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Sample records for biolistics

  1. Dynamics Modelling of Biolistic Gene Guns

    SciTech Connect

    Zhang, M.; Tao, W.; Pianetta, P.A.

    2009-06-04

    The gene transfer process using biolistic gene guns is a highly dynamic process. To achieve good performance, the process needs to be well understood and controlled. Unfortunately, no dynamic model is available in the open literature for analysing and controlling the process. This paper proposes such a model. Relationships of the penetration depth with the helium pressure, the penetration depth with the acceleration distance, and the penetration depth with the micro-carrier radius are presented. Simulations have also been conducted. The results agree well with experimental results in the open literature. The contribution of this paper includes a dynamic model for improving and manipulating performance of the biolistic gene gun.

  2. Biolistic transformation of cotton zygotic embryo meristem

    USDA-ARS?s Scientific Manuscript database

    Biolistic transformation of cotton meristems, isolated from mature seed is detailed in this book chapter. This method is simple and avoids the necessity to use genotype-dependent regenerable cell cultures. However, identification of germ line transformation using this method is laborious and time-c...

  3. Biolistic mediated production of transgenic oil palm.

    PubMed

    Kadir Ahmad Parveez, Ghulam

    2008-01-01

    Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.

  4. Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

    PubMed

    Mialhe, E; Miller, L H

    1994-05-01

    To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

  5. Bombarding Cancer: Biolistic Delivery of therapeutics using Porous Si Carriers

    NASA Astrophysics Data System (ADS)

    Zilony, Neta; Tzur-Balter, Adi; Segal, Ester; Shefi, Orit

    2013-08-01

    A new paradigm for an effective delivery of therapeutics into cancer cells is presented. Degradable porous silicon carriers, which are tailored to carry and release a model anti-cancer drug, are biolistically bombarded into in-vitro cancerous targets. We demonstrate the ability to launch these highly porous microparticles by a pneumatic capillary gene gun, which is conventionally used to deliver cargos by heavy metal carriers. By optimizing the gun parameters e.g., the accelerating gas pressure, we have successfully delivered the porous carriers, to reach deep targets and to cross a skin barrier in a highly spatial resolution. Our study reveals significant cytotoxicity towards the target human breast carcinoma cells following the delivery of drug-loaded carriers, while administrating empty particles results in no effect on cell viability. The unique combination of biolistics with the temporal control of payload release from porous carriers presents a powerful and non-conventional platform for designing new therapeutic strategies.

  6. Bombarding Cancer: Biolistic Delivery of therapeutics using Porous Si Carriers

    PubMed Central

    Zilony, Neta; Tzur-Balter, Adi; Segal, Ester; Shefi, Orit

    2013-01-01

    A new paradigm for an effective delivery of therapeutics into cancer cells is presented. Degradable porous silicon carriers, which are tailored to carry and release a model anti-cancer drug, are biolistically bombarded into in-vitro cancerous targets. We demonstrate the ability to launch these highly porous microparticles by a pneumatic capillary gene gun, which is conventionally used to deliver cargos by heavy metal carriers. By optimizing the gun parameters e.g., the accelerating gas pressure, we have successfully delivered the porous carriers, to reach deep targets and to cross a skin barrier in a highly spatial resolution. Our study reveals significant cytotoxicity towards the target human breast carcinoma cells following the delivery of drug-loaded carriers, while administrating empty particles results in no effect on cell viability. The unique combination of biolistics with the temporal control of payload release from porous carriers presents a powerful and non-conventional platform for designing new therapeutic strategies. PMID:23975675

  7. Generation of Transgenic C. elegans by Biolistic Transformation

    PubMed Central

    Hochbaum, Daniel; Ferguson, Annabel A.; Fisher, Alfred L.

    2010-01-01

    The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection. PMID:20811328

  8. Generation of transgenic C. elegans by biolistic transformation.

    PubMed

    Hochbaum, Daniel; Ferguson, Annabel A; Fisher, Alfred L

    2010-08-23

    The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

  9. Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.).

    PubMed

    King, Zachary R; Bray, Adam L; Lafayette, Peter R; Parrott, Wayne A

    2014-02-01

    Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 μm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.

  10. Preliminary attempts to biolistic inoculation of grapevine fanleaf virus.

    PubMed

    Valat, L; Mode, F; Mauro, M C; Burrus, M

    2003-03-01

    Biolistics has been studied to inoculate grapevine fanleaf virus (GFLV), a Nepovirus, to its natural woody host, Vitis sp., and its herbaceous host, Chenopodium quinoa. At first, bombardment conditions for in vitro and greenhouse grown plants were set using the uidA reporter gene. The infectious feature of the cartridges was then evaluated by studying infection of C. quinoa plants. Systemic infection was obtained with either GFLV particles or RNA extracts in experimental conditions which gave also the highest transient uidA gene expression. Concerning grapevine, our results indicate that extrapolation to this plant is difficult. In only 1 out of 8 independent bombardment experiments done with GFLV and 41B, we were able to detect the virus in freshly bombarded leaves. Similarly, later after bombardment, Pol mRNAs were detected once, at days 7 and 14 only. Incubating the plants in darkness, as suggested in the literature, or using Rupestris Saint Georges, an indicator for GFLV presence, did not yield any improvement. Finely, our observations suggest that detection of GFLV in bombarded grapevine tissues by immunological or molecular techniques remains a limiting factor, probably due to an excess of inhibitory compounds released during the biolistic process.

  11. Proteolistics: a biolistic method for intracellular delivery of proteins.

    PubMed

    Martin-Ortigosa, Susana; Wang, Kan

    2014-10-01

    In this work, an intracellular protein delivery methodology termed "proteolistics" is described. This method utilizes a biolistic gun apparatus and involves a simple protein/projectile preparation step. The protein to be delivered is mixed with a gold particle microprojectile suspension and is placed onto a gene gun cartridge, where it is dehydrated using either lyophilization or room-temperature air-drying. Subsequent intracellular protein delivery is achieved in plant and mammalian tissues upon bombardment. Because the method does not require modification of delivery agents or cargo biomolecules and involves a simple physical deposition of the protein onto the microprojectiles, there is no restriction on protein type in terms of molecular weight, isoelectric point or tertiary structure. Because the method delivers protein through the widely used gene gun system, it can be readily applied to any tissue or organism amenable to biolistics. A variety of proteins with molecular weight ranging from 24 to 68 kDa and isoelectric point from 4.8 to 10.1 were tested in this work. It is anticipated that this simple and versatile technique will offer biologists a powerful tool for basic research in areas such as understanding of cell and gene functions and for biotechnological applications such as genome editing.

  12. An in planta biolistic method for stable wheat transformation.

    PubMed

    Hamada, Haruyasu; Linghu, Qianyan; Nagira, Yozo; Miki, Ryuji; Taoka, Naoaki; Imai, Ryozo

    2017-09-13

    The currently favoured method for wheat (Triticum aestivum L.) transformation is inapplicable to many elite cultivars because it requires callus culture and regeneration. Here, we developed a simple, reproducible, in planta wheat transformation method using biolistic DNA delivery without callus culture or regeneration. Shoot apical meristems (SAMs) grown from dry imbibed seeds were exposed under a microscope and subjected to bombardment with different-sized gold particles coated with the GFP gene construct, introducing DNA into the L2 cell layer. Bombarded embryos were grown to mature, stably transformed T0 plants and integration of the GFP gene into the genome was determined at the fifth leaf. Use of 0.6-µm particles and 1350-psi pressure resulted in dramatically increased maximum ratios of transient GFP expression in SAMs and transgene integration in the fifth leaf. The transgene was integrated into the germ cells of 62% of transformants, and was therefore inherited in the next generation. We successfully transformed the model wheat cultivar 'Fielder', as well as the recalcitrant Japanese elite cultivar 'Haruyokoi'. Our method could potentially be used to generate stable transgenic lines for a wide range of commercial wheat cultivars.

  13. Shock Wave Based Biolistic Device for DNA and Drug Delivery

    NASA Astrophysics Data System (ADS)

    Nakada, Mutsumi; Menezes, Viren; Kanno, Akira; Hosseini, S. Hamid R.; Takayama, Kazuyoshi

    2008-03-01

    A shock wave assisted biolistic (biological ballistic) device has been developed to deliver DNA/drug-coated micro-projectiles into soft living targets. The device consists of an Nd:YAG laser, an optical setup to focus the laser beam and, a thin aluminum (Al) foil (typically 100 µm thick) which is a launch pad for the micro-projectiles. The DNA/drug-coated micro-particles to be delivered are deposited on the anterior surface of the foil and the posterior surface of the foil is ablated using the laser beam with an energy density of about 32×109 W/cm2. The ablation launches a shock wave through the foil that imparts an impulse to the foil surface, due to which the deposited particles accelerate and acquire sufficient momentum to penetrate soft targets. The device has been tested for particle delivery by delivering 1 µm size tungsten particles into liver tissues of experimental rats and in vitro test models made of gelatin. The penetration depths of about 90 and 800 µm have been observed in the liver and gelatin targets, respectively. The device has been tested for in vivo DNA [encoding β-glucuronidase (GUS) gene] transfer by delivering plasmid DNA-coated, 1-µm size gold (Au) particles into onion scale, tobacco leaf and soybean seed cells. The GUS activity was detected in the onion, tobacco and soybean cells after the DNA delivery. The present device is totally non-intrusive in nature and has a potential to get miniaturized to suit the existing medical procedures for DNA and/or drug delivery.

  14. Optimized conditions for biolistic-mediated transformation of Lilium longilforum 'Nellie White'

    USDA-ARS?s Scientific Manuscript database

    A variety of tissues were used for biolistic-mediated transformation of Lilum longiflorum 'Nellie White'. Transgenic plants were not recovered from five-month-old, non-embryogenic callus or suspension cells that had been bombarded with pDM327 that contains the bar-uidA fusion gene under control the ...

  15. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    PubMed Central

    2011-01-01

    Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that

  16. Plastid transformation in cabbage (Brassica oleracea L. var. capitata L.) by the biolistic process.

    PubMed

    Tseng, Menq-Jiau; Yang, Ming-Te; Chu, Wan-Ru; Liu, Cheng-Wei

    2014-01-01

    Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide. Scientists are using biotechnology in addition to traditional breeding methods to develop new cabbage varieties with desirable traits. Recent biotechnological advances in chloroplast transformation technology have opened new avenues for crop improvement. In 2007, we developed a stable plastid transformation system for cabbage and reported the successful transformation of the cry1Ab gene into the cabbage chloroplast genome. This chapter describes the methods for cabbage transformation using biolistic procedures. The following sections are included in this protocol: preparation of donor materials, coating gold particles with DNA, biolistic bombardment, as well as the regeneration and selection of transplastomic cabbage plants. The establishment of a plastid transformation system for cabbage offers new possibilities for introducing new agronomic and horticultural traits into Brassica crops.

  17. Biolistic transfection of neuronal cultures using a hand-held gene gun

    PubMed Central

    O'Brien, John A; Lummis, Sarah C R

    2009-01-01

    Biolistic transfection is a technique in which subcellular-sized particles coated with DNA are accelerated to high velocity to propel them into cells. This method is applicable to tissues, cells and organelles, and can be used for both in vitro and in vivo transformations; with the right equipment, it is simple, rapid and efficient. Here we provide a detailed protocol for biolistic transfection of plasmids into cultured human embryonic kidney (HEK) 293 cells and organotypic brain slices using a hand-held gene gun. There are three major steps: (i) coating microcarriers with DNA, (ii) transferring the microcarriers into a cartridge to make a ‘bullet’, and (iii) firing the DNA-coated microcarriers into cells using a pulse of helium gas. The method can be readily adapted to other cell types and tissues. The protocol can be completed in 1–2 h. PMID:17406333

  18. Plastid transformation in lettuce (Lactuca sativa L.) by biolistic DNA delivery.

    PubMed

    Ruhlman, Tracey A

    2014-01-01

    The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa L.). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide an ideal platform for the production of biopharmaceuticals.

  19. Plasmid-mediated gene transfer in neurons using the biolistics technique.

    PubMed

    Biewenga, J E; Destrée, O H; Schrama, L H

    1997-01-01

    Biolistics has been developed as a system for gene delivery into plant cells, but has recently been introduced for transfection into mammalian tissue, including few attempts in neural cells. Basically, in this system the plasmid DNA of interest is coated onto small particles, that are accelerated by a particular driving force. The combination of several so-called 'ballistic' parameters and tissue parameters determine the transfection efficiency. The main advantage of the system is that it is, unlike other available transfection methods, a mechanical way to cross the plasma membrane and therefore less dependent on target cell characteristics. In terms of transfection efficiency, biolistics seems favorable above conventional techniques, like calcium phosphate precipitation and lipofection. Compared to viral techniques biolistics may be less efficient, but is quicker and easier to handle and seems to produce fewer complications for in vivo gene delivery. Therefore, although the technique is only in a developmental stage, preliminary results seem promising, and optimalization of the method may prove useful in scientific research and/or clinical use.

  20. Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.).

    PubMed

    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.

  1. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    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.

  2. Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus neoformans.

    PubMed

    Taylor, Tonya; Bose, Indrani; Luckie, Taylor; Smith, Kerry

    2015-03-19

    The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide. Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only biolistic delivery provides an effective means to transform linear DNA that can be integrated into the genome by homologous recombination.  Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known. A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into C. neoformans. We then confirm integration of the ACK-mCherry fusion into the ACK locus.

  3. Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics.

    PubMed

    Djulic, Alma; Schmid, Annette; Lenz, Heike; Sharma, Pia; Koch, Christin; Wirsel, Stefan G R; Voegele, Ralf T

    2011-07-01

    Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host-parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (β-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  4. Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method

    SciTech Connect

    Martin-Ortigosa, Susana; Valenstein, Justin S.; Lin, Victor S.-Y.; Trewyn, Brian G.; Wang, Kan

    2012-09-11

    The synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au-MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green fluorescent protein (eGFP) are investigated. As a proof-of-concept demonstration, Au-MSN with large average pore diameters (10 nm) are shown to deliver and subsequently release proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denatured proteins to plant cells. This advance represents the first example of biolistic-mediated codelivery of proteins and plasmid DNA to plant cells via gold-functionalized MSN and provides a powerful tool for both fundamental and applied research of plant sciences.

  5. Parameters affecting the efficient delivery of mesoporous silica nanoparticle materials and gold nanorods into plant tissues by the biolistic method.

    PubMed

    Martin-Ortigosa, Susana; Valenstein, Justin S; Sun, Wei; Moeller, Lorena; Fang, Ning; Trewyn, Brian G; Lin, Victor S-Y; Wang, Kan

    2012-02-06

    Applying nanotechnology to plant science requires efficient systems for the delivery of nanoparticles (NPs) to plant cells and tissues. The presence of a cell wall in plant cells makes it challenging to extend the NP delivery methods available for animal research. In this work, research is presented which establishes an efficient NP delivery system for plant tissues using the biolistic method. It is shown that the biolistic delivery of mesoporous silica nanoparticle (MSN) materials can be improved by increasing the density of MSNs through gold plating. Additionally, a DNA-coating protocol is used based on calcium chloride and spermidine for MSN and gold nanorods to enhance the NP-mediated DNA delivery. Furthermore, the drastic improvement of NP delivery is demonstrated when the particles are combined with 0.6 μm gold particles during bombardment. The methodology described provides a system for the efficient delivery of NPs into plant cells using the biolistic method. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

    PubMed Central

    Arsenault, Jason; Nagy, Andras; Henderson, Jeffrey T.; O'Brien, John A.

    2014-01-01

    Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues. New modifications to the accelerator chamber have enhanced the gene gun's firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene's effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun. PMID:25407047

  7. Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus.

    PubMed

    Yepes, L M; Mittak, V; Pang, S Z; Gonsalves, C; Slightom, J L; Gonsalves, D

    1995-08-01

    In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.

