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Sample records for biolistics

  1. Barley Transformation Using Biolistic Techniques

    NASA Astrophysics Data System (ADS)

    Harwood, Wendy A.; Smedley, Mark A.

    Microprojectile bombardment or biolistic techniques have been widely used for cereal transformation. These methods rely on the acceleration of gold particles, coated with plasmid DNA, into plant cells as a method of directly introducing the DNA. The first report of the generation of fertile, transgenic barley plants used biolistic techniques. However, more recently Agrobacterium-mediated transformation has been adopted as the method of choice for most cereals including barley. Biolistic procedures are still important for some barley transformation applications and also provide transient test systems for the rapid checking of constructs. This chapter describes methods for the transformation of barley using biolistic procedures and also highlights the use of the technology in transient assays.

  2. 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.

  3. Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells.

    PubMed

    Smith, F D; Harpending, P R; Sanford, J C

    1992-01-01

    Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 10(4) per plate per 0.8 micrograms DNA. Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.

  4. Biolistic transformation of Scoparia dulcis L.

    PubMed

    Srinivas, Kota; Muralikrishna, Narra; Kumar, Kalva Bharath; Raghu, Ellendula; Mahender, Aileni; Kiranmayee, Kasula; Yashodahara, Velivela; Sadanandam, Abbagani

    2016-01-01

    Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant. PMID:27186019

  5. 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

  6. 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.

  7. Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

    PubMed

    Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

    2012-01-01

    After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

  8. Biolistic transformation of wheat with centrophenoxine as a synthetic auxin.

    PubMed

    Ismagul, Ainur; Iskakova, Gulnur; Harris, John C; Eliby, Serik

    2014-01-01

    Cereal crops, including bread wheat (Triticum aestivum L.), are an important staple food worldwide. With a growing global population, it is evident that current crop production will not meet the rising demands being placed on modern agriculture. Efforts to improve crop yield and stress-tolerance by traditional breeding are labor intensive, time consuming, and highly dependent upon the ability to capture existing and novel genetic variation from a restricted genetic pool. Genetic engineering of crop species is one of several alternatives to traditional breeding for the introduction of novel genetic variation. This recently established technology has proved useful for the introduction of novel traits like pest resistance and herbicide tolerance. As a universal tool for genetic transformation, the Biolistic Gene Gun allows for the genomic integration of novel gene sequences from various sources into a whole host of living organisms.In this chapter, we present a novel and detailed protocol for the Biolistic Transformation of bread wheat that uses the pharmaceutical compound, Centrophenoxine (CPX). The application of CPX as the main auxin-like plant growth regulator in cereal genetic transformation replaces the potent but more toxic herbicide 2,4-D.

  9. 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. PMID:25092532

  10. 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.

  11. Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

    PubMed Central

    Woods, Georgia; Zito, Karen

    2008-01-01

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices. PMID:19066564

  12. Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.

    PubMed

    Woods, Georgia; Zito, Karen

    2008-02-13

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  14. 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

  15. 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.

  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. PMID:24599866

  17. Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response.

    PubMed

    Mitrasinovic, Olivera M; Robinson, Christopher C; Tenen, Daniel G; Lee, Yuen Ling; Poon, Clara; Murphy, Greer M

    2004-08-01

    The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.

  18. 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

    2012-02-01

    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. PMID:22174078

  19. Method for Biolistic Site-Specific Integration in Plants Catalyzed by Bxb1 Integrase.

    PubMed

    Li, Ruyu; Han, Zhiguo; Hou, Lili; Kaur, Gurminder; Yin, Qian; Ow, David W

    2016-01-01

    Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al., Mol Plant 7:1756-1765, 2014). Being able to stack DNA to a previous location ensures that the number of genetic loci does not increase with each new round of transgene addition. Whereas the previous demonstration was conducted through polyethylene glycol to mediate uptake of DNA into tobacco protoplasts, we now describe protocols for using biolistic transformation to stack DNA in tobacco and rice. PMID:27557683

