform other species of Cercospora. Images PMID:16349469
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Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun
2016-01-01
Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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Uptake and Transformation of Methylated and Inorganic Antimony in Plants.
Ji, Ying; Mestrot, Adrien; Schulin, Rainer; Tandy, Susan
2018-01-01
Used as a hardening agent in lead bullets, antimony (Sb) has become a major contaminant in shooting range soils of some countries including Switzerland. Soil contamination by Sb is also an environmental problem in countries with Sb-mining activities such as China and Bolivia. Because of its toxicity and relatively high mobility, there is concern over the risk of Sb transfer from contaminated soils into plants, and thus into the food chain. In particular there is very little information on the environmental behavior of methylated antimony, which can be produced by microbial biomethylation of inorganic Sb in contaminated soils. Using a new extraction and high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method, we investigated antimony speciation in roots and shoots of wheat, fescue, rye, and ryegrass plants exposed to trimethyl antimony(V) (TMSb), antimonite (Sb(III)), and antimonate (Sb(V)) in hydroponics. The total root Sb concentrations followed the order Sb(III) treatment > Sb(V) treatment > TMSb treatment, except for fescue. Shoot Sb concentrations, however, did not differ among the three treatments. In the Sb(V) treatment small quantities of TMSb were found in the roots, whereas no TMSb was detected in the roots of Sb(III)-treated plants. In contrast, similar concentrations of TMSb were found in the shoots in both inorganic Sb treatments. The results indicate that biomethylation of Sb may occur in plants. In the TMSb treatment TMSb was the major Sb species, but the two inorganic Sb species were also found both in shoots and roots along with some unknown Sb species, suggesting that also TMSb demethylation may occur within plant tissues. The results furthermore indicate that methylated Sb is more mobile in plants than inorganic Sb species. Knowledge about this is important in risk assessments of Sb-contaminated sites, as methylation may render Sb more toxic than inorganic Sb, as it is known for arsenic (As).
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Uptake and Transformation of Methylated and Inorganic Antimony in Plants
Ji, Ying; Mestrot, Adrien; Schulin, Rainer; Tandy, Susan
2018-01-01
Used as a hardening agent in lead bullets, antimony (Sb) has become a major contaminant in shooting range soils of some countries including Switzerland. Soil contamination by Sb is also an environmental problem in countries with Sb-mining activities such as China and Bolivia. Because of its toxicity and relatively high mobility, there is concern over the risk of Sb transfer from contaminated soils into plants, and thus into the food chain. In particular there is very little information on the environmental behavior of methylated antimony, which can be produced by microbial biomethylation of inorganic Sb in contaminated soils. Using a new extraction and high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method, we investigated antimony speciation in roots and shoots of wheat, fescue, rye, and ryegrass plants exposed to trimethyl antimony(V) (TMSb), antimonite (Sb(III)), and antimonate (Sb(V)) in hydroponics. The total root Sb concentrations followed the order Sb(III) treatment > Sb(V) treatment > TMSb treatment, except for fescue. Shoot Sb concentrations, however, did not differ among the three treatments. In the Sb(V) treatment small quantities of TMSb were found in the roots, whereas no TMSb was detected in the roots of Sb(III)-treated plants. In contrast, similar concentrations of TMSb were found in the shoots in both inorganic Sb treatments. The results indicate that biomethylation of Sb may occur in plants. In the TMSb treatment TMSb was the major Sb species, but the two inorganic Sb species were also found both in shoots and roots along with some unknown Sb species, suggesting that also TMSb demethylation may occur within plant tissues. The results furthermore indicate that methylated Sb is more mobile in plants than inorganic Sb species. Knowledge about this is important in risk assessments of Sb-contaminated sites, as methylation may render Sb more toxic than inorganic Sb, as it is known for arsenic (As). PMID
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Heterozygote deficits in cyst plant-parasitic nematodes: possible causes and consequences.