  8. Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

    PubMed Central

    Thomas, Jean-luc

    2003-01-01

    There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473–479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B. mori tissues. Abbreviation: GFP Green Fluorescent Protein CCD Charged Coupled Device PMID:15841233

  9. A microarray MEMS device for biolistic delivery of vaccine and drug powders

    PubMed Central

    Pirmoradi, Fatemeh Nazly; Pattekar, Ashish V; Linn, Felicia; Recht, Michael I; Volkel, Armin R; Wang, Qian; Anderson, Greg B; Veiseh, Mandana; Kjono, Sandra; Peeters, Eric; Uhland, Scott A; Chow, Eugene M

    2015-01-01

    We report a biolistic technology platform for physical delivery of particle formulations of drugs or vaccines using parallel arrays of microchannels, which generate highly collimated jets of particles with high spatial resolution. Our approach allows for effective delivery of therapeutics sequentially or concurrently (in mixture) at a specified target location or treatment area. We show this new platform enables the delivery of a broad range of particles with various densities and sizes into both in vitro and ex vivo skin models. Penetration depths of ∼1 mm have been achieved following a single ejection of 200 µg high-density gold particles, as well as 13.6 µg low-density polystyrene-based particles into gelatin-based skin simulants at 70 psi inlet gas pressure. Ejection of multiple shots at one treatment site enabled deeper penetration of ∼3 mm in vitro, and delivery of a higher dose of 1 mg gold particles at similar inlet gas pressure. We demonstrate that particle penetration depths can be optimized in vitro by adjusting the inlet pressure of the carrier gas, and dosing is controlled by drug reservoirs that hold precise quantities of the payload, which can be ejected continuously or in pulses. Future investigations include comparison between continuous versus pulsatile payload deliveries. We have successfully delivered plasmid DNA (pDNA)-coated gold particles (1.15 µm diameter) into ex vivo murine and porcine skin at low inlet pressures of ∼30 psi. Integrity analysis of these pDNA-coated gold particles confirmed the preservation of full-length pDNA after each particle preparation and jetting procedures. This technology platform provides distinct capabilities to effectively deliver a broad range of particle formulations into skin with specially designed high-speed microarray ejector nozzles. PMID:26090875

  10. A microarray MEMS device for biolistic delivery of vaccine and drug powders.

    PubMed

    Pirmoradi, Fatemeh Nazly; Pattekar, Ashish V; Linn, Felicia; Recht, Michael I; Volkel, Armin R; Wang, Qian; Anderson, Greg B; Veiseh, Mandana; Kjono, Sandra; Peeters, Eric; Uhland, Scott A; Chow, Eugene M

    2015-01-01

    We report a biolistic technology platform for physical delivery of particle formulations of drugs or vaccines using parallel arrays of microchannels, which generate highly collimated jets of particles with high spatial resolution. Our approach allows for effective delivery of therapeutics sequentially or concurrently (in mixture) at a specified target location or treatment area. We show this new platform enables the delivery of a broad range of particles with various densities and sizes into both in vitro and ex vivo skin models. Penetration depths of ∼1 mm have been achieved following a single ejection of 200 µg high-density gold particles, as well as 13.6 µg low-density polystyrene-based particles into gelatin-based skin simulants at 70 psi inlet gas pressure. Ejection of multiple shots at one treatment site enabled deeper penetration of ∼3 mm in vitro, and delivery of a higher dose of 1 mg gold particles at similar inlet gas pressure. We demonstrate that particle penetration depths can be optimized in vitro by adjusting the inlet pressure of the carrier gas, and dosing is controlled by drug reservoirs that hold precise quantities of the payload, which can be ejected continuously or in pulses. Future investigations include comparison between continuous versus pulsatile payload deliveries. We have successfully delivered plasmid DNA (pDNA)-coated gold particles (1.15 µm diameter) into ex vivo murine and porcine skin at low inlet pressures of ∼30 psi. Integrity analysis of these pDNA-coated gold particles confirmed the preservation of full-length pDNA after each particle preparation and jetting procedures. This technology platform provides distinct capabilities to effectively deliver a broad range of particle formulations into skin with specially designed high-speed microarray ejector nozzles.

  11. Highly efficient virus-induced gene silencing in apple and soybean by apple latent spherical virus vector and biolistic inoculation.

    PubMed

    Yamagishi, Noriko; Yoshikawa, Nobuyuki

    2013-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the analysis of the gene function in plants within a short time. However, in woody fruit tree like apple, some of Solanum crops, and soybean, it is generally difficult to inoculate virus vector by conventional inoculation methods. Here, we show efficient VIGS in apple and soybean by Apple latent spherical virus (ALSV) vector and biolistic inoculation. The plants inoculated with ALSV vectors by particle bombardment showed uniform silenced phenotypes of target genes within 2-3 weeks post inoculation.

  12. Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers.

    PubMed

    De Guglielmo-Cróquer, Z; Altosaar, I; Zaidi, M; Menéndez-Yuffá, A

    2010-05-01

    The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

  13. Molecular and FISH analyses of a 53-kbp intact DNA fragment inserted by biolistics in wheat (Triticum aestivum L.) genome.

    PubMed

    Partier, A; Gay, G; Tassy, C; Beckert, M; Feuillet, C; Barret, P

    2017-06-30

    A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.

  14. 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).

  15. A new biolistic intradermal injector

    NASA Astrophysics Data System (ADS)

    Brouillette, M.; Doré, M.; Hébert, C.; Spooner, M.-F.; Marchand, S.; Côté, J.; Gobeil, F.; Rivest, M.; Lafrance, M.; Talbot, B. G.; Moutquin, J.-M.

    2016-01-01

    We present a novel intradermal needle-free drug delivery device which exploits the unsteady high-speed flow produced by a miniature shock tube to entrain drug or vaccine particles onto a skin target. A first clinical study of pain and physiological response of human subjects study is presented, comparing the new injector to intramuscular needle injection. This clinical study, performed according to established pain assessment protocols, demonstrated that every single subject felt noticeably less pain with the needle-free injector than with the needle injection. Regarding local tolerance and skin reaction, bleeding was observed on all volunteers after needle injection, but on none of the subjects following powder injection. An assessment of the pharmacodynamics, via blood pressure, of pure captopril powder using the new device on spontaneously hypertensive rats was also performed. It was found that every animal tested with the needle-free injector exhibited the expected pharmacodynamic response following captopril injection. Finally, the new injector was used to study the delivery of an inactivated influenza vaccine in mice. The needle-free device induced serum antibody response to the influenza vaccine that was comparable to that of subcutaneous needle injection, but without requiring the use of an adjuvant. Although no effort was made to optimize the formulation or the injection parameters in the present study, the novel injector demonstrates great promise for the rapid, safe and painless intradermal delivery of systemic drugs and vaccines.

  16. Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization.

    PubMed

    Sudowe, Stephan; Dominitzki, Sabine; Montermann, Evelyn; Bros, Matthias; Grabbe, Stephan; Reske-Kunz, Angelika B

    2009-09-01

    Gene gun-mediated biolistic DNA vaccination with beta-galactosidase (betaGal)-encoding plasmid vectors efficiently modulated antigen-induced immune responses in an animal model of type I allergy, including the inhibition of immunoglobulin E (IgE) production. Here we show that CD4(+) as well as CD8(+) T cells from mice biolistically transfected with a plasmid encoding betaGal under the control of the fascin promoter (pFascin-betaGal) are capable of inhibiting betaGal-specific IgE production after adoptive transfer into naïve recipients. Moreover, suppression of IgE production was dependent on interferon (IFN)-gamma. To analyse the modalities of activation of CD4(+) and CD8(+) T cells regarding the localization of antigen synthesis following gene gun-mediated DNA immunization, we used the fascin promoter and the keratin 5 promoter (pK5-betaGal) to direct betaGal production mainly to dendritic cells (DCs) and to keratinocytes, respectively. Gene gun-mediated DNA immunization with each vector induced considerable activation of betaGal-specific CD8(+) cytotoxic T cells. Cytokine production by re-stimulated CD4(+) T cells in draining lymph nodes and immunoglobulin isotype profiles in sera of immunized mice indicated that immunization with pFascin-betaGal induced a T helper type 1 (Th1)-biased immune response, whereas immunization with pK5-betaGal generated a mixed Th1/Th2 immune response. Nevertheless, DNA vaccination with pFascin-betaGal and pK5-betaGal, respectively, efficiently inhibited specific IgE production in the mouse model of type I allergy. In conclusion, our data show that uptake of exogenous antigen produced by keratinocytes and its presentation by untransfected DCs as well as the presentation of antigen synthesized endogenously in DCs represent equivalent pathways for efficient priming of cellular immune responses.

  17. 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...

  18. Chemokine overexpression in the skin by biolistic DNA delivery.

    PubMed

    Jalili, Ahmad

    2013-01-01

    Chemokines are a family of small, secreted proteins that function in leukocyte and tumor cell trafficking and recruiting. CC chemokine ligand 21 (CCL21)/secondary lymphoid chemokine (SLC) belongs to the inflammatory subgroup of chemokines and is expressed by stromal cells in the T-cell-rich zones of peripheral lymph nodes, afferent lymphatic endothelial cells and high endothelial venules. CCR7 (both in human and mouse) and CXCR3 (in mouse) are expressed by the most potent antigen-presenting cells (dendritic cells), naïve/central memory, and effector T cells, respectively. Inflammation in the skin can induce expression of CCL21 which is subsequently drained into loco-regional lymph nodes responsible for co-localization of antigen-presenting cells and T lymphocytes, a prerequisite for induction of adaptive immune responses. Here, skin functions as a remote control for induction of targeted cell migration in vivo. This chapter describes Gene Gun administration of plasmid DNA expressing functionally active CCL21 (as an example of a chemokine) into the skin in mice and subsequent functional evaluation of the transgene expression in vivo.

  19. Biolistic-mediated production of transgenic oil palm.

    PubMed

    Parveez, Ghulam Kadir Ahmad; Bahariah, Bohari

    2012-01-01

    The effectiveness of mannose (using phosphomannose isomerase [pmi] gene) as a positive selection agent to preferably allow the growth of transformed oil palm embryogenic calli was successfully evaluated. Using the above selection agent in combination with the previously optimized physical and biological parameters and the best constitutive promoter, oil palm embryogenic calli were transformed with pmi gene for producing transgenic plants. Bombarded embryogenic calli were exposed to embryogenic calli medium containing 30:0 g/L mannose to sucrose 3 weeks postbombardment. Selectively, proliferating embryogenic calli started to emerge around 6 months on the above selection medium. The proliferated embryogenic calli were individually isolated once they reached a specific size and regenerated to produce complete plantlets. The complete regenerated plantlets were evaluated for the presence of transgenes by PCR and Southern analyses.

  20. A New Biolistic Intradermal Injector Based on a Miniature Shock Tube

    NASA Astrophysics Data System (ADS)

    Brouillette, M.

    Intradermal powder injection is an emerging technology for the needlefree delivery of a potentially wide array of drugs and vaccines. Although needle injection of liquids is widespread principally because of its low cost, this delivery method is painful, generates dangerous medical waste and can cause contamination. Various technologies have been developed to address these shortcomings, amongst them creams, patches, inhalers and liquid jet injectors, each with their own severe limitations.

  1. Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves.

    PubMed

    Ivic-Haymes, Snezana D; Smigocki, Ann C

    2005-03-01

    Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.

  2. Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5.

    PubMed

    Wirth, Marcus J; Brun, Annika; Grabert, Jochen; Patz, Silke; Wahle, Petra

    2003-12-01

    Neurotrophins are candidate molecules for regulating dendritogenesis. We report here on dendritic growth of rat visual cortex pyramidal and interneurons overexpressing 'brain-derived neurotrophic factor' BDNF and 'neurotrophin 4/5' NT4/5. Neurons in organotypic cultures were transfected with plasmids encoding either 'enhanced green fluorescent protein' EGFP, BDNF/EGFP or NT4/5/EGFP either at the day of birth with analysis at 5 days in vitro, or at 5 days in vitro with analysis at 10 days in vitro. In pyramidal neurons, both TrkB ligands increased dendritic length and number of segments without affecting maximum branch order and number of primary dendrites. In the early time window, only infragranular neurons were responsive. Neurons in layers II/III became responsive to NT4/5, but not BDNF, during the later time window. BDNF and NT4/5 transfectants at 10 days in vitro had still significantly shorter dendrites than adult pyramidal neurons, suggesting a massive growth spurt after 10 days in vitro. However, segment numbers were already in the range of adult neurons. Although this suggested a role for BDNF, long-term activity-deprived, and thus BDNF-deprived, pyramidal cells developed a dendritic complexity not different from neurons in active cultures except for higher spine densities on neurons of layers II/III and VI. Neutralization of endogenous NT4/5 causes shorter and less branched dendrites at 10 days in vitro suggesting an essential role for NT4/5. Neutralization of BDNF had no effect. Transfected multipolar interneurons became identifiable during the second time window. Both TrkB ligands significantly increased number of segments and branch order towards the adult state with little effects on dendritic length. The results suggested that early in development BDNF and NT4/5 probably accelerate dendritogenesis in an autocrine fashion. In particular, branch formation was advanced towards the adult pattern in pyramidal cells and interneurons.

  3. Study on transient expression of gus gene in Chlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Li, Wen-Bin; Bai, Qin-Hua; Sun, Yong-Ru

    1998-03-01

    Study on the transient expression of GUS gene at different growing stage of Chlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed that C. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.

  4. Micro-shock Wave Assisted Plant Transformation

    NASA Astrophysics Data System (ADS)

    Gnanadhas, Divya Prakash; Datey, Akshay; Chakravortty, Dipshikha; Gopalan, Jagadeesh

    Genetically modified (GM) crops are developed by transforming the desired DNA to plant. There are various methods employed to achieve the required transformation in plants. Agrobacterium mediated transformation and Biolistics or particle bombardment method are the most commonly used methods.

  5. A Transgenic Durum Wheat Line that is Free of Marker Genes and Expresses 1dy10

    USDA-ARS?s Scientific Manuscript database

    We used a combination of “clean gene” technology and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments exc...

  6. Improvement of peanut (Arachis hypogaea L.) transformation efficiency and determination of transgene copy number by relative quantitative real-time PCR

    USDA-ARS?s Scientific Manuscript database

    The biolistic method is reliable for delivering genes of interest into various species. Low transformation efficiency has been a limiting factor for its application. The DNA coating agent protamine was shown to improve transformation efficiency in rice, while a reduction of plasmid DNA in the bomb...

  7. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    PubMed Central

    2012-01-01

    Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns. PMID:22853641

  8. Progress on genotyping and phenotyping recombinant inbred line populations of peanut

    USDA-ARS?s Scientific Manuscript database

    The biolistic method is reliable for delivering genes of interest into various species. Low transformation efficiency has been a limiting factor for its application. The DNA coating agent protamine was shown to improve transformation efficiency in rice, while a reduction of plasmid DNA in the bomb...

  9. Genetic transformation of grapevine cells using the minimal cassette technology: the need of 3'-end protection.

    PubMed

    Sanjurjo, Laura; Vidal, José Ramón; Segura, Antonio; de la Torre, Francisco

    2013-02-20

    The use of minimal cassettes (linear DNA comprising promoter+open reading frame+terminator) for genetic transformation offers a means of transforming plant cells by biolistics without introducing unwanted sequences. However, a species-specific approach of the factors involved in successful transformation with this technology is advisable. Protection of the minimal cassette upstream promoter and downstream terminator may be necessary due to the nuclease activity of target plant material. Genetic transformation by biolistics followed by expression analysis was used to evaluate different DNA constructs in 110 Richter (Vitis sp.) cell suspensions. Our results suggest the importance of 3'-end cassette protection for successful protein expression using the minimal cassette technology. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun

    PubMed Central

    O'Brien, John A; Lummis, Sarah CR

    2009-01-01

    Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h. PMID:17406443

  11. A transformation model for Laminaria Japonica (Phaeophyta, Laminariales)

    NASA Astrophysics Data System (ADS)

    Qin, Song; Jiang, Peng; Li, Xin-Ping; Wang, Xi-Hua; Zeng, Cheng-Kui

    1998-03-01

    A genetic transformation model for the seaweed Laminaria japonica mainly includes the following aspects: 1. The method to introduce foreign genes into the kelp, L. japonica Biolistic bombardment has been proved to be an effective method to bombard foreign DNA through cell walls into intact cells of both sporophytes and gametophytes. The expression of cat and lacZ was detected in regenerated sporophytes, which suggests that this method could induce random integration of foreign genes. Promoters to drive gene expression

  12. Bellis perennis: a useful tool for protein localization studies.

    PubMed

    Jaedicke, Katharina; Rösler, Jutta; Gans, Tanja; Hughes, Jon

    2011-10-01

    Fluorescent fusion proteins together with transient transformation techniques are commonly used to investigate intracellular protein localisation in vivo. Biolistic transfection is reliable, efficient and avoids experimental problems associated with producing and handling fragile protoplasts. Onion epidermis pavement cells are frequently used with this technique, their excellent properties for microscopy resulting from their easy removal from the underlying tissues and large size. They also have advantages over mesophyll cells for fluorescence microscopy, as they are devoid of chloroplasts whose autofluorescence can pose problems. The arrested plastid development is peculiar to epidermal cells, however, and stands in the way of studies on protein targeting to plastids. We have developed a system enabling studies of in vivo protein targeting to organelles including chloroplasts within a photosynthetically active plant cell with excellent optical properties using a transient transformation procedure. We established biolistic transfection in epidermal pavement cells of the lawn daisy (Bellis perennis L., cultivar "Galaxy red") which unusually contain a moderate number of functional chloroplasts. These cells are excellent objects for fluorescence microscopy using current reporters, combining the advantages of the ease of biolistic transfection, the excellent optical properties of a single cell layer and access to chloroplast protein targeting. We demonstrate chloroplast targeting of plastid-localised heme oxygenase, and two further proteins whose localisation was equivocal. We also demonstrate unambiguous targeting to mitochondria, peroxisomes and nuclei. We thus propose that the Bellis system represents a valuable tool for protein localisation studies in living plant cells.