  20. Transient Gene Expression in Epidermal Cells of Plant Leaves by Biolistic DNA Delivery

    PubMed Central

    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. PMID:23104330

  1. High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants.

    PubMed

    Rech, Elibio L; Vianna, Giovanni R; Aragão, Francisco J L

    2008-01-01

    This protocol describes a method for high-frequency recovery of transgenic soybean, bean and cotton plants, by combining resistance to the herbicide imazapyr as a selectable marker, multiple shoot induction from embryonic axes of mature seeds and biolistics techniques. This protocol involves the following stages: plasmid design, preparation of soybean, common bean and cotton apical meristems for bombardment, microparticle-coated DNA bombardment of apical meristems and in vitro culture and selection of transgenic plants. The average frequencies (the total number of fertile transgenic plants divided by the total number of bombarded embryonic axes) of producing germline transgenic soybean and bean and cotton plants using this protocol are 9, 2.7 and 0.55%, respectively. This protocol is suitable for studies of gene function as well as the production of transgenic cultivars carrying different traits for breeding programs. This protocol can be completed in 7-10 months.

  2. Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase.

    PubMed

    Aswath, Chenna Reddy; Mo, Sung Youn; Kim, Doo Hwan; Park, S Won

    2006-03-01

    A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. PMID:16211408

  3. 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.

  4. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

    PubMed

    Joyce, Priya; Hermann, Scott; O'Connell, Anthony; Dinh, Quang; Shumbe, Leonard; Lakshmanan, Prakash

    2014-05-01

    Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.

  5. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

    PubMed

    Joyce, Priya; Hermann, Scott; O'Connell, Anthony; Dinh, Quang; Shumbe, Leonard; Lakshmanan, Prakash

    2014-05-01

    Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques. PMID:24330327

  6. Performance analysis of a new biolistic gun using high power laser irradiation

    NASA Astrophysics Data System (ADS)

    Han, Tae-Hee; Lee, Hyunhee; Choi, Soojin; Gojani, Ardian B.; Yoh, Jack J.

    2010-11-01

    Impingement of a high power laser pulse (above 109 W/cm2) on a metal foil causes ablation, which is characterized by a rapid expulsion of matter and initiation of a strong shock wave inside the solid metal. The shock propagates through the foil and reverberates on the rear side causing instant deformation of the foil, whose surface is treated with micro particles prior to ablation. Based on this principle of micro particle ejection, we develop a new biolistic gun with improved controllability, stability, efficiency of our previous system, and perform characterization of the penetration shapes at varying confinements and energy levels. The confinement media include BK7 glass, water, and succulent jelly (ultrasound gel). Biological tissue was replicated by a gelatin-water solution at a 3% weight ratio. Present data show that confinement effect results in a conspicuous enhancement of penetration reached by 5 μm cobalt micro particles. Also, there exists an optimal thickness at each energy level when using liquid confinement for enhanced particle delivery.

  7. 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

  8. A helium burst biolistic device adapted to penetrate fragile insect tissues

    PubMed Central

    Thomas, Jean-Luc; Bardou, Jérôme; L'hoste, Sebastien; Mauchamp, Bernard; Chavancy, Gérard

    2001-01-01

    To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolistic® device and the deleterious blast effect, design modifications have been made to the launching module. These modifications were evaluated on Bombyx mori embryos and fragile tissues, such as oocytes and imaginal wing disks. The original floppy macrocarrier was replaced by a rigid macrocarrier to avoid the effects of the helium blast. The efficiency of the gene gun bombardment was reinforced by the addition of a focusing nozzle. The reduced blast effect allowed us to carry out high-pressure shootings to small organs with improved penetration. This system allowed potentially all the internal embryonic tissues to be transfected with optimal survival rates. The new module was effective on tissues that are difficult to transfect, such as the epithelial wing disk that is covered by a peripodial membrane, and the ovarian follicle cells that lie under the ovariole cell membrane. The new macrocarrier allowed both an aqueous delivery of particles and an ethanolic dry delivery. No significant differences were noted between these two modes of delivery. The major improvement is the possibility of high pressure shooting correlated with appreciable penetration and a weak blast effect. PMID:15455069

  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. PMID:26090875

  11. The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease.

    PubMed

    Ariyo, O A; Atiri, G I; Dixon, A G O; Winter, S

    2006-10-01

    Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.