Montarry, Josselin; Jan, Pierre-Loup; Gracianne, Cecile; Overall, Andrew D J; Bardou-Valette, Sylvie; Olivier, Eric; Fournet, Sylvain; Grenier, Eric; Petit, Eric J
2015-04-01
Deviations of genotypic frequencies from Hardy-Weinberg equilibrium (HWE) expectations could reveal important aspects of the biology of populations. Deviations from HWE due to heterozygote deficits have been recorded for three plant-parasitic nematode species. However, it has never been determined whether the observed deficits were due (i) to the presence of null alleles, (ii) to a high level of consanguinity and/or (iii) to a Wahlund effect. The aim of the present work was, while taking into the possible confounding effect of null alleles, to disentangle consanguinity and Wahlund effect in natural populations of those three economically important cyst nematodes using microsatellite markers: Globodera pallida, G. tabacum and Heterodera schachtii, pests of potato, tobacco and sugar beet, respectively. The results show a consistent pattern of heterozygote deficiency in the three nematode species sampled at the spatial scale of the host plant. We demonstrate that the prevalence of null alleles is weak and that heterozygote deficits do not have a single origin. Our results suggested that it is restricted dispersal that leads to heterozygote deficits through both consanguinity and substructure, which effects can be linked to soil movement, cyst density, and the number of generations per year. We discuss potential implications for the durability of plant resistances that are used to protect crops against parasites in which mating between relatives occur. While consanguineous mating leads to homozygosity at all loci, including loci governing avirulence/virulence, which favours the expression of virulence when recessive, the Wahlund effect is expected to have no particular effect on the adaptation of nematodes to resistances. © 2015 John Wiley & Sons Ltd.
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Chiyoda, Shota; Ishizaki, Kimitsune; Kataoka, Hideo; Yamato, Katsuyuki T; Kohchi, Takayuki
2008-09-01
The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3''-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.
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Transformation of soil and vegetable conditions at oil production territories
NASA Astrophysics Data System (ADS)
Gatina, Evgeniia
2017-04-01
On the territory of modern oil production soil, vegetation, ecosystem conditions of the environment are significantly transformed. Researches have been conducted on the oil production territories located in a boreal coniferous forest natural zone from 2005 to 2015. Standard geobotanical and soil methods are used. Mechanical destruction of a plant cover, change of the water conditions, intake of oil products and salty waters in ecosystems, pollution of the atmosphere are considered as the major technology-related factors defining transformation of land ecosystems at operation of the oil field. Under the mechanical destruction of a plant cover the pioneer plant communities are formed. These communities are characterized by most reduced specific wealth with prevalence of types of meadow groups of plants and presence of types of wetland groups of plants. The biodiversity of biocenosis which are affected linear infrastructure facilities of oil production territories and change of the water conditions, decreases. It is observed decrease in species wealth, simplification of structure of communities. Under the salting of soils in ecosystems there is a decrease species diversity of communities to prevalence nitrophilous and meadow plant species. At the increased content of organic substances in the soils that is a consequence of intake of oil products, is characteristic increase in specific richness of communities, introduction of types of wetland and oligotrophic groups of plants in forest communities. Influence depends on distance to an influence source. In process of removal from a source of atmospheric pollution in forest communities there is a decrease in species diversity and complication of structure of community. It is caused by introduction of types of meadow groups of plants in ecotone sites of the forest communities located near a source of influence and restoration of structural features of forest communities in process of removal from an influence source
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Macroclimatic change expected to transform coastal wetland ecosystems this century
Gabler, Christopher A.; Osland, Michael J.; Grace, James B.; Stagg, Camille L.; Day, Richard H.; Hartley, Stephen B.; Enwright, Nicholas M.; From, Andrew; McCoy, Meagan L.; McLeod, Jennie L.