  13. 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.

  14. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene

    SciTech Connect

    Carrer, H.; Maliga, P.

    1995-08-01

    To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in {approx}20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene. 26 refs., 5 figs., 1 tab.

  15. 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.

  16. Transformation and strain engineering of Tetrahymena.

    PubMed

    Chalker, Douglas L

    2012-01-01

    Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Simulations of transient shock motion within a biological contoured-shock-tube system

    NASA Astrophysics Data System (ADS)

    Liu, Y.

    2008-02-01

    This study is motivated by the author’s interest in developing needle-free powdered vaccine/drug delivery systems. One system configuration is called the Contoured Shock Tube (CST). Of great importance is the behaviour of a transonic gas flow with a strongly nonlinear starting process, which accelerates powdered vaccines in micro-form to a sufficient momentum to penetrate the outer layer of human skin or mucosal tissue. In this paper, an established Modified Implicit Flux Vector Splitting (MIFVS) solver for the Navier-Stokes equations is extended to numerically study these transient transonic gas flows. A low Reynolds number k-ɛ turbulence model, with the compressibility effect considered, is integrated into the MIFVS solver to predict the turbulent structures and interactions with inherent shock systems. The MIFVS is first calibrated for NASA validation case, NPARC, and the resulting flow characteristic are compared with experimental date and simulations published. The MIFVS calculation with the modified k-ɛ model shows the best agreement. Subsequently, the MIFVS is applied to model the transient gas flow within a biolistic CST prototype. Comparison with experimental pressure traces shows the MIFVS captures gas flow mechanics with more accuracy than calculations with a commercial code (Fluent). This illustrates that the MIFVS is well-suited to model the strongly nonlinear fluid dynamics associated with the CST biolistic particle delivery system.

  18. Mucosal deformation from an impinging transonic gas jet and the ballistic impact of microparticles

    NASA Astrophysics Data System (ADS)

    Hardy, M. P.; Kendall, M. A. F.

    2005-10-01

    By means of a transonic gas jet, gene guns ballistically deliver microparticle formulations of drugs and vaccines to the outer layers of the skin or mucosal tissue to induce unique physiological responses for the treatment of a range of conditions. Reported high-speed imaging experiments show that the mucosa deforms significantly while subjected to an impinging gas jet from a biolistic device. In this paper, the effect of this tissue surface deformation on microparticle impact conditions is simulated with computational fluid dynamics (CFD) calculations. The microparticles are idealized as spheres of diameters 26.1, 39 and 99 µm and a density of 1050 kg m-3. Deforming surface calculations of particle impact conditions are compared directly with an immobile surface case. The relative velocity and obliquity of the deforming surface decrease the normal component of particle impact velocity by up to 30% at the outer edge of the impinging gas jet. This is qualitatively consistent with reported particle penetration profiles in the tissue. It is recommended that these effects be considered in biolistic studies requiring quantified particle impact conditions.

  19. Cloning of the complete infectious cDNA of the plum pox virus strain PPV-Rec.

    PubMed

    Predajňa, L; Nagyová, A; Glasa, M; Subr, Z W

    2012-01-01

    Plum pox virus (PPV) is the causal agent of Sharka, considered to be the most detrimental viral disease of Prunus spp. worldwide. So far, several PPV strains have been recognized, three of them (PPV-D, PPV-M, and PPV-Rec) having shown serious economic impact in the European area. Infectious cDNA clones of plant RNA viruses are excellent tools for functional studies of viral genomes. Preparation and use of PPV-D and PPV-M infectious clones have been previously reported. Here we describe the construction of an infectious cDNA clone of the strain PPV-Rec (isolate BOR-3) by the strategy involving the subsequent exchanges of homologous BOR-3 genome parts in the backbone of the previously prepared PPV-D infectious construct. The infectivity of each intermediate chimeric cDNA as well as that of the final construct (pIC-PPV-Rec) was confirmed by biolistic transfection of Nicotiana benthamiana plants. Complete sequence of the cloned viral BOR-3 cDNA revealed 0.14% of difference at the nucleotide level compared to original BOR-3 sequence, resulting in four amino acid changes. This slight inequality was related to the population heterogeneity of the initial BOR-3 isolate; no difference in the amino acid sequence resulted from the cloning steps performed. inter-strain chimera; biolistics; genome sequence.

  20. [Features of development and reproduction of transgenic flax].

    PubMed

    Lemesh, V A; Samatadze, T E; Guzenko, E V; Zhelezniakova, E V; Amosova, A V; Zelenin, A V; Muravenko, O V

    2014-01-01

    Primary transformants carrying a genetic construct with the chimeric gfp-tua6 gene were obtained using biolistic transformation of hypocotyl explants of flax variety Vasilek. Viable modified plants were used as a basis for the production of inbred lines with confirmed inheritance of introduced genetic construct in three generations. The characteristics of phenological growth stages, plant height, number of bolls and meiosis were studied for transgenic plants. A comparison of transformed lines based on reproduction years revealed a significant decrease of seed production in one line. Meiotic analysis of this line at metaphase I and anaphase I stages was conducted. The percentage of cells with impaired meiosis was highest in transgenic plants of the line with the lowest seed production. Thus, the nonspecific incorporation of genetic construct into the flax genome using biolistic transformation impairs meiosis to a different extent and it is the main reason for unequal reproducibility of transgenic flax. The production of stably reproducing transgenic lines requires systematic analysis of meiosis.

  1. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.

    PubMed

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-10-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.

  2. A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha.

    PubMed

    Boehm, Christian R; Ueda, Minoru; Nishimura, Yoshiki; Shikanai, Toshiharu; Haseloff, Jim

    2016-02-01

    Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  3. A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    PubMed Central

    Boehm, Christian R.; Ueda, Minoru; Nishimura, Yoshiki; Shikanai, Toshiharu; Haseloff, Jim

    2016-01-01

    Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity. PMID:26634291

  4. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

    PubMed Central

    Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

    1990-01-01

    Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

  5. Molecular characterization and an infectious clone construction of sweet potato leaf curl virus (SPLCV) isolated from Korea.

    PubMed

    Choi, E; Lee, G; Park, J; Lee, T K; Choi, H S; Lee, S

    2012-01-01

    Sweet potato leaf curl disease (SPLCD) was primarily identified in sweet potato fields in Korea in 2003, and the complete genomic sequence of sweet potato leaf curl virus (SPLCV) has been cloned. The genome of the Korean SPLCV isolate (SPLCV-KR) comprises 2,828 nucleotides with six open reading frames in DNA-A, similar to a monopartite begomovirus. Additionally, neither the genome B genomic component nor the DNA beta sequence was detected. The results of phylogenetic analysis using the maximum parsimony method showed that SPLCV-KR is more closely related to SPLCV-US (US) than SPLCV-CN (China) and SPLCV-JP (Japan). A tandem repeat dimer of SPLCV-KR was cloned and found to be infectious in sweet potatoes (Ipomoea batatas) via biolistic inoculation. The SPLCV-infected sweet potatoes exhibited mild leaf curl symptoms of SPLCD, and the newly-replicated viral DNA was detected via Southern blot analysis. Results of biotic, molecular, and phylogenetic characterization suggest that SPLCV-KR is a new strain of SPLCV and is importantly placed in the evolutionary progression from curtoviruses to begomoviruses. sweet potato leaf curl virus; sweet potato leaf curl disease; phylogenetic analysis; infectious clone; biolistic infection.

  6. Mesoporous Silica Nanoparticle-Mediated Intracellular Cre Protein Delivery for Maize Genome Editing via loxP Site Excision1,2[W][OPEN

    PubMed Central

    Martin-Ortigosa, Susana; Peterson, David J.; Valenstein, Justin S.; Lin, Victor S.-Y.; Trewyn, Brian G.; Lyznik, L. Alexander; Wang, Kan

    2014-01-01

    The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method. PMID:24376280

  7. Molecular characterization of a new begomovirus that infects Euphorbia heterophylla and Solanum lycopersicum in Venezuela.

    PubMed

    Zambrano, Karla; Fernández-Rodríguez, Thaly; Marys, Edgloris

    2012-02-01

    We report the complete nucleotide sequence of a begomovirus isolate infecting Euphorbia heterophylla and tomato (Solanum lycopersicum) in central Venezuela. Based on the current taxonomic criteria for the genus Begomovirus, the isolate was shown to represent a novel species, tentatively named Euphorbia mosaic Venezuela virus (EuMVV). Its DNA-A is most closely related to those of Euphorbia-infecting begomoviruses from the Caribbean and Central America. The DNA B component forms a phylogenetic cluster with Euphorbia and Sida-infecting begomoviruses from the squash leaf curl virus (SLCV) cluster. EuMVV is transmissible to S. lycopersicum and Capsicum annuum by biolistics of infectious cloned DNA-A and DNA-B components and induces characteristic leaf downward curling and yellowing in S. lycopersicum and and yellowing and leaf distortion in Capsicum annuum.

  8. Plastid transformation in eggplant.

    PubMed

    Bansal, Kailash C; Singh, Ajay K

    2014-01-01

    Eggplant (Solanum melongena L.) is an important vegetable crop of tropical and temperate regions of the world. Here we describe a procedure for eggplant plastid transformation, which involves preparation of explants, biolistic delivery of plastid transformation vector into green stem segments, selection procedure, and identification of the transplastomic plants. Shoot buds appear from cut ends of the stem explants following 5-6 weeks of spectinomycin selection after bombardment with the plastid transformation vector containing aadA gene as selectable marker. Transplastomic lines are obtained after the regenerated shoots are subjected to several rounds of spectinomycin selection over a period of 9 weeks. Homoplasmic transplastomic lines are further confirmed by spectinomycin and streptomycin double selection. The transplastomic technology development in this plant species will open up exciting possibilities for improving crop performance, metabolic engineering, and the use of plants as factories for producing biopharmaceuticals.

  9. Genetic immunization is a simple method for eliciting an immune response

    NASA Astrophysics Data System (ADS)

    Tang, De-Chu; Devit, Michael; Johnston, Stephen A.

    1992-03-01

    To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system1-4 which can propel DNA-coated gold micro-projectiles directly into cells in the living animal3,5,6. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.

  10. Plastid transformation in eggplant (Solanum melongena L.).

    PubMed

    Singh, A K; Verma, S S; Bansal, K C

    2010-02-01

    We have developed a method for plastid transformation in eggplant (Solanum melongena L.), a solanaceous plant species. Plastid transformation in eggplant was achieved by bombardment of green stem segments with pPRV111A plastid expression vector carrying the aadA gene encoding aminoglycoside 3''-adenylyltransferase. Biolistic delivery of the pPRV111A plasmid yielded transplastomic plants at a frequency of two per 21 bombarded plates containing 25 stem explants each. Integration of the aadA gene in the plastome was verified by PCR analysis and also by Southern blotting using 16S rDNA (targeting sequence) and the aadA gene as a probe. Transplastomic expression of the aadA gene was verified by RT-PCR. The development of transplastomic technology in eggplant may open up exciting possibilities for novel gene introduction and expression in the engineered plastome for agronomic or pharmaceutical traits.

  11. Production of green fluorescent protein in transgenic rice seeds.

    PubMed

    Li, Ding; Gao, Jing; Shen, Chunxiu; Fang, Zhen; Xia, Yumei; Yuan, Longping; Cao, Mengliang

    2013-03-01

    Immature embryos from immature seeds of rice (Oryza sativa L.) were transformed by biolistic bombardment with the plasmid carrying the coding region of the hygromycin phosphotransferase gene under the control of the 5' region of the cauliflower mosaic virus 35S promoter and the synthetic green fluorescence protein gene (sgfp) under the control of the maize ubiquitine promoter. Southern blot analysis confirmed the stable integration of hpt and sgfp genes in transformants. Subsequently leaves from regenerated plants were resistant to hygromycin, and microscopic observation of the green fluorescence and immunoblotting analysis revealed that green fluorescence protein was not only detected in the leaf and pollen of primary transformants but also in mature seeds. The results bear out the importance of the suitability of GFP as an in vivo marker to follow the processes of selection of somatic hybrid embryos and plants.

  12. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

    PubMed Central

    Svitashev, Sergei; Schwartz, Christine; Lenderts, Brian; Young, Joshua K.; Mark Cigan, A.

    2016-01-01

    Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species. PMID:27848933

  13. AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

    PubMed

    Tsuboyama, Shoko; Kodama, Yutaka

    2014-01-01

    The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

  14. Nanoparticle-Mediated Recombinase Delivery into Maize.

    PubMed

    Martin-Ortigosa, Susana; Trewyn, Brian G; Wang, Kan

    2017-01-01

    We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants. Using the experimental procedures and conditions described here, as high as 20% of bombarded embryos can produce regenerable recombinant callus events. This nanomaterial-mediated, DNA-free methodology has potential to become an effective tool for plant genome editing.

  15. Complete nucleotide sequence and construction of an infectious clone of Chinese yam necrotic mosaic virus suggest that macluraviruses have the smallest genome among members of the family Potyviridae.

    PubMed

    Kondo, Toru; Fujita, Takashi

    2012-12-01

    The complete nucleotide sequence of Chinese yam necrotic mosaic virus (CYNMV) was determined from cloned virus cDNA. The CYNMV genomic RNA is 8224 nucleotides in length, excluding the poly(A) tail, and contains one long open reading frame encoding a large polyprotein of 2620 amino acids. CYNMV has no counterpart to the P1 cistron and a short HC-Pro cistron located at the 5' side of the potyvirus genome. A full-length cDNA clone, pCYNMV, was assembled under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. Biolistic inoculation of Nagaimo plants with cDNA resulted in systemic necrotic mosaic symptoms typical of CYNMV infection. To our knowledge, this is the first report of the complete nucleotide sequence and construction of an infectious cDNA clone of a member of the genus Macluravirus.

  16. Plastid transformation in sugar beet: Beta vulgaris.

    PubMed

    De Marchis, Francesca; Bellucci, Michele

    2014-01-01

    Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of utmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories; conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

  17. Infectivity of Deinbollia mosaic virus, a novel weed-infecting begomovirus in East Africa.

    PubMed

    Kyallo, Martina; Ateka, Elijah Miinda; Sseruwagi, Peter; Ascencio-Ibáñez, José Trinidad; Ssemakula, Mildred-Ochwo; Skilton, Robert; Ndunguru, Joseph

    2017-08-09

    Weed-infecting begomoviruses play an important role in the epidemiology of crop diseases because they can potentially infect crops and contribute to the genetic diversity of crop-infecting begomoviruses. Despite the important epidemiological role that weed-infecting begomoviruses play, they remain insufficiently studied in Africa. Recently, we identified Deinbollia mosaic virus (DMV), a distinct begomovirus found naturally infecting the weed host Deinbollia borbonica (Sapindaceae) in Kenya and Tanzania. In this study, we investigated the capacity of DMV to infect a restricted host range of Solanaceae and Euphorbiaceae species. Biolistic inoculation of Nicotiana benthamiana with concatemeric DNAs resulted in systemic infection associated with yellow mosaic symptoms, while DNA partial dimers caused asymptomatic systemic infection. DMV was not infectious to cassava (Manihot esculenta Crantz), suggesting host resistance to the virus. Here, we demonstrate the first experimental infectivity analysis of DMV in N. benthamiana and cassava.

  18. Transformation of oil palm using Agrobacterium tumefaciens.

    PubMed

    Izawati, Abang Masli Dayang; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul

    2012-01-01

    Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.

  19. Transient expression of exogenous gus gene in Porphyra yezoensis (Rhodophyta)

    NASA Astrophysics Data System (ADS)

    Kuang, Mei; Wang, Su-Juan; Li, Yao; Shen, Da-Leng; Zeng, Cheng-Kui

    1998-03-01

    Electroporation, PEC, PEG plus electroporation and Biolistics methods were tested in gene transformation of P. yezoensis. The exogenous gus was from plasmid of pBI121 and pCAMBIA1301, both contain the CaMV35S promoter. The receptors included the protoplasts, tissues and free-living conchocelis filaments of P. yezoensis. Several factors, for example, the voltage, capacitance and bivalent cations, etc., were studied. Results show that these four methods are all efficient for gene transformation in P. yezoensis; and that PEG is the best one, with transformation efficiency of up to 4×10-5. GUS activity was detected 26 days after transformation by using PEG method.

  20. Molecular and biological characterization of corchorus mottle virus, a new begomovirus from Brazil.

    PubMed

    Blawid, Rosana; Fontenele, Rafaela S; Lacorte, Cristiano; Ribeiro, Simone G

    2013-12-01

    A begomovirus infecting Orinoco jute (Corchorus hirtus) from Brazil was characterized. Molecular analysis revealed a bipartite genomic organization, which is typical of the New World begomoviruses. Sequence analysis and phylogenetic data showed that both genomic components have the closest relationship with abutilon mosaic Brazil virus, with an identity of 87.3 % for DNA-A, indicating that this virus is a member of a new begomovirus species for which the name "Corchorus mottle virus" (CoMoV) is proposed. Sida rhombifolia plants inoculated by biolistics with an infectious clone of CoMoV showed systemic vein chlorosis, mottling and leaf deformation symptoms, while Nicotiana benthamiana and tomato plants had symptomless infection. CoMoV is the first corchorus-infecting begomovirus reported in Brazil.

  1. pSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

    PubMed Central

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3′rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses. PMID:24897541

  2. Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge).

    PubMed

    Sandhu, Sukhpreet; Altpeter, Fredy

    2008-11-01

    Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1-4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.

  3. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans

    PubMed Central

    Arras, Samantha D. M.; Fraser, James A.

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well. PMID:27643854

  4. Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas).

    PubMed

    Khanna, H K; Raina, S K

    2002-08-01

    Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic or Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (approximately 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.

  5. Generation of transgenic energy cane plants with integration of minimal transgene expression cassette.

    PubMed

    Fouad, Walid M; Hao, Wu; Xiong, Yuan; Steeves, Cody; Sandhu, Surinder K; Altpeter, Fredy

    2015-01-01

    Lignocellulosic biomass has the potential to serve as feedstock and direct replacement for petrochemicals in the fuel, chemical, pharmaceutical and material industries. Energy cane has been identified by the U.S. Department of Energy (DOE) as prime lignocellulosic feedstock as it produces record biomass yields and is able to grow on low-value land with reduced inputs. Molecular improvement of energy cane is an essential step toward the development of a high-value crop and may contribute to improved biomass conversion to value added products. Such improvements require a development of an efficient regeneration and transformation system for the vegetatively propagated energy cane varieties. In this report, an efficient biolistic gene delivery protocol for energy canes (genotype L 79-1002 and Ho 00-961) has been established with immature leaf rolls as explants. Embryonic calli, developed approximately 6 weeks after culture initiation and was used as target for biolistic transfer of a minimum expression cassette of P-ubi::nptII::35S polyA derived from plasmid pJFNPTII. Putative transgenic clones of callus were obtained after selection on callus induction medium supplemented with 30 mg l(-1) geneticin. Regeneration was carried out on NB medium, which is modified from MS supplemented with 1.86 mg l(-1) naphthaleneacetic acid (NAA) and 0.1mg l(-1), 6- benzylaminopurine (BAP) and 20mg l(-1) paromomycin. Shoots growing on selection media were transferred to hormone free medium with 20 mg l(-1) paromomycin. Putative transgenic lines were first analyzed by PCR. Transgene integration was confirmed by Southern blot analysis. ELISA (Enzyme-Linked Immunosorbent Assay) and Immunochromathography assays confirmed transgene expression.

  6. Generation of Transgenic Energy Cane Plants with Integration of Minimal Transgene Expression Cassette.

    PubMed

    Fouad, Walid M; Hao, W U; Xiong, Yuan; Steeves, Cody; Sandhu, Surinder K; Altpeter, Fredy

    2015-03-03

    Lignocellulosic biomass has the potential to serve as feedstock and direct replacement for petrochemicals in the fuel, chemical, pharmaceutical and material industries. Energy cane has been identified by the U.S. Department of Energy (DOE) as prime lignocellulosic feedstock as it produces record biomass yields and is able to grow on low-value land with reduced inputs. Molecular improvement of energy cane is an essential step toward the development of a high-value crop and may contribute to improved biomass conversion to value added products. Such improvements require a development of an efficient regeneration and transformation system for the vegetatively propagated energy cane varieties. In this report, an efficient biolistic gene delivery protocol for energy canes (genotype L 79-1002 and Ho 00-961) has been established with immature leaf rolls as explants. Embryonic calli, developed approximately 6 weeks after culture initiation and was used as target for biolistic transfer of a minimum expression cassette of P-ubi::nptII::35S polyA derived from plasmid pJFNPTII. Putative transgenic clones of callus were obtained after selection on callus induction medium supplemented with 30 mg l-1 geneticin. Regeneration was carried out on NB medium, which is modified from MS supplemented with 1.86 mg l-1 naphthaleneacetic acid (NAA) and 0.1mg l-1, 6-benzylaminopurine (BAP) and 20mg l-1 paromomycin. Shoots growing on selection media were transferred to hormone free medium with 20 mg l-1 paromomycin. Putative transgenic lines were first analyzed by PCR. Transgene integration was confirmed by Southern blot analysis. ELISA (Enzyme-Linked Immunosorbent Assay) and Immunochromathography assays confirmed transgene expression.

  7. Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

    PubMed Central

    Guillet, Carine; Aboul-Soud, Mourad A. M.; Le Menn, Aline; Viron, Nicolas; Pribat, Anne; Germain, Véronique; Just, Daniel; Baldet, Pierre; Rousselle, Patrick; Lemaire-Chamley, Martine; Rothan, Christophe

    2012-01-01

    The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars. PMID:22615815

  8. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    PubMed

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.

  9. Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

    PubMed Central

    Idnurm, Alexander; Reedy, Jennifer L.; Nussbaum, Jesse C.; Heitman, Joseph

    2004-01-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their

  10. Human Granulocyte Colony-Stimulating Factor (hG-CSF) Expression in Plastids of Lactuca sativa

    PubMed Central

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Background: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. Methods: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. Results: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. Conclusions: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment. PMID:23748895

  11. The contribution of translesion synthesis polymerases on geminiviral replication.

    PubMed

    Richter, Kathrin S; Götz, Monika; Winter, Stephan; Jeske, Holger

    2016-01-15

    Geminiviruses multiply primarily in the plant phloem, but never in meristems. Their Rep protein can activate DNA synthesis in differentiated cells. However, when their single-stranded DNA is injected into the phloem by insects, no Rep is present for inducing initial complementary strand replication. Considering a contribution of translesion synthesis (TLS) polymerases in plants, four of them (Polη, Polζ, Polκ, Rev1) are highly and constitutively expressed in differentiated tissues like the phloem. Two geminiviruses (Euphorbia yellow mosaic virus, Cleome leaf crumple virus), inoculated either biolistically or by whiteflies, replicated in Arabidopsis thaliana mutant lines of these genes to the same extent as in wild type plants. Comparative deep sequencing of geminiviral DNAs, however, showed a high exchange rate (10(-4)-10(-3)) similar to the phylogenetic variation described before and a significant difference in nucleotide substation rates if Polη and Polζ were absent, with a differential response to the viral DNA components. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing.

    PubMed

    Vazquez Rovere, C; Asurmendi, S; Hopp, H E

    2001-07-01

    Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

  13. Biotechnology tools in agriculture: recent patents involving soybean, corn and sugarcane.

    PubMed

    Hansen, Daiane; Nakahata, Adriana M; Haraguchi, Mitsue; Alonso, Antonio

    2011-05-01

    The technological opportunities opened up by biotechnology in agriculture are diverse, including plant breeding, the partial or total relief of pesticides chemicals usage, the improvement of soil fertility, the improvement of the quality attributes of various foods. Specifically, various tricks of biotechnology can be used for higher seed yield, resistance to diseases and insects, better stems and roots, tolerance to drought and heat, and better agronomic quality. A number of recent works considerably widen the potential of plant biotechnology where transformation methods and studies of molecular genomics have been described. For example, transformation techniques and search for new selectable markers involving biolistic technique, gene transfer technique using the soil bacterium Agrobacterium tumefaciens, selection technique based on the use of mannose, utilization of genes promoting endogenous hormone production under the control of chemical stimulants, further more, engineering the nuclear genome without antibiotic resistance genes and engineering the plastid genome. We are presenting in this paper some of the recent patents on methods and techniques involving genes coding proteins and breeding techniques with possible agronomic applicability on crops economically important, such as soybean, corn and sugarcane.

  14. Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.

    PubMed

    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.

  15. Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation[OPEN

    PubMed Central

    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

  16. 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

    PubMed Central

    Stern, D B; Kindle, K L

    1993-01-01

    Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms. Images PMID:8455609

  17. Using melanopsin to study G protein signaling in cortical neurons.

    PubMed

    McGregor, K M; Bécamel, C; Marin, P; Andrade, R

    2016-09-01

    Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons. Copyright © 2016 the American Physiological Society.

  18. Identification and properties of insect resistance-associated maize anionic peroxidases.

    PubMed

    Dowd, Patrick F; Johnson, Eric T; Pinkerton, T Scott

    2010-08-01

    Previous studies with transgenic plants have indicated a tobacco anionic peroxidase can confer enhanced resistance to a variety of insects when expressed in different plant species. Tissue that expresses high levels of this enzyme often browns rapidly when damaged. Maize roots damaged under sterile conditions browned and had an anionic peroxidase induced. When introduced biolistically, maize callus transformants expressing a maize peroxidase gene with a predicted isoelectric point of ca. 5.1 produced browner callus compared to a corresponding beta-glucuronidase (GUS) transformant as callus aged. Higher production of only one isozyme of ca. pI 4.5 was noted. When the callus was fed to two maize pest caterpillar species, growth rates were slower (as reflected by weights) relative to the GUS callus. Based on examination of published information and electrophoretic properties, this gene appears to code for Px11, a peroxidase isozyme that is primarily produced in root tissue and callus. When sequence of the gene in several inbreds was examined, coding variations were noted, and abilities to utilize ferulic and p-coumaric acids differed. These coding differences may influence the ability of corresponding forms of the peroxidase to promote resistance. In addition to potential use in marker assisted breeding, enhanced expression of this anionic peroxidase through breeding or genetic engineering may lead to enhanced insect or disease resistance. Published by Elsevier Ltd.

  19. Indica rice (Oryza sativa, BR29 and IR64).

    PubMed

    Datta, Karabi; Datta, Swapan Kumar

    2006-01-01

    Rice is the world's most important food crop. Indica-type rice provides the staple food for more than half of the world population. To satisfy the growing demand of the ever-increasing population, more sustained production of indica-type rice is needed. In addition, because of the high per capita consumption of indica rice, improvement of any traits including its nutritive value may have a significant positive health outcome for the rice-consuming population. Rice yield productivity is greatly affected by different biotic stresses, like diseases and insect pests, and abiotic stresses like drought, cold, and salinity. Attempts to improve resistance in rice to these stresses by conventional breeding through introgression of traits have limited success owing to a lack of resistance germplasm in the wild relatives. Gene transfer technology with genes from other sources can be used to make rice plants resistant or tolerant to insect pests, diseases, and different environmental stresses. For improving the nutritional value of the edible endosperm part of the rice, genes for increasing iron, beta-carotene, or better quality protein can be introduced in rice plants by genetic engineering. Different crops have been transformed using various gene transfer methods, such as protoplast transformation, biolistic, and Agrobacterium-mediated transformation. This chapter describes the Agrobacterium-mediated transformation protocol for indica-type rice. The selectable marker genes used are hygromycin phosphotransferase (hpt), neomycin phosphotransferase (nptII), or phosphomannose isomerase (pmi), and, accordingly, the selection agents are hygromycin, kanamycin (G418), or mannose, respectively.

  20. A Perspective on Hypericum perforatum Genetic Transformation.

    PubMed

    Hou, Weina; Shakya, Preeti; Franklin, Gregory

    2016-01-01

    Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering.

  1. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

    PubMed

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

  2. Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs

    PubMed Central

    Dalakouras, Athanasios; Wassenegger, Michèle; McMillan, John N.; Cardoza, Vinitha; Maegele, Ira; Dadami, Elena; Runne, Miriam; Krczal, Gabi; Wassenegger, Michael

    2016-01-01

    In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves. PMID:27625678

  3. pORE: a modular binary vector series suited for both monocot and dicot plant transformation.

    PubMed

    Coutu, Catherine; Brandle, James; Brown, Dan; Brown, Kirk; Miki, Brian; Simmonds, John; Hegedus, Dwayne D

    2007-12-01

    We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an 'open' set for general plant transformation, a 'reporter' set for promoter analysis and an 'expression' set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P (HPL), P (ENTCUP2) and P (TAPADH)), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).

  4. Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.

    PubMed

    Kamo, Kathryn; Gera, Abed; Cohen, Jacob; Hammond, John; Blowers, Alan; Smith, Franzine; Van Eck, Joyce

    2005-02-01

    Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV.

  5. Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11.

    PubMed

    Yang, Mai Lee; Uhrig, John; Vu, Kiem; Singapuri, Anil; Dennis, Michael; Gelli, Angie; Thompson, George R

    2015-12-07

    Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.

  6. Bioengineering of the Plant Culture of Capsicum frutescens with Vanillin Synthase Gene for the Production of Vanillin.

    PubMed

    Chee, Marcus Jenn Yang; Lycett, Grantley W; Khoo, Teng-Jin; Chin, Chiew Foan

    2017-01-01

    Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.

  7. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L)

    PubMed Central

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

    2012-01-01

    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1-1 mannose without sucrose. Transgenic plants were verified using PCR analysis. Abbreviations PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene. PMID:22368388

  8. Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants.

    PubMed

    Jain, Mukesh; Chengalrayan, Kudithipudi; Abouzid, Ahmed; Gallo, Maria

    2007-05-01

    For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.

  9. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

    PubMed

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

    2012-01-01

    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

  10. [Molecular, genetic and physiological analysis of photoinhibition and photosynthetic]. Progress report, June 1991--November 1992

    SciTech Connect

    Not Available

    1992-12-01

    A major goal of this project is to use a combined molecular genetic, biochemical and physiological approach to understand the relationship between photosynthetic performance and the structure of the multifunctional D1 reaction center protein of Photosystem II encoded by the chloroplast psbA gene. Relative to other chloroplast proteins, turover of D1 is rapid and highly light dependent and de novo synthesis of D1 is required for a plant`s recovery from short term exposure to irradiances which induce photoinhibitory damage. These observations have led to models for a damage/repair cycle of PSII involving the targeted degradation and replacement of photodamaged D1. To investigate the effects of perturbing the D1 cycle on photosynthesis and autotrophic growth under high and low irradiance, we have examined the consequences of site-specific mutations of the psbA and 16S rRNA genes affecting synthesis, maturation and function/stability of the D1 protein introduced into the chloroplast genome of wildtype strain of the green alga Chlamydomonas reinhardtii using biolistic transformation.

  11. [Molecular, genetic and physiological analysis of photoinhibition and photosynthetic

    SciTech Connect

    Not Available

    1992-01-01

    A major goal of this project is to use a combined molecular genetic, biochemical and physiological approach to understand the relationship between photosynthetic performance and the structure of the multifunctional D1 reaction center protein of Photosystem II encoded by the chloroplast psbA gene. Relative to other chloroplast proteins, turover of D1 is rapid and highly light dependent and de novo synthesis of D1 is required for a plant's recovery from short term exposure to irradiances which induce photoinhibitory damage. These observations have led to models for a damage/repair cycle of PSII involving the targeted degradation and replacement of photodamaged D1. To investigate the effects of perturbing the D1 cycle on photosynthesis and autotrophic growth under high and low irradiance, we have examined the consequences of site-specific mutations of the psbA and 16S rRNA genes affecting synthesis, maturation and function/stability of the D1 protein introduced into the chloroplast genome of wildtype strain of the green alga Chlamydomonas reinhardtii using biolistic transformation.

  12. Development of plant regeneration and transformation protocols for the desiccation-sensitive weeping lovegrass Eragrostis curvula.