2017-01-01
Coastal wetlands, existing at the interface between land and sea, are highly vulnerable to climate change. Macroclimate (for example, temperature and precipitation regimes) greatly influences coastal wetland ecosystem structure and function. However, research on climate change impacts in coastal wetlands has concentrated primarily on sea-level rise and largely ignored macroclimatic drivers, despite their power to transform plant community structure and modify ecosystem goods and services. Here, we model wetland plant community structure based on macroclimate using field data collected across broad temperature and precipitation gradients along the northern Gulf of Mexico coast. Our analyses quantify strongly nonlinear temperature thresholds regulating the potential for marsh-to-mangrove conversion. We also identify precipitation thresholds for dominance by various functional groups, including succulent plants and unvegetated mudflats. Macroclimate-driven shifts in foundation plant species abundance will have large effects on certain ecosystem goods and services. Based on current and projected climatic conditions, we project that transformative ecological changes are probable throughout the region this century, even under conservative climate scenarios. Coastal wetland ecosystems are functionally similar worldwide, so changes in this region are indicative of potential future changes in climatically similar regions globally.
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ENZYMATIC PROCESSES USED BY PLANTS TO DEGRADE ORGANIC COMPOUNDS
This is a review of recent plant enzyme systems that have been studied in uptake and transformation of organic contaminants. General procedures of plant preparation and enzyme isolation are covered. Six plant enzyme systems have been investigated for activity with selected pollut...
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Hieber, A David; Bugos, Robert C; Verhoeven, Amy S; Yamamoto, Harry Y
2002-01-01
Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.
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Chen, Dan; Deng, Yingtian; Zhao, Jie
2012-01-15
Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.
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[Study on mechanism of precursors transforming into indigo and indirubin in blue-genera plants].
Yang, Ming; Liu, Zeyu; Su, Zhetong; Zou, Wenquan
2010-04-01
Accessed to literatures at home and abroad, we introduced the process of indigo naturalis transforming from dyestuff industry into pharmaceutical industry. It is affirmed that the precursors of indigo and indirubin are isatan A, isatan B, isatan C and indicant. Meanwhile, author clarified the mechanism of transformation for these precursors transforming into indigo and indirubin. And we summarized methods of determination for these precursors. In summary, these references provide us accordance of study on processing principle of Indigo naturalis, and lay the foundation for technics of making indigo and indirubin entering into modern industry.
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Ca2+ transport in plant cells and mechanisms of transformation of phytochrome-induced photosignals
NASA Astrophysics Data System (ADS)
Volotovski, Igor D.
1995-01-01
The recent data on the influence of phytochrome on the efficiency of Ca2+ translocation across the membranes of oat protoplasts are given. Ca2+ uptake in the protoplasts was shown to be influenced by the red light (R) illumination. This effect was reverted by the following far-red light (FR) illumination. To elucidate the sensitivity to phytochrome-controlling action the screening between the mechanisms of Ca2+ transport across the plasma membranes of oat protoplasts, Na+/Ca2+ and Ca2+/H+ exchangers, Ca2+-pump and Ca2+-channel was done. It was established that phytochrome modulated the activity of Na+/Ca2+-exchanger and Ca2+-pump. The light-mediated oscillations of cytoplasmic Ca2+ concentration in the oat protoplasts were demonstrated using fluorescence probe quin2 loaded into the cells and laser monitoring of fluorescence signal. The evidences were obtained that the oscillations were not the result of the elevation of cytoplasmic Ca2+ concentration and had no connection with Ca2+ pool of mitochondria. The possibility of the relation between the Ca2+ oscillations and phosphoinositide metabolism in plant cell membranes is analyzed. The mechanisms of transformation of primary phytochrome signal into biological effects were discussed.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Agarwal, Vivek; Lybeck, Nancy J.; Pham, Binh
Research and development efforts are required to address aging and reliability concerns of the existing fleet of nuclear power plants. As most plants continue to operate beyond the license life (i.e., towards 60 or 80 years), plant components are more likely to incur age-related degradation mechanisms. To assess and manage the health of aging plant assets across the nuclear industry, the Electric Power Research Institute has developed a web-based Fleet-Wide Prognostic and Health Management (FW-PHM) Suite for diagnosis and prognosis. FW-PHM is a set of web-based diagnostic and prognostic tools and databases, comprised of the Diagnostic Advisor, the Asset Faultmore » Signature Database, the Remaining Useful Life Advisor, and the Remaining Useful Life Database, that serves as an integrated health monitoring architecture. The main focus of this paper is the implementation of prognostic models for generator step-up transformers in the FW-PHM Suite. One prognostic model discussed is based on the functional relationship between degree of polymerization, (the most commonly used metrics to assess the health of the winding insulation in a transformer) and furfural concentration in the insulating oil. The other model is based on thermal-induced degradation of the transformer insulation. By utilizing transformer loading information, established thermal models are used to estimate the hot spot temperature inside the transformer winding. Both models are implemented in the Remaining Useful Life Database of the FW-PHM Suite. The Remaining Useful Life Advisor utilizes the implemented prognostic models to estimate the remaining useful life of the paper winding insulation in the transformer based on actual oil testing and operational data.« less
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Wound-induced expression of horseradish peroxidase.
Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A
1994-01-01
Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.
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Bedoya, Leonor C.; Martínez, Fernando; Orzáez, Diego; Daròs, José-Antonio
2012-01-01
Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method. PMID:22238422
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Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.
Chin, Dong Poh; Mishiba, Kei-ichiro; Mii, Masahiro
2007-06-01
Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.
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Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.).
Wu, Hao; Acanda, Yosvanis; Jia, Hongge; Wang, Nian; Zale, Janice
2016-09-01
The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.
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Bekiaris, Georgios; Triolo, Jin M; Peltre, Clément; Pedersen, Lene; Jensen, Lars S; Bruun, Sander
2015-12-01
Biochemical methane potential (BMP) is a very important characteristic of a given feedstock for optimisation of its use in biogas production. However, the long digestion time needed to determine BMP is the main limitation for the use of this assay during the operation of anaerobic digesters to produce biogas. Fourier transform mid-infrared photoacoustic spectroscopy (FTIR-PAS) was used to predict the BMP of 87 plant biomasses. The developed calibration model was able to explain 81% of the variance in the measured BMP of a selected test set with a root mean square error (RMSE) of 40NLCH4kg(-1) of volatile solids (VS) and a ratio of performance to deviation (RPD) of 2.38. The interpretation of the regression coefficients used in the calibration revealed a positive correlation of BMP with easily degradable compounds (amorphous cellulose, hemicellulose and aliphatic compounds) and a negative correlation with inhibitors of cellulose hydrolysis (lignin, hemicellulose). Copyright © 2015 Elsevier Ltd. All rights reserved.
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Micah E. Stevens; Paula M. Pijut
2014-01-01
Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to...
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Wuriyanghan, Hada; Falk, Bryce W.
2013-01-01
The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will
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Zhao, Jian; Ren, Wenting; Dai, Yanhui; Liu, Lijiao; Wang, Zhenyu; Yu, Xiaoyu; Zhang, Junzhe; Wang, Xiangke; Xing, Baoshan
2017-07-05
Engineered nanoparticles (NPs) are being released into aquatic environments with their increasing applications. In this work, we investigated the interaction of CuO NPs with a floating plant, water hyacinth (Eichhornia crassipes). CuO NPs (50 mg/L) showed significant growth inhibition on both roots and shoots of E. crassipes after 8-day exposure, much higher than that of the bulk CuO particles (50 mg/L) and their corresponding dissolved Cu 2+ ions (0.30 mg/L). Scanning electron and light microscopic observations showed that the root caps and meristematic zone of E. Crassipes were severely damaged after CuO NP exposure, with disordered cell arrangement and a destroyed elongation zone of root tips. It is confirmed that CuO NPs could be translocated to shoot from both roots and submerged leaves. As detected by X-ray absorption near-edge spectroscopy analysis (XANES), CuO NPs were observed in roots, submerged leaves, and emerged leaves. Cu 2 S and other Cu species were also detected in these tissues, providing solid evidence of the transformation of CuO NPs. In addition, stomatal closure was observed during CuO NPs-leaf contact, which was induced by the production of H 2 O 2 and increased Ca level in leaf guard cells. These findings are helpful for better understanding the fate of NPs in aquatic plants and related biological responses.