    PubMed

    Ncanana, Sandile; Brandt, Wolf; Lindsey, George; Farrant, Jill

    2005-08-01

    A tissue culture protocol, suitable for transformation, was established for the pasture grass Eragrostis curvula. Callus was generated in the dark from leaf and seed tissues on a medium comprising MS salts supplemented with 2 mg/l 2,4-D, 0.01 mg/l BAP and 2% sucrose. Plant regeneration occurred via organogenesis on the same medium with 6% and 3% sucrose for shoot and root formation, respectively. Optimal regeneration (50 plantlets per callus) occurred when light of 45 micromol/m2 per s was used. The yeast Saccharomyces cerevisiae Hsp12 gene was cloned into the Sac1 of the pCAMBIAUbeeQ vector and callus was transformed by biolistic bombardment. Best transformation (30%) occurred when the target tissue was bombarded twice at a distance of 70 mm using a bombardment pressure of 9,100 kPa. Although successful transformation and transcription of the Hsp12 gene occurred, no Hsp12 protein was found present in tissue extracts of transformed grass.

  13. Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species.

    PubMed

    Maghuly, Fatemeh; da Câmara Machado, Artur; Leopold, Stephan; Khan, Mahmood Ali; Katinger, Hermann; Laimer, Margit

    2007-01-01

    Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.

  14. A study of weeds as potential inoculum sources for a tomato-infecting begomovirus in central Brazil.

    PubMed

    Barreto, S S; Hallwass, M; Aquino, O M; Inoue-Nagata, A K

    2013-05-01

    Tomato severe rugose virus (ToSRV) is the most important begomovirus species in Brazilian tomato production. Many weeds are associated with tomato, and some are hosts of begomoviruses. Only one species of weed, Nicandra physaloides, has been found to be infected with ToSRV. In this study, four weed species were investigated for their capacity to be infected by ToSRV and serve as a potential source of inoculum for tomato. Begomoviruses from naturally infected Crotalaria spp., Euphorbia heterophylla, N. physaloides, and Sida spp. were successfully transferred to tomato plants by biolistic inoculation. ToSRV was the major virus transferred to tomato. In contrast, other begomoviruses were transferred to weeds, such as Sida micrantha mosaic virus and Euphorbia yellow mosaic virus. Furthermore, a new strain of Sida micrantha mosaic virus is reported. We also confirmed that Crotalaria spp., E. heterophylla, and Sida spp. are infected with ToSRV but at low viral titers and in mixed infections with weed-infecting begomoviruses. Thus, it was demonstrated that weeds are potential sources of ToSRV for tomato in central Brazil.

  15. The Late Developmental Pattern of Mu Transposon Excision Is Conferred by a Cauliflower Mosaic Virus 35S –Driven MURA cDNA in Transgenic Maize

    PubMed Central

    Raizada, Manish N.; Walbot, Virginia

    2000-01-01

    The MuDR element responsible for Mutator activities in maize encodes two genes, mudrA and mudrB. Each encodes multiple transcripts hypothesized to regulate, directly or indirectly, the unique late timing and switch in transposition mechanism during maize development. mudrA, which encodes the MURA transposase, is unstable in bacterial plasmids, a technical problem solved by using phage M13 as a vector to prepare DNA for biolistic transformation. In transgenic maize, a single 2.7-kb mudrA cDNA predicted to encode an 823–amino acid protein is sufficient to catalyze late somatic excisions, despite removal of the native promoter, alternative transcription start sites, known introns, polymorphic 5′ and 3′ untranslated sequences, and the mudrB gene. These results suggest that post-translational regulation confers Mu excision timing. The transgene is active in lines containing silencing MuDR elements. This suggests that endogenous MuDR transposons do not measurably immunize the host against expression of a homologous transgene. PMID:10634904

  16. Spermine either delays or promotes cell death in Nicotiana tabacum L. corolla depending on the floral developmental stage and affects the distribution of transglutaminase.

    PubMed

    Cai, Giampiero; Della Mea, Massimiliano; Faleri, Claudia; Fattorini, Laura; Aloisi, Iris; Serafini-Fracassini, Donatella; Del Duca, Stefano

    2015-12-01

    The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. A Small Chloroplast-Encoded Protein as a Novel Architectural Component of the Light-Harvesting Antenna

    PubMed Central

    Ruf, Stephanie; Biehler, Klaus; Bock, Ralph

    2000-01-01

    A small conserved open reading frame in the plastid genome, ycf9, encodes a putative membrane protein of 62 amino acids. To determine the function of this reading frame we have constructed a knockout allele for targeted disruption of ycf9. This allele was introduced into the tobacco plastid genome by biolistic transformation to replace the wild-type ycf9 allele. Homoplasmic ycf9 knockout plants displayed no phenotype under normal growth conditions. However, under low light conditions, their growth rate was significantly reduced as compared with the wild-type, due to a lowered efficiency of the light reaction of photosynthesis. We show that this phenotype is caused by the deficiency in a pigment–protein complex of the light-harvesting antenna of photosystem II and hence by a reduced efficiency of photon capture when light availability is limiting. Our results indicate that, in contrast to the current view, light-harvesting complexes do not only consist of the classical pigment-binding proteins, but may contain small structural subunits in addition. These subunits appear to be crucial architectural factors for the assembly and/or maintenance of stable light-harvesting complexes. PMID:10769029

  18. Replacement of two non-adjacent amino acids in the S.cerevisiae bi2 intron-encoded RNA maturase is sufficient to gain a homing-endonuclease activity.

    PubMed Central

    Szczepanek, T; Lazowska, J

    1996-01-01

    Two homologous group I introns, the second intron of the cyt b gene, from related Saccharomyces species differ in their mobility. The S.capensis intron is mobile and encodes the I-ScaI endonuclease promoting intron homing, whilst the homologous S.cerevisiae intron is not mobile, but functions as an RNA maturase promoting splicing. These two intron-encoded proteins differ by only four amino acid substitutions. Taking advantage of the remarkable similarity of the two intron open reading frames and using biolistic transformation of mitochondria, we show that the replacement of only two non-adjacent residues in the S.cerevisiae maturase carboxy-terminal sequence is sufficient to induce a homing-endonuclease activity without losing the splicing function. Also, we demonstrate that these two activities reside in the S.capensis bi2-encoded protein which functions in both splicing and intron mobility in the wild-type cells. These results provide new insight into our understanding of the activity and the evolution of group I intron-encoded proteins. Images PMID:8670880

  19. Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer.

    PubMed

    Lin, Chi-Chen; Yen, Meng-Chi; Lin, Chiu-Mei; Huang, Shih-Shien; Yang, Huei-Jiun; Chow, Nan-Haw; Lai, Ming-Derg

    2008-06-01

    DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. Plasmid DNA expression was mainly in the epidermis. Noncarrier naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 microg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of naked DNA vaccine.

  20. Subchronic feeding study of high oleic acid soybeans (Event DP-3Ø5423-1) in Sprague-Dawley rats.

    PubMed

    Delaney, Bryan; Appenzeller, Laura M; Munley, Susan M; Hoban, Denise; Sykes, Greg P; Malley, Linda A; Sanders, Craig

    2008-12-01

    DP-3Ø5423-1 (305423) is a genetically-modified (GM) soybean that was produced by biolistic insertion of a gm-fad2-1 gene fragment and the gm-hra gene into the germline of soybean seeds. The gm-fad2-1 gene fragment cosuppresses expression of the endogenous FAD2-1 gene encoding the seed-specific omega-6 fatty acid desaturase resulting in higher concentrations of oleic acid (18:1) relative to linoleic acid (18:2). The gm-hra gene encoding a modified acetolactate synthase (ALS) enzyme was used as a selectable marker. In the current study, processed fractions (meal, hulls, and oil) from 305423 soybeans, non-GM soybeans with a similar genetic background (near isoline control) and three commercially-available non-GM varieties were used to formulate diets that were nutritionally comparable to PMI Certified Rodent LabDiet 5002. Diets were fed to young adult Crl:CD(SD) rats (12/sex/group) for approximately 90 days. Compared with rats fed the non-GM control diet, no biologically relevant differences were observed in rats fed the 305423 diet with respect to body weight/gain, food consumption/efficiency, mortality, clinical signs of toxicity, or ophthalmological observations. No test diet-related effects were observed on neurobehavioral assessments, organ weights, or clinical or anatomic pathology. These results demonstrated that 305423 soybeans are as safe and wholesome as non-GM soybeans.

  1. Opto-injection into single living cells by femtosecond near-infrared laser

    NASA Astrophysics Data System (ADS)

    Peng, Cheng

    This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

  2. Genetic transformation of major cereal crops.

    PubMed

    Ji, Qing; Xu, Xing; Wang, Kan

    2013-01-01

    Of the more than 50,000 edible plant species in the world, at least 10,000 species are cereal grains. Three major cereal crops, rice (Oryza sativa), maize (Zea mays), and wheat (Triticum sp.), provide two-thirds of the world's food energy intake. Although crop yields have improved tremendously thanks to technological advances in the past 50 years, population increases and climate changes continue to threaten the sustainability of current crop productions. Whereas conventional and marker-assisted breeding programs continue to play a major role in crop improvement, genetic engineering has drawn an intense worldwide interest from the scientific community. In the past decade, genetic transformation technologies have revolutionized agricultural practices and millions of hectares of biotech crops have been cultured. Because of its unique ability to insert well-characterized gene sequences into the plant genome, genetic engineering can also provide effective tools to address fundamental biological questions. This technology is expected to continue to be an indispensable approach for both basic and applied research. Here, we overview briefly the development of the genetic transformation in the top seven cereals, namely maize, rice, wheat, barley (Hordeum vulgare), sorghum (Sorghum sp.), oat (Avena sativa), and millets. The advantages and disadvantages of the two major transformation methods, Agrobacterium tumefaciens-mediated and biolistic methods, are also discussed.

  3. The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

    NASA Technical Reports Server (NTRS)

    Krishnamurthy, Konduru; Heppler, Marty; Mitra, Ruchira; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

    2003-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

  4. Reuteran and levan as carbohydrate sinks in transgenic sugarcane.

    PubMed

    Bauer, Rolene; Basson, Carin E; Bekker, Jan; Eduardo, Iban; Rohwer, Johann M; Uys, Lafras; van Wyk, Johannes H; Kossmann, Jens

    2012-12-01

    The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03 mg/g FW), sucrose content (ca 60 mg/g FW) was not affected, while starch content (<0.4 mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01 mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.

  5. Stable transformation of ferns using spores as targets: Pteris vittata and Ceratopteris thalictroides.

    PubMed

    Muthukumar, Balasubramaniam; Joyce, Blake L; Elless, Mark P; Stewart, C Neal

    2013-10-01

    Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid 'C-fern Express' (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.

  6. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

    PubMed

    Benediktsson, Adrienne M; Schachtele, Scott J; Green, Steven H; Dailey, Michael E

    2005-01-30

    Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

  7. Genetic transformation of the sugar beet plastome.

    PubMed

    De Marchis, Francesca; Wang, Yongxin; Stevanato, Piergiorgio; Arcioni, Sergio; Bellucci, Michele

    2009-02-01

    It is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome; this technology provides new opportunities for the genetic improvement of this crop and for social acceptance of genetically modified sugar beet plants.

  8. A Small GTP-Binding Host Protein Is Required for Entry of Powdery Mildew Fungus into Epidermal Cells of Barley1

    PubMed Central

    Schultheiss, Holger; Dechert, Cornelia; Kogel, Karl-Heinz; Hückelhoven, Ralph

    2002-01-01

    Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions. PMID:11950993

  9. Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.

    PubMed

    Kumar, S; Arul, L; Talwar, D

    2010-01-01

    We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

  10. Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach 'Genpei' bearing variegated and fully pigmented flowers.

    PubMed

    Uematsu, Chiyomi; Katayama, Hironori; Makino, Izumi; Inagaki, Azusa; Arakawa, Osamu; Martin, Cathie

    2014-03-01

    Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis.

  11. Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves

    SciTech Connect

    Howard, Amanda R.; Heppler, Marty L.; Ju, Ho-Jong; Krishnamurthy, Konduru; Payton, Mark E.; Verchot-Lubicz, Jeanmarie . E-mail: verchot@okstate.edu

    2004-10-25

    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, whereas 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.

  12. Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach ‘Genpei’ bearing variegated and fully pigmented flowers

    PubMed Central

    Uematsu, Chiyomi; Inagaki, Azusa

    2014-01-01

    Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis. PMID:24453228

  13. Involvement of the P1 cistron in overcoming eIF4E-mediated recessive resistance against Clover yellow vein virus in pea.

    PubMed

    Nakahara, Kenji S; Shimada, Ryoko; Choi, Sun-Hee; Yamamoto, Haruko; Shao, Jun; Uyeda, Ichiro

    2010-11-01

    Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance.

  14. Virus-induced gene silencing in Rauwolfia species.

    PubMed

    Corbin, Cyrielle; Lafontaine, Florent; Sepúlveda, Liuda Johana; Carqueijeiro, Ines; Courtois, Martine; Lanoue, Arnaud; Dugé de Bernonville, Thomas; Besseau, Sébastien; Glévarec, Gaëlle; Papon, Nicolas; Atehortúa, Lucia; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; St-Pierre, Benoit; Oudin, Audrey; Courdavault, Vincent

    2017-07-01

    Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

  15. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    PubMed

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

  16. Transformation of an edible crop with the pagA gene of Bacillus anthracis.

    PubMed

    Aziz, Mohammad Azhar; Sikriwal, Deepa; Singh, Samer; Jarugula, Sridhar; Kumar, P Anand; Bhatnagar, Rakesh

    2005-09-01

    Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here.

  17. Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes†

    PubMed Central

    Trinks, Daniela; Rajeswaran, R.; Shivaprasad, P. V.; Akbergenov, Rashid; Oakeley, Edward J.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing. PMID:15681452

  18. Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

    PubMed

    Trinks, Daniela; Rajeswaran, R; Shivaprasad, P V; Akbergenov, Rashid; Oakeley, Edward J; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-02-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

  19. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  20. Production of early flowering transgenic barley expressing the early flowering allele of Cryptochrome2 gene.

    PubMed

    El-Assal, Salah El-Din; Abd-Alla, Samir M; El-Tarras, Adel A; El-Awady, Mohamed A

    2011-01-01

    This work was carried out in order to develop early flowering barley lines. These lines will be useful to producers by enabling multiple crops within a single season and increasing production. Transgenic barley plants containing the natural early flowering time AtCRY2 allele from the Cape Verde Island (Cvi) ecotype of Arabidopsis have been generated using biolistic transformation. Immature embryo derived calli of two commercially important barley cultivars (El-Dwaser and El-Taif), were transformed using a pCAMBIA-2300 plasmid harboring a genomic fragment containing the AtCRY2-Cvi allele. Transformation was performed utilizing 600 immature embryos for each cultivar. Stable transformation was confirmed in T 0 and T 1 plants by using genomic PCR, RT-PCR and western blot analysis with AtCRY2 specific primers and antibodies, respectively. The transformation efficiency was 5.6% and 3.4% for El-Dwaser and El-Taif cultivars, respectively. Seeds from several T 1 lines were germinated on kanamycin plates and the lines that contained a single locus were selected for further evaluation. The transformed barley plants showed the specific AtCRY2-Cvi flowering phenotype, i.e. early flowering and day length insensitivity, compared to the non transgenic plants. The time to flowering in transgenic T 1 plants was assessed and two lines exhibited flowering more than 25 days earlier than the parental cultivars under short day conditions.

  1. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

    PubMed Central

    Shin, Sanghyun; Mackintosh, Caroline A.; Lewis, Janet; Heinen, Shane J.; Radmer, Lorien; Dill-Macky, Ruth; Baldridge, Gerald D.; Zeyen, Richard J.; Muehlbauer, Gary J.

    2008-01-01

    Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions. PMID:18467324

  2. The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

    NASA Technical Reports Server (NTRS)

    Krishnamurthy, Konduru; Heppler, Marty; Mitra, Ruchira; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

    2003-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

  3. Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period

    PubMed Central

    2010-01-01

    Background Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years. Results Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control. Conclusion Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape. PMID:20525262

  4. Developing nanotechnology for biofuel and plant science applications

    SciTech Connect

    Valenstein, Justin

    2012-01-01

    This dissertation presents the research on the development of mesoporous silica based nanotechnology for applications in biofuels and plant science. Mesoporous silica nanoparticles (MSNs) have been the subject of great interest in the last two decades due to their unique properties of high surface area, tunable pore size and particle morphology. The robust nature of the silica framework is easily functionalized to make the MSNs a promising option for selective separations. Also, the independent channels that form the pores of MSN have been exploited in the use of particles as platforms for molecular delivery. Pore size and organic functionality are varied to identify the ideal adsorbent material for free fatty acids (FFAs). The resulting material is able to sequester FFAs with a high degree of selectivity from a simulated solution and microalgal oil. The recyclability and industrial implications are also explored. A continuation of the previous material, further tuning of MSN pore size was investigated. Particles with a smaller diameter selectively sequester polyunsaturated free fatty acids (PUFAs) over monounsaturated FFAs and saturated FFAs. The experimental results were verified with molecular modeling. Mesoporous silica nanoparticle materials with a pore diameter of 10 nm (MSN-10) were decorated with small gold nanoparticles. The resulting materials were shown to deliver proteins and DNA into plant cells using the biolistic method.

  5. High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants

    PubMed Central

    Klahre, Ulrich; Crété, Patrice; Leuenberger, Sabrina A.; Iglesias, Victor A.; Meins, Frederick

    2002-01-01

    Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs. PMID:12181491

  6. A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

    PubMed Central

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

  7. Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11

    PubMed Central

    Yang, Mai Lee; Uhrig, John; Vu, Kiem; Singapuri, Anil; Dennis, Michael

    2015-01-01

    Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37°C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance. PMID:26643330

  8. Heterologous Acidothermus cellulolyticus 1,4-β-Endoglucanase E1 Produced Within the Corn Biomass Converts Corn Stover Into Glucose

    NASA Astrophysics Data System (ADS)

    Ransom, Callista; Balan, Venkatesh; Biswas, Gadab; Dale, Bruce; Crockett, Elaine; Sticklen, Mariam

    Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced, heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active from.

  9. Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg.

    PubMed

    Bordat, Amandine; Houvenaghel, Marie-Christine; German-Retana, Sylvie

    2015-06-14

    Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context. Three overlapping long distance PCR fragments were amplified and assembled in a single-step process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein. The Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg. This is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyvirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyvirus.

  10. The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri.

    PubMed

    Jakobiak, Thomas; Mages, Wolfgang; Scharf, Birgit; Babinger, Patrick; Stark, Klaus; Schmitt, Rüdiger

    2004-12-01

    The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3'-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker--paromomycin resistance (PmR)--for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3' UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable PmR transformants ranged about 15 per 106 target cells. Due to rapid and sharp selection, PmR transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of PmR transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.

  11. Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides1[W][OPEN

    PubMed Central

    Muthukumar, Balasubramaniam; Joyce, Blake L.; Elless, Mark P.; Stewart, C. Neal

    2013-01-01

    Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns. PMID:23933990

  12. Carbohydrate profiling in seeds and seedlings of transgenic triticale modified in the expression of sucrose:sucrose-1-fructosyltransferase (1-SST) and sucrose:fructan-6-fructosyltransferase (6-SFT).

    PubMed

    Diedhiou, Calliste; Gaudet, Denis; Liang, Yehong; Sun, Jinyue; Lu, Zhen-Xing; Eudes, François; Laroche, André

    2012-10-01

    Constructs with sucrose-sucrose 1-fructosyltransferase (1-SST) from rye and or sucrose-fructan 6-fructosyltransferase (6-SFT) from wheat were placed under the control of wheat aleurone-specific promoter and expressed in triticale using biolistic and microspore transformation. Transgenic lines expressing one or both the 1-SST and the 6-SFT accumulated 50% less starch and 10-20 times more fructan, particularly 6-kestose, in the dry seed compared to the untransformed wild-type (WT) triticale; other fructans ranged in size from DP 4 to DP 15. During germination from 1 to 4 days after imbibition (dai), fructans were rapidly metabolized and only in transgenic lines expressing both 1-SST and 6-SFT were fructan contents significantly higher than in the untransformed controls after 4 days. In situ hybridization confirmed expression of 6-SFT in the aleurone layer in imbibed seeds of transformed plants. When transgenic lines were subjected to a cold stress of 4°C for 2 days, synthesis of fructan increased compared to untransformed controls during low-temperature germination. The increase of fructan in dry seed and germinating seedling was generally associated with transcript expression levels in transformed plants but total gene expression was not necessarily correlated with the time course accumulation of fructan during germination. This is the first report of transgenic modification of cereals to achieve production of fructans in cereal seeds and during seed germination. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  13. Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.

    PubMed

    Soria-Guerra, Ruth Elena; Alpuche-Solís, Angel G; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; Bendik, Elise M; Martínez-González, Luzmila; Korban, Schuyler S

    2009-05-01

    Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.

  14. Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

    PubMed

    Abouseadaa, Heba H; Osman, Gamal H; Ramadan, Ahmed M; Hassanein, Sameh E; Abdelsattar, Mohamed T; Morsy, Yasser B; Alameldin, Hussien F; El-Ghareeb, Doaa K; Nour-Eldin, Hanan A; Salem, Reda; Gad, Adel A; Elkhodary, Soheir E; Shehata, Maher M; Mahfouz, Hala M; Eissa, Hala F; Bahieldin, Ahmed

    2015-07-22

    Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. Transgenic wheat plants had improved resistance to Sitophilus granarius.

  15. The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon.

    PubMed

    Thilmony, Roger; Guttman, Mara E; Lin, Jeanie W; Blechl, Ann E

    2014-01-01

    The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for β-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.

  16. Diversity of begomoviruses associated with mosaic disease of cultivated cassava (Manihot esculenta Crantz) and its wild relative (Manihot glaziovii Mull. Arg.) in Uganda.

    PubMed

    Sserubombwe, W S; Briddon, R W; Baguma, Y K; Ssemakula, G N; Bull, S E; Bua, A; Alicai, T; Omongo, C; Otim-Nape, G W; Stanley, J

    2008-07-01

    Cassava (Manihot esculenta) growing in Uganda during 2001-2002 has been screened for the presence of begomoviruses using PCR-RFLP, cloning full-length genomic components and nucleotide sequence analysis. In contrast with a recent survey in neighbouring Kenya, which identified three distinct strains of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2) as well as East African cassava mosaic Zanzibar virus and the new species East African cassava mosaic Kenya virus, only EACMV-UG and, to a lesser extent, African cassava mosaic virus (ACMV) were found associated with cassava in Uganda. The integrity of the cloned genomic components of representative virus isolates was confirmed by demonstrating their infectivity in Nicotiana benthamiana and cassava using biolistic inoculation, providing a convenient means to screen cassava varieties for disease resistance. Both EACMV-UG and ACMV were also associated with Manihot glaziovii. Infectivity studies using cloned components confirmed that viruses from one host could infect the other, suggesting that this wild relative of cassava might be a reservoir host for the disease. The relatively low level of diversity of begomoviruses associated with cassava mosaic disease in Uganda is consistent with reports that EACMV-UG has displaced other begomovirus species and strains during the recent epidemic that swept through the country.

  17. Development of transgenic imazapyr-tolerant cowpea (Vigna unguiculata).

    PubMed

    Citadin, C T; Cruz, A R R; Aragão, F J L

    2013-04-01

    Here we present the development of cowpea lines tolerant to a herbicide from imidazoline class (imazapyr). Plants presented tolerance to fourfold the commercial recommended dose for weed control. Cowpea is one of the most important and widely cultivated legumes in many parts of the world. Its cultivation is drastically affected by weeds, causing damages during growth and development of plants, competing for light, nutrients and water. Consequently, weed control is critical, especially using no-tillage farming systems. In tropical regions, no-till farming is much easier with the use of herbicides to control weeds. This study was conducted to evaluate the possibility of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during the summer season. The biolistic process was used to insert a mutated acetohydroxyacid synthase coding gene (Atahas) which confers tolerance to imazapyr. The transgene integration was confirmed by Southern blot analysis. Out of ten lines tested for tolerance to 100 g ha(-1) imazapyr, eight presented some tolerance. One line (named 59) revealed high herbicide tolerance and developmental growth comparable to non-transgenic plants. This line was further tested for tolerance to higher herbicide concentrations and presented tolerance to 400 g ha(-1) imazapyr (fourfold the commercial recommended dose) with no visible symptoms. Line 59 will be the foundation for generating imidazolinone-tolerant cowpea varieties, which will facilitate cultivation of this crop in large areas.

  18. A Perspective on Hypericum perforatum Genetic Transformation

    PubMed Central

    Hou, Weina; Shakya, Preeti; Franklin, Gregory

    2016-01-01

    Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering. PMID:27446112

  19. A Novel Nucleus-Targeted Protein Is Expressed in Barley Leaves during Senescence and Pathogen Infection1

    PubMed Central

    Krupinska, Karin; Haussühl, Kirsten; Schäfer, Anke; van der Kooij, Tom A.W.; Leckband, Gunhild; Lörz, Horst; Falk, Jon

    2002-01-01

    The barley (Hordeum vulgare) cDNA HvS40 represents a gene with enhanced mRNA level during leaf senescence. Biolistic transformation of onion (Allium cepa) epidermal cell layers with a glucuronidase fusion protein construct provided evidence that the 15.4-kD protein encoded by HvS40 is localized to the nucleus. Expression of the gene is induced by jasmonate and salicylic acid; both are known to act as signaling compounds during senescence and defense toward pathogens. Transcript levels of HvS40 were observed to be particularly high in leaf sectors that undergo necrosis and chlorosis after infection with Pyrenophora teres. This pathogen-related expression is, in contrast, clearly reduced in transgenic barley plants expressing a stilbene synthase from grape (Vitis vinifera), whereas the mRNA level of a gene encoding the pathogen-related protein HvPR1 is unaffected. In situ hybridization with HvS40 antisense RNA revealed that during leaf senescence, the HvS40 transcript predominantly accumulates in the mesophyll. Taken together, the findings suggest a connection between the nuclear protein encoded by HvS40 and the degeneration of chloroplasts occurring during senescence and during infection of barley wild-type plants with P. teres. PMID:12427984

  20. Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments.

    PubMed

    Zienkiewicz, Maksymilian; Krupnik, Tomasz; Drożak, Anna; Golke, Anna; Romanowska, Elżbieta

    2017-01-01

    We have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism. Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae-an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi: 10.1007/s00709-015-0936-9 ). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae-a new model organism.

  1. Stable integration and expression of a plant defensin in tomato confers resistance to fusarium wilt.

    PubMed

    Abdallah, Naglaa A; Shah, Dilip; Abbas, Dina; Madkour, Magdy

    2010-01-01

    Plant defensins are small cysteine-rich peptides which belong to a group of pathogenasis related defense mechanism proteins. The proteins inhibit the growth of a broad range of microbes and are highly stable under extreme environmental stresses. Tomato cultivation is affected by fungal disease such as Fusarium wilt. In order to overcome fungal damages, transgenic tomato plants expressing the Medicago sativa defensin gene MsDef1 under the control of the CaMV 35S promoter were developed. The Fusarium-susceptible tomato (Lycobersicum esculentum Mill) cultivar CastleRock was used for transformation to acquire fungal resistance. Hypocotyl with a part of cotyledon (hypocotyledonary) for young tomato seedlings were used as an explant material and transformation was performed using the biolistic delivery system. Bombarded shoots were selected on regeneration medium supplemented with hygromycin and suitable concentrations of BA, zeatin ripozide and AgNO(3). Putative transgenic plantlets of T(0) were confirmed by PCR analysis using primers specific for the transgene and the transformation frequency obtained was 52.3%. Transformation and transcription of transgenes were confirmed in T(1) by PCR, Southern hybridizations, and reverse-transcription PCR (RT-PCR). The copy numbers of integrated transgene into tomato genome ranged between 1-3 copies. Greenhouse bioassay was performed on the transgenic T(1) and T(2) young seedlings and non-transgenic controls by challenging with a vigorous isolate of the fungal pathogen Fusarium oxysporum f. sp. Lycopersici. The level of fungal infectivity was determined using RT-PCR with tomatinase specific primers. Transgenic lines were more resistant to infection by fusarium than the control plants. These results indicated that overexpressing defensins in transgenic plants confer resistance to fungal pathogens.

  2. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology.

    PubMed

    Li, Jian-Feng; Li, Li; Sheen, Jen

    2010-01-14

    Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  3. Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Neu, R; Goffart, S; Wolf, K; Schäfer, B

    1998-05-01

    In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

  4. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes

    PubMed Central

    2010-01-01

    Background High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. Results Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). Conclusion We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable. PMID:20626919

  5. Brain-derived neurotrophic factor mediates activity-dependent dendritic growth in nonpyramidal neocortical interneurons in developing organotypic cultures.

    PubMed

    Jin, Xiaoming; Hu, Hang; Mathers, Peter H; Agmon, Ariel

    2003-07-02

    Brain-derived neurotrophic factor (BDNF) promotes postnatal maturation of GABAergic inhibition in the cerebral and cerebellar cortices, and its expression and release are enhanced by neuronal activity, suggesting that it acts in a feedback manner to maintain a balance between excitation and inhibition during development. BDNF promotes differentiation of cerebellar, hippocampal, and neostriatal inhibitory neurons, but its effects on the dendritic development of neocortical inhibitory interneurons remain unknown. Here, we show that BDNF mediates depolarization-induced dendritic growth and branching in neocortical interneurons. To visualize inhibitory interneurons, we biolistically transfected organotypic cortical slice cultures from neonatal mice with green fluorescent protein (GFP) driven by the glutamic acid decarboxylase (GAD)67 promoter. Nearly all GAD67-GFP-expressing neurons were nonpyramidal, many contained GABA, and some expressed markers of neurochemically defined GABAergic subtypes, indicating that GAD67-GFP-expressing neurons were GABAergic. We traced dendritic trees from confocal images of the same GAD67-GFP-expressing neurons before and after a 5 d growth period, and quantified the change in total dendritic length (TDL) and total dendritic branch points (TDBPs) for each neuron. GAD67-GFP-expressing neurons growing in control medium exhibited a 20% increase in TDL, but in 200 ng/ml BDNF or 10 mm KCl, this increase nearly doubled and was accompanied by a significant increase in TDBPs. Blocking action potentials with TTX did not prevent the BDNF-induced growth, but antibodies against BDNF blocked the growth-promoting effect of KCl. We conclude that BDNF, released by neocortical pyramidal neurons in response to depolarization, enhances dendritic growth and branching in nearby inhibitory interneurons.

  6. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner

    PubMed Central

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1−/− GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  7. Progress towards the 'Golden Age' of biotechnology.

    PubMed

    Gartland, K M A; Bruschi, F; Dundar, M; Gahan, P B; Viola Magni, M p; Akbarova, Y

    2013-07-01

    Biotechnology uses substances, materials or extracts derived from living cells, employing 22 million Europeans in a € 1.5 Tn endeavour, being the premier global economic growth opportunity this century. Significant advances have been made in red biotechnology using pharmaceutically and medically relevant applications, green biotechnology developing agricultural and environmental tools and white biotechnology serving industrial scale uses, frequently as process feedstocks. Red biotechnology has delivered dramatic improvements in controlling human disease, from antibiotics to overcome bacterial infections to anti-HIV/AIDS pharmaceuticals such as azidothymidine (AZT), anti-malarial compounds and novel vaccines saving millions of lives. Green biotechnology has dramatically increased food production through Agrobacterium and biolistic genetic modifications for the development of 'Golden Rice', pathogen resistant crops expressing crystal toxin genes, drought resistance and cold tolerance to extend growth range. The burgeoning area of white biotechnology has delivered bio-plastics, low temperature enzyme detergents and a host of feedstock materials for industrial processes such as modified starches, without which our everyday lives would be much more complex. Biotechnological applications can bridge these categories, by modifying energy crops properties, or analysing circulating nucleic acid elements, bringing benefits for all, through increased food production, supporting climate change adaptation and the low carbon economy, or novel diagnostics impacting on personalized medicine and genetic disease. Cross-cutting technologies such as PCR, novel sequencing tools, bioinformatics, transcriptomics and epigenetics are in the vanguard of biotechnological progress leading to an ever-increasing breadth of applications. Biotechnology will deliver solutions to unimagined problems, providing food security, health and well-being to mankind for centuries to come. Copyright © 2013

  8. Bioengineered 'golden' indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems.

    PubMed

    Datta, Karabi; Baisakh, Niranjan; Oliva, Norman; Torrizo, Lina; Abrigo, Editha; Tan, Jing; Rai, Mayank; Rehana, Sayda; Al-Babili, Salim; Beyer, Peter; Potrykus, Ingo; Datta, Swapan K

    2003-03-01

    Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene beta-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition.

  9. Stable expression of AtGA2ox1 in a low-input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improves turf quality under field conditions.

    PubMed

    Agharkar, Mrinalini; Lomba, Paula; Altpeter, Fredy; Zhang, Hangning; Kenworthy, Kevin; Lange, Theo

    2007-11-01

    Bahiagrass (Paspalum notatum Flugge) is a prime candidate for molecular improvement of turf quality. Its persistence and low input characteristics made it the dominant utility turfgrass along highways in the south-eastern USA. However, the comparatively poor turf quality due to reduced turf density and prolific production of unsightly inflorescences currently limits the widespread use of bahiagrass as residential turf. Alteration of endogenous gibberellin (GA) levels by application of growth regulators or transgenic strategies has modified plant architecture in several crops. GA catabolizing AtGA2ox1 was subcloned under the control of the constitutive maize ubiquitin promoter and Nos 3'UTR. A minimal AtGA2ox1 expression cassette lacking vector backbone sequences was stably introduced into apomictic bahiagrass by biolistic gene transfer as confirmed by Southern blot analysis. Expression of AtGA2ox1 in bahiagrass as indicated by reverse transcription-polymerase chain reaction and Northern blot analysis resulted in a significant reduction of endogenous bioactive GA(1) levels compared to wild type. Interestingly, transgenic plants displayed an increased number of vegetative tillers which correlated with the level of AtGA2ox1 expression and enhanced turf density under field conditions. This indicates that GAs contribute to signalling the outgrowth of axillary buds in this perennial grass. Transgenic plants also showed decreased stem length and delayed flowering under controlled environment and field conditions. Consequently, turf quality following weekly mowing was improved in transgenic bahiagrass. Transgene expression and phenotype were transmitted to seed progeny. Argentine bahiagrass produces seeds asexually by apomixis, which reduces the risk of unintended transgene dispersal by pollen and results in uniform progeny.

  10. Physical methods of nucleic acid transfer: general concepts and applications

    PubMed Central

    Villemejane, Julien; Mir, Lluis M

    2009-01-01

    Physical methods of gene (and/or drug) transfer need to combine two effects to deliver the therapeutic material into cells. The physical methods must induce reversible alterations in the plasma membrane to allow the direct passage of the molecules of interest into the cell cytosol. They must also bring the nucleic acids in contact with the permeabilized plasma membrane or facilitate access to the inside of the cell. These two effects can be achieved in one or more steps, depending upon the methods employed. In this review, we describe and compare several physical methods: biolistics, jet injection, hydrodynamic injection, ultrasound, magnetic field and electric pulse mediated gene transfer. We describe the physical mechanisms underlying these approaches and discuss the advantages and limitations of each approach as well as its potential application in research or in preclinical and clinical trials. We also provide conclusions, comparisons, and projections for future developments. While some of these methods are already in use in man, some are still under development or are used only within clinical trials for gene transfer. The possibilities offered by these methods are, however, not restricted to the transfer of genes and the complementary uses of these technologies are also discussed. As these methods of gene transfer may bypass some of the side effects linked to viral or biochemical approaches, they may find their place in specific clinical applications in the future. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19154421

  11. Molecular cloning of a novel pathogen-inducible cDNA encoding a putative acyl-CoA synthetase from Capsicum annuum L.

    PubMed

    Lee, S J; Suh, M C; Kim, S; Kwon, J K; Kim, M; Paek, K H; Choi, D; Kim, B D

    2001-08-01

    By means of differential display, a pool of salicylic acid (SA)-induced mRNAs were identified and subsequently their cDNAs were isolated from a cDNA library prepared from SA-induced leaf tissues of hot pepper. One of these cDNA clones, designated CaSIG4, was 1900 bp and contained an open reading frame encoding 523 amino acids with a calculated molecular mass of 56.3 kDa. The predicted amino acid sequence of CaSIG4 showed high sequence similarity to the AMP-binding protein family of both prokaryotic and eukaryotic acyl-CoA synthetases. CaSIG4 transcripts accumulated rapidly after SA treatment and in response to both incompatible and compatible interactions with Xanthomonas campestris pv. vesicatoria race 1. To investigate the cis-acting elements mediating CaSIG4 expression, the CaSIG4 5'-flanking region was isolated by inverse PCR. Database searches indicated that a potential cis-regulatory element is almost identical to the consensus core sequences ACC(A/T)ACC(A/C) which are conserved among promoters of other phenylpropanoid biosynthetic genes. The subcellular localization of the CaSIG4 protein was studied by using a soluble modified GFP gene fusion delivered into epidermal cells of onion by biolistic bombardment. The CaSIG4-smGFP fusion protein was localized to the plasma membrane. Taken together, CaSIG4 encoding a putative acyl-CoA synthetase could function as a plasma membrane-bound protein with a role in signaling in plant defense.

  12. Watermelon chlorotic stunt virus (WmCSV): a serious disease threatening watermelon production in Jordan.

    PubMed

    Al-Musa, A; Anfoka, G; Al-Abdulat, A; Misbeh, S; Haj Ahmed, F; Otri, I

    2011-08-01

    The incidence of watermelon chlorotic stunt disease and the molecular characterization of the Jordanian isolate of Watermelon chlorotic stunt virus (WmCSV-[JO]) are described in this study. Symptomatic leaf samples obtained from watermelon (Citrullus lanatus Thunb.), melon (Cucumis melo L.), squash (Cucurbita pepo), cucumber (Cucumis sativus L.), and bottle gourd (Lagenaria siceraria) plants were tested for WmCSV-[JO] infection by PCR. The virus could be detected in 8 melon and 87 watermelon samples obtained from Ghor Assafi (southern part of Jordan Valley). Three samples collected from Mafraq (eastern part of Jordan) were found mixed infected with WmCSV-[JO] and Squash leaf curl virus. The full-length DNA-A and DNA-B genomes of WmCSV-[JO] were amplified, and sequences were deposited in the GenBank under accession numbers EU561237 and EU561236, respectively. Sequence analysis reveals that WmCSV-[JO] is closely related to other virus isolates from Israel (WmCSV-[IL]), Yemen (WmCSV-[YE]), Iran (WmCSV-[IR]), Lebanon (WmCSV-[LB]), and Sudan (WmCSV-[SD]). DNA-A of WmCSV-[JO] showed highest nucleotide identity (99.42%) with WmCSV-[IL], while DNA-B had highest nucleotide identity (95.52%) with WmCSV-[YE]. Data of this study demonstrate that digestion of DNA-B genome of WmCSV isolates with ApaI enzyme can discriminate between these isolates at the molecular level. Infectious clones of WmCSV-[JO] were constructed and agroinoculated to Nicotiana benthamiana plants. Inoculated plants developed mild disease symptoms 4 weeks post inoculation, while watermelon plants biolistically inoculated with WmCSV-[JO] developed characteristic mottling, yellowing and severe leaf curling symptoms 3 weeks post inoculation.

  13. Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.

    PubMed

    Möller, Ralf; McDonald, Armando G; Walter, Christian; Harris, Philip J

    2003-09-01

    Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCl. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography-mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state (13)C cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactogluco-mannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression.

  14. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.

    PubMed

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1 (flox/flox) mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1 (-/-) GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  15. Mutations in SYNGAP1 cause intellectual disability, autism, and a specific form of epilepsy by inducing haploinsufficiency.

    PubMed

    Berryer, Martin H; Hamdan, Fadi F; Klitten, Laura L; Møller, Rikke S; Carmant, Lionel; Schwartzentruber, Jeremy; Patry, Lysanne; Dobrzeniecka, Sylvia; Rochefort, Daniel; Neugnot-Cerioli, Mathilde; Lacaille, Jean-Claude; Niu, Zhiyv; Eng, Christine M; Yang, Yaping; Palardy, Sylvain; Belhumeur, Céline; Rouleau, Guy A; Tommerup, Niels; Immken, Ladonna; Beauchamp, Miriam H; Patel, Gayle Simpson; Majewski, Jacek; Tarnopolsky, Mark A; Scheffzek, Klaus; Hjalgrim, Helle; Michaud, Jacques L; Di Cristo, Graziella

    2013-02-01

    De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.

  16. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    NASA Astrophysics Data System (ADS)

    Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

    2006-12-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  17. Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield

    PubMed Central

    Yadav, Dinesh; Shavrukov, Yuri; Bazanova, Natalia; Chirkova, Larissa; Borisjuk, Nikolai; Kovalchuk, Nataliya; Ismagul, Ainur; Parent, Boris; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2015-01-01

    Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1–T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20–30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance. PMID:26220082

  18. Antibodies to ovine herpesvirus 2 glycoproteins decrease virus infectivity and prevent malignant catarrhal fever in rabbits.

    PubMed

    Cunha, Cristina W; Knowles, Donald P; Taus, Naomi S; O'Toole, Donal; Nicola, Anthony V; Aguilar, Hector C; Li, Hong

    2015-02-25

    Ovine herpesvirus-2 (OvHV-2) is the etiological agent of sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease of many species in the order Artiodactyla. Development of a vaccine is critical to prevent mortality. Because OvHV-2 has not been cultured in vitro, SA-MCF research is hindered by the lack of in vitro tools to study viral constituents and specific host immune responses. As an alternative, in this study the neutralizing activity of antibodies against OvHV-2 glycoproteins gB and gH/gL was evaluated in vivo using rabbits. OvHV-2-specific antibodies were developed in rabbits by immunization using biolistic delivery of plasmids expressing the genes of interest. A lethal dose of OvHV-2 was incubated with the antisera and then nebulized into rabbits. Virus neutralization was assessed by measuring infection parameters associated with the virus infectious dose. Anti-gB or anti-gH/gL antibodies alone blocked infection in five out of six rabbits (83%), while a combination of anti-gB and anti-gH/gL antibodies protected all six rabbits (100%) from infection. These results indicate that antibodies to OvHV-2 gB and gH/gL are capable of neutralizing virions, and consequently, reduce virus infectivity and prevent SA-MCF in rabbits. Thus, OvHV-2 gB and gH/gL are suitable targets to be tested in a SA-MCF vaccine aimed at stimulating neutralizing antibody responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

    PubMed

    Gil-Humanes, Javier; Wang, Yanpeng; Liang, Zhen; Shan, Qiwei; Ozuna, Carmen V; Sánchez-León, Susana; Baltes, Nicholas J; Starker, Colby; Barro, Francisco; Gao, Caixia; Voytas, Daniel F

    2017-03-01

    The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

  20. Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay

    PubMed Central

    Esparza-Araiza, Mayra J.; Bañuelos-Hernández, Bernardo; Argüello-Astorga, Gerardo R.; Lara-Ávila, José P.; Goodwin, Paul H.; Isordia-Jasso, María I.; Castillo-Collazo, Rosalba; Rougon-Cardoso, Alejandra; Alpuche-Solís, Ángel G.

    2015-01-01

    Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in ~61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI-silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid-dependent defense responses. PMID:26734014

  1. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii.

    PubMed

    Wannathong, Thanyanan; Waterhouse, Janet C; Young, Rosanna E B; Economou, Chloe K; Purton, Saul

    2016-06-01

    In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species-Chlamydomonas reinhardtii-and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5'UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient ΔpsbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.

  2. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

    PubMed

    Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

    2017-01-12

    Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2(-ΔΔCt) analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g(-1) fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g(-1) fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

  3. Characterization and Functional Identification of a Novel Plant 4,5-Extradiol Dioxygenase Involved in Betalain Pigment Biosynthesis in Portulaca grandiflora

    PubMed Central

    Christinet, Laurent; Burdet, Frédéric X.; Zaiko, Maïa; Hinz, Ursula; Zrÿd, Jean-Pierre

    2004-01-01

    Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism. PMID:14730069

  4. Evaluation of North American isolates of Soybean mosaic virus for gain of virulence on Rsv-genotype soybeans with special emphasis on resistance-breaking determinants on Rsv4.

    PubMed

    Khatabi, B; Fajolu, O L; Wen, R-H; Hajimorad, M R

    2012-12-01

    Resistance to Soybean mosaic virus (SMV) in soybean is conferred by three dominant genes: Rsv1, Rsv3 and Rsv4. Over the years, scientists in the USA have utilized a set of standard pathotypes, SMV-G1 to SMV-G7, to study interaction with Rsv-genotype soybeans. However, these pathotypes were isolated from a collection of imported soybean germplasm over 30 years ago. In this study, 35 SMV field isolates collected in recent years from 11 states were evaluated for gain of virulence on soybean genotypes containing individual Rsv genes. All isolates were avirulent on L78-379 (Rsv1), whereas 19 were virulent on L29 (Rsv3). On PI88788 (Rsv4), 14 of 15 isolates tested were virulent; however, only one was capable of systemically infecting all of the inoculated V94-5152 (Rsv4). Nevertheless, virulent variants from 11 other field isolates were rapidly selected on initial inoculation onto V94-5152 (Rsv4). The P3 cistrons of the original isolates and their variants on Rsv4-genotype soybeans were sequenced. Analysis showed that virulence on PI88788 (Rsv4) was not associated, in general, with selection of any new amino acid, whereas Q1033K and G1054R substitutions were consistently selected on V94-5152 (Rsv4). The role of Q1033K and G1054R substitutions, individually or in combination, in virulence on V94-5152 (Rsv4) was confirmed on reconstruction in the P3 cistron of avirulent SMV-N, followed by biolistic inoculation. Collectively, our data demonstrate that SMV has evolved virulence towards Rsv3 and Rsv4, but not Rsv1, in the USA. Furthermore, they confirm that SMV virulence determinants on V94-5152 (Rsv4) reside on P3.

  5. Genetic transformation of Indian bread (T. aestivum) and pasta (T. durum) wheat by particle bombardment of mature embryo-derived calli

    PubMed Central

    Patnaik, Debasis; Khurana, Paramjit

    2003-01-01

    Background Particle bombardment has been successfully employed for obtaining transgenics in cereals in general and wheat in particular. Most of these procedures employ immature embryos which are not available throughout the year. The present investigation utilizes mature seeds as the starting material and the calli raised from the hexaploid Triticum aestivum and tetraploid Triticum durum display a high regeneration response and were therefore used as the target tissue for genetic transformation by the biolistic approach. Results Mature embryo-derived calli of bread wheat (Triticum aestivum, cv. CPAN1676) and durum wheat (T. durum, cv. PDW215) were double bombarded with 1.1 gold microprojectiles coated with pDM302 and pAct1-F at a target distance of 6 cm. Southern analysis using the bar gene as a probe revealed the integration of transgenes in the T0 transformants. The bar gene was active in both T0 and T1 generations as evidenced by phosphinothricin leaf paint assay. Approximately 30% and 33% primary transformants of T. aestivum and T. durum, respectively, were fertile. The transmission of bar gene to T1 progeny was demonstrated by PCR analysis of germinated seedlings with primers specific to the bar gene. Conclusions The transformation frequency obtained was 8.56% with T. aestivum and 10% with T. durum. The optimized protocol was subsequently used for the introduction of the barley gene encoding a late embryogenesis abundant protein (HVA1) in T. aestivum and T. durum. The presence of the HVA1 transgene was confirmed by Southern analysis in the T0 generation in case of Triticum aestivum, and T0 and T1 generation in Triticum durum. PMID:12952555

  6. Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum.

    PubMed

    Meziadi, Chouaib; Blanchet, Sophie; Richard, Manon M S; Pilet-Nayel, Marie-Laure; Geffroy, Valérie; Pflieger, Stéphanie

    2016-08-01

    Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus-induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)-derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4-β-D-glucanase, respectively. The features of the 'one-step' BPMV-derived VIGS vector include (i) the ease of rub-inoculation, without any need for biolistic or agro-inoculation procedures, (ii) simple cost-effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low- to high-throughput VIGS studies. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  7. The coat protein of tobamovirus acts as elicitor of both L2 and L4 gene-mediated resistance in Capsicum.

    PubMed

    Gilardi, P; García-Luque, I; Serra, M T

    2004-07-01

    In Capsicum, the resistance conferred by the L(2) gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L(4) gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L(2) gene confers resistance to Paprika mild mottle virus (PaMMV), while the L(4) gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L(2)L(2)) and Capsicum chacoense (L(4)L(4)) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L(2)L(2)) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L(2) resistance gene. In C. chacoense (L(4)L(4)) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L(4) gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L(2)L(2)) leaves and PMMoV CP into C. chacoense (L(4)L(4)) leaves by biolistic co-bombardment with a beta-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L(2) and L(4) gene-mediated hypersensitive response.

  8. Evaluation of a SUMO E2 Conjugating Enzyme Involved in Resistance to Clavibacter michiganensis Subsp. michiganensis in Solanum peruvianum, Through a Tomato Mottle Virus VIGS Assay.

    PubMed

    Esparza-Araiza, Mayra J; Bañuelos-Hernández, Bernardo; Argüello-Astorga, Gerardo R; Lara-Ávila, José P; Goodwin, Paul H; Isordia-Jasso, María I; Castillo-Collazo, Rosalba; Rougon-Cardoso, Alejandra; Alpuche-Solís, Ángel G

    2015-01-01

    Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianum. Previous research showed up-regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus-induced gene-silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in ~61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI-silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid-dependent defense responses.

  9. Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus.

    PubMed

    Park, Chang-Jin; Shin, Ryoung; Park, Jeong Mee; Lee, Gil-Je; You, Jin-Sam; Paek, Kyung-Hee

    2002-02-01

    Pepper (Capsicum annuum) plants exhibit hypersensitive response (HR) against infection by many tobamoviruses. A clone encoding a putative nonspecific lipid transfer protein (CaLTP1) was isolated by differential screening of a cDNA library from resistant pepper leaves when inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaLTP1 is highly similar to that of the other plant LTPs. Southern blot analysis showed that a small gene family of LTP-related sequences was present in the pepper genome. Transcripts homologous to CaLTP1 accumulated abundantly in old leaves and flowers. CaLTP1 expression was induced in the incompatible interaction with TMV-P0 but was not induced in the compatible interaction with TMV-P1.2. In correlation with the temporal progression of HR in the inoculated leaves, CaLTP1 transcripts started to accumulate at 24 h after TMV-P0 inoculation, reaching a maximal level at 48 h. A strain of Xanthomonas campestris pv. vesicatoria (Xcv) that carries the bacterial avirulence gene, avrBs2, was infiltrated into leaves of a pepper cultivar containing the Bs2 resistance gene. A marked induction of CaLTP1 expression was observed in Xcv-infiltrated leaves. Effects of exogenously applied abiotic elicitors on CaLTP1 expression were also examined. Salicylic acid caused a rapid accumulation of CaLTP1 transcripts in pepper leaves and ethephon treatment also induced the expression of the CaLTP1 gene. Transient expression in the detached pepper leaves by biolistic gene bombardment indicated that CaLTP1 is localized mostly at the plant cell surface, possibly in the cell wall. These results suggest possible role(s) for LTPs in plant defense against pathogens including viruses.

  10. Single Cell Electroporation Method for Mammalian CNS Neurons in Organotypic Slice Cultures

    NASA Astrophysics Data System (ADS)

    Uesaka, Naofumi; Hayano, Yasufumi; Yamada, Akito; Yamamoto, Nobuhiko

    Axon tracing is an essential technique to study the projection pattern of neurons in the CNS. Horse radish peroxidase and lectins have contributed to revealing many neural connection patterns in the CNS (Itaya and van Hoesen, 1982; Fabian and Coulter, 1985; Yoshihara, 2002). Moreover, a tracing method with fluorescent dye has enabled the observation of growing axons in living conditions, and demon strated a lot of developmental aspects in axon growth and guidance (Harris et al., 1987; O'Rourke and Fraser, 1990; Kaethner and Stuermer, 1992; Halloran and Kalil, 1994; Yamamoto et al., 1997). More recently, genetically encoded fluores cent proteins can be used as a powerful tool to observe various biological events. Several gene transfer techniques such as microinjection, biolistic gene gun, viral infection, lipofection and transgenic technology have been developed (Feng et al., 2000; Ehrengruber et al., 2001; O'Brien et al., 2001; Ma et al., 2002; Sahly et al., 2003). In particular, the electroporation technique was proved as a valuable tool, since it can be applied to a wide range of tissues and cell types with little toxicity and can be performed with relative technical easiness. Most methods, including a stand ard electroporation technique, are suitable for gene transfer to a large number of cells. However, this is not ideal for axonal tracing, because observation of individ ual axons is occasionally required. To overcome this problem, we have developed an electroporation method using glass micropipettes containing plasmid solutions and small current injection. Here we introduce the method in detail and exemplified results with some example applications and discuss its usefulness.

  11. Sequences enhancing cassava mosaic disease symptoms occur in the cassava genome and are associated with South African cassava mosaic virus infection.

    PubMed

    Maredza, A T; Allie, F; Plata, G; Rey, M E C

    2016-06-01

    Cassava is an important food security crop in Sub-Saharan Africa. Two episomal begomovirus-associated sequences, named Sequences Enhancing Geminivirus Symptoms (SEGS1 and SEGS2), were identified in field cassava affected by the devastating cassava mosaic disease (CMD). The sequences reportedly exacerbated CMD symptoms in the tolerant cassava landrace TME3, and the model plants Arabidopsis thaliana and Nicotiana benthamiana, when biolistically co-inoculated with African cassava mosaic virus-Cameroon (ACMV-CM) or East African cassava mosaic virus-UG2 (EACMV-UG2). Following the identification of small SEGS fragments in the cassava EST database, the intention of this study was to confirm their presence in the genome, and investigate a possible role for these sequences in CMD. We report that multiple copies of varying lengths of both SEGS1 and SEGS2 are widely distributed in the sequenced cassava genome and are present in several other cassava accessions screened by PCR. The endogenous SEGS1 and SEGS2 are in close proximity or overlapping with cassava genes, suggesting a possible role in regulation of specific biological processes. We confirm the expression of SEGS in planta using EST data and RT-PCR. The sequence features of endogenous SEGS (iSEGS) are unique but resemble non-autonomous transposable elements (TEs) such as MITEs and helitrons. Furthermore, many SEGS-associated genes, some involved in virus-host interactions, are differentially expressed in susceptible (T200) and tolerant TME3) cassava landraces infected by South African cassava mosaic virus (SACMV) of susceptible (T200) and tolerant (TME3) cassava landraces. Abundant SEGS-derived small RNAs were also present in mock-inoculated and SACMV-infected T200 and TME3 leaves. Given the known role of TEs and associated genes in gene regulation and plant immune responses, our observations are consistent with a role of these DNA elements in the host's regulatory response to geminiviruses.

  12. Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas.

    PubMed Central

    Rolland, N; Dorne, A J; Amoroso, G; Sültemeyer, D F; Joyard, J; Rochaix, J D

    1997-01-01

    The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts. PMID:9362486

  13. Characterization of a sugarcane (Saccharum spp.) gene homolog to the brassinosteroid insensitive1-associated receptor kinase 1 that is associated to sugar content.

    PubMed

    Vicentini, Renato; Felix, Juliana de Maria; Dornelas, Marcelo Carnier; Menossi, Marcelo

    2009-03-01

    The present article reports on the characterization of ScBAK1, a leucine-rich repeat receptor-like kinase from sugarcane (Saccharum spp.), expressed predominantly in bundle-sheath cells of the mature leaf and potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. In this report, it was shown that the ScBAK1 sequence was similar to the brassinosteroid insensitive1-associated receptor kinase1 (BAK1). The putative cytoplasmatic domain of ScBAK1 contains all the amino acids characteristic of protein kinases, and the extracellular domain contains five leucine-rich repeats and a putative leucine zipper. Transcripts of ScBAK1 were almost undetectable in sugarcane roots or in any other sink tissue, but accumulated abundantly in the mature leaves. The ScBAK1 expression was higher in the higher sugar content individuals from a population segregating for sugar content throughout the growing season. In situ hybridization in sugarcane leaves showed that the ScBAK1 mRNA accumulated at much higher levels in bundle-sheath cells than in mesophyll cells. In addition, using biolistic bombardment of onion epidermal cells, it was shown that ScBAK1-GFP fusions were localized in the plasma membrane as predicted for a receptor kinase. All together, the present data indicate that ScBAK1 might be a receptor involved in the regulation of specific processes in bundle-sheath cells and in sucrose synthesis in mature sugarcane leaves.

  14. Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes

    PubMed Central

    O'Donnell, John C.; Jackson, Joshua G.

    2016-01-01

    Recently, mitochondria have been localized to astrocytic processes where they shape Ca2+ signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca2+ channel blocker), two inhibitors of reversed Na+/Ca2+ exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca2+ indicator Lck-GCaMP-6S, we observed two types of Ca2+ signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca2+ through reversed Na+/Ca2+ exchange triggers mitochondrial loss and dramatic increases in Ca2+ signaling in astrocytic processes. SIGNIFICANCE STATEMENT Astrocytes, the most abundant cell type in the

  15. Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies.

    PubMed

    Abdollahi, M R; Memari, H Rajabi; van Wijnen, A J

    2011-11-01

    The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24°C, 38°C and 55°C) were investigated. To avoid this problem in microspores, two incubation temperatures (38°C and 55°C) at different periods after GUS assay (4, 24 and 48h) and in the presence of 1mM potassium ferricyanide and 1mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1mM potassium ferricyanide and 1mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55°C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2μM gibberellic acid

  16. Melon chlorotic leaf curl virus: characterization and differential reassortment with closest relatives reveal adaptive virulence in the squash leaf curl virus clade and host shifting by the host-restricted bean calico mosaic virus.

    PubMed

    Idris, A M; Mills-Lujan, K; Martin, K; Brown, J K

    2008-02-01

    The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at approximately 90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the "wild-type" (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.

  17. Molecular characterization and experimental host range of an isolate of Wissadula golden mosaic St. Thomas virus.

    PubMed

    Collins, A M; Mujaddad-ur-Rehman, Malik; Brown, J K; Reddy, C; Wang, A; Fondong, V; Roye, M E

    2009-12-01

    Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field

  18. Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

    PubMed Central

    2010-01-01

    Background Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of

  19. Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

    PubMed Central

    2012-01-01

    Background Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding. Results The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria) or a plant DOD (from Portulaca grandiflora), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis) lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production. Conclusions The fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an

  20. A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease.

    PubMed

    Beyene, Getu; Chauhan, Raj Deepika; Taylor, Nigel J

    2017-03-07

    Cassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12-18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12-22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene. A VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2-4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants. A rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2-4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for

  1. Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

    PubMed Central

    Guan, Dongxu; Armstrong, William E; Foehring, Robert C

    2013-01-01

    The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold ‘bullets’ coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3–7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from –78 to –2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation

  2. Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield.

    PubMed

    Yadav, Dinesh; Shavrukov, Yuri; Bazanova, Natalia; Chirkova, Larissa; Borisjuk, Nikolai; Kovalchuk, Nataliya; Ismagul, Ainur; Parent, Boris; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2015-11-01

    Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1-T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20-30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes.

    PubMed

    O'Donnell, John C; Jackson, Joshua G; Robinson, Michael B

    2016-07-06

    Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes. Astrocytes, the most abundant cell type in the brain

  4. Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

    PubMed Central

    2011-01-01

    Background The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. Results PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. Conclusions The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots. PMID:21745390

  5. Recombination and pseudorecombination driving the evolution of the begomoviruses Tomato severe rugose virus (ToSRV) and Tomato rugose mosaic virus (ToRMV): two recombinant DNA-A components sharing the same DNA-B

    PubMed Central

    2014-01-01

    Background Begomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens since the introduction of a new biotype of the insect vector in the mid-1990’s. Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete sequence of the DNA-B components of ToSRV and ToRMV show an identity of 98.2%. Additionally, the high nucleotide identity (96.2%) between their common regions indicates that these two viruses may share the same DNA-B. Methods Tomato seedlings were biolistically inoculated with ToSRV (DNA-A and DNA-B) and ToRMV (DNA-A and DNA-B) infectious clones in every possible combination of single or mixed infection. Symptom expression was evaluated for up to 35 days post-inoculation (dpi). DNA was extracted at 28 dpi and the presence of each viral genomic component was examined by rolling circle amplification (RCA) followed by digestion, as well as by quantitative, real-time PCR. Sequence comparisons, recombination and phylogenetic analyzes were performed using EMBOSS needle, RDP program and maximum likelihood inference, respectively. Results Symptoms in tomato plants inoculated with the different combinations of ToRMV and ToSRV DNA-A and DNA-B components consisted of a typical mosaic in all combinations. Pseudorecombinants were formed in all possible combinations. When two DNA-A or two DNA-B components were inoculated simultaneously, the ToRMV components were detected preferentially in relation to the ToSRV components. The combination of minor changes in both the Rep protein and the CR may be involved in the preferential replication of ToRMV components. Recombination and phylogenetic analyzes support the exchange of genetic material between ToRMV and ToSRV. Conclusions ToRMV and ToSRV form viable pseudorecombinants in their natural host (Solanum

  6. Developing molecular tools for Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Noor-Mohammadi, Samaneh

    nicotinamide cofactor NAD(P)H plays a pivotal role in many biochemical oxidation and reduction reactions, thus this enzyme would allow regeneration of NAD(P)H in a microalgae strain over-expressing a NAD(P)H-dependent oxidoreductase. A phosphite dehydrogenase gene was introduced into the chloroplast genome (codon optimized) and nuclear genome of C. reinhardtii by biolistic transformation and electroporation in separate events, respectively. Successful expression of the heterologous protein was confirmed by transcript analysis and protein analysis. In conclusion, this new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast or nuclear genome of microalgae, and this should aid current efforts to engineer algae for recombinant protein expression, biofuels production and production of other desirable natural products.

  7. Selective Vulnerability of Specific Retinal Ganglion Cell Types and Synapses after Transient Ocular Hypertension

    PubMed Central

    Jo, Rebecca E.; Ullian, Erik M.; Wong, Rachel O.L.

    2016-01-01

    Key issues concerning ganglion cell type-specific loss and synaptic changes in animal models of experimental glaucoma remain highly debated. Importantly, changes in the structure and function of various RGC types that occur early, within 14 d after acute, transient intraocular pressure elevation, have not been previously assessed. Using biolistic transfection of individual RGCs and multielectrode array recordings to measure light responses in mice, we examined the effects of laser-induced ocular hypertension on the structure and function of a subset of RGCs. Among the α-like RGCs studied, αOFF-transient RGCs exhibited higher rates of cell death, with corresponding reductions in dendritic area, dendritic complexity, and synapse density. Functionally, OFF-transient RGCs displayed decreases in spontaneous activity and receptive field size. In contrast, neither αOFF-sustained nor αON-sustained RGCs displayed decreases in light responses, although they did exhibit a decrease in excitatory postsynaptic sites, suggesting that synapse loss may be one of the earliest signs of degeneration. Interestingly, presynaptic ribbon density decreased to a greater degree in the OFF sublamina of the inner plexiform layer, corroborating the hypothesis that RGCs with dendrites stratifying in the OFF sublamina may be damaged early. Indeed, OFF arbors of ON-OFF RGCs lose complexity more rapidly than ON arbors. Our results reveal type-specific differences in RGC responses to injury with a selective vulnerability of αOFF-transient RGCs, and furthermore, an increased susceptibility of synapses in the OFF sublamina. The selective vulnerability of specific RGC types offers new avenues for the design of more sensitive functional tests and targeted neuroprotection. SIGNIFICANCE STATEMENT Conflicting reports regarding the selective vulnerability of specific retinal ganglion cell (RGC) types in glaucoma exist. We examine, for the first time, the effects of transient intraocular pressure

  8. In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.

    PubMed

    Kim, Jae Yoon; Nong, Guang; Rice, John D; Gallo, Maria; Preston, James F; Altpeter, Fredy

    2017-03-01

    Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity

  9. Accelerated Stem Growth Rates and Improved Fiber Properties of Loblolly Pine: Functional Analysis Of CyclinD from Pinus taeda

    SciTech Connect

    Dr. John Cairney, School of Biology and Institute of Paper Science and Technology @ Georgia Tech, Georgia Institute of Technology; Dr. Gary Peter, University of Florida; Dr. Ulrika Egertsdotter, Dept. of Forestry, Virgina Tech; Dr. Armin Wagner, New Zealand Forest Research Institute Ltd.

    2005-11-30

    divisions in the cambial meristem as expected. We isolated a promoter from a cambial specific gene and commenced development of transformation protocols for loblolly pine. Since our results show that cyclin D expression correlates with increased growth we continued with experiments to demonstrate the effect of cyclin overexpression upon tree growth. Vectors which constitutively express the cyclin D cDNA were constructed and transformed into a transgenic pine system through the collaboration with Forest Research, New Zealand. The transformation system for Pinus radiata is well established and we hoped to gain phenotypic information in a closely related pine, rather than await development of a robust loblolly pine transformation method. Transformation experiments were conducted by a biolistic method developed at Forest Research, NZ. A total of 78 transgenic embryogenic lines were generated and bulked up with a good representation of transgenic lines per construct. Transformed calli were originally identified by resistance to the antibiotic Geneticin contained in the medium. The transgenic nature of the selected lines was subsequently confirmed using histochemical GUS staining. To date, 10 out of 13 selected transgenic lines have produced embryos and we are currently harvesting the first transgenic plantlets. At present time 22 of those plantlets have been moved to GMO facilities. We will soon develop a strategy for assessing potential phenotypic differences between the transclones and non-transformed controls. Transgenic plants are being grown to a stage (approx. 1 year) when meaningful phenotypic evaluation can be conducted. The recent availability of 10,000 element loblolly pine cDNA microarray will permit the evaluation of cyclinD overexpression upon gene expression in transgenic Pinus.