  12. Development of a rapid biolistic assay to determine changes in relative levels of intracellular calcium in leaves following tetracycline uptake by pinto bean plants.

    PubMed

    Farkas, Michael H; Mojica, Elmer-Rico E; Patel, Minesh; Aga, Diana S; Berry, James O

    2009-08-01

    Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.

  13. 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).

  14. 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.

  15. 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. PMID:22351007

  16. Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei.

    PubMed

    Christiansen, S K; Knudsen, S; Giese, H

    1995-12-01

    Particle gun acceleration appears to be a possible way to transform mycelium cells of obligate plant parasites growing on host surfaces. GUS expression was obtained in E. graminis f.sp. hordei cells after bombardment with the GUS gene under the control of the E. graminis f.sp. hordei β-tubulin promoter. Three heterologous promoters, onefrom Aspergillus nidulans and two from Cochliobolus heterostrophus, gave very low or no expression of GUS.

  17. Biolistic-mediated gene expression in guinea pigs and cattle tissue in vivo.

    PubMed

    Rech, E L; De-Bem, A R; Aragão, F J

    1996-10-01

    Foreign genes were introduced and expressed in vivo in guinea pigs and cattle utilizing a new hand-held device based on high-pressure helium gas to accelerate DNA-coated microparticles. Guinea pigs were used to evaluate the physical parameters to introduce and express the exogenous DNA. The best conditions were applied to conduct bombardments in cattle. The results showed a high frequency of gene expression in all the bombarded cattle. This procedure could be used to study the immune responses in cattle and in a wide variety of animals through genetic immunization. PMID:9181095

  18. 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.

  19. 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.

  20. High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.

    PubMed

    Remacle, Claire; Cardol, Pierre; Coosemans, Nadine; Gaisne, Mauricette; Bonnefoy, Nathalie

    2006-03-21

    Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I. PMID:16537419

  1. 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.

  2. Using a Handheld Gene Gun for Genetic Transformation of Tetrahymena thermophila.

    PubMed

    Gotesman, Michael; Williams, Selwyn A

    2016-01-01

    This chapter describes protocols for using a handheld gene gun to deliver transformation vectors for overexpression of genes or gene replacement in the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

    PubMed

    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  6. Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos

    NASA Astrophysics Data System (ADS)

    Huixia, Wu; Angela, Doherty; Jones, Huw D.

    Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

  7. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    PubMed

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  8. Selection of Transformed Plants

    NASA Astrophysics Data System (ADS)

    Jones, Huw D.; Sparks, Caroline A.

    The low frequency and randomness of transgene integration into host cells, combined with the significant challenges of recovering whole plants from those rare events, makes the use of selectable marker genes routine in plant transformation experiments. For research applications that are unlikely to be grown in the field, strong herbicide- or antibiotic resistance is commonly used. Here we use genes conferring resistance to glufosinate herbicides as an example of a selectable marker in wheat transformation by either Agrobacterium or biolistics.

  9. 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

  10. Assaying effector function in planta using double-barreled particle bombardment.

    PubMed

    Kale, Shiv D; Tyler, Brett M

    2011-01-01

    The biolistic transient gene expression assay is a beneficial tool for studying gene function in vivo. However, biolistic transient assay systems have inherent pitfalls that often cause experimental inaccuracies such as poor transformation efficiency, which can be confused with biological phenomena. The double-barreled gene gun device is an inexpensive and highly effective attachment that enables statistically significant data to be obtained with one-tenth the number of experimental replicates compared to conventional biolistic assays. The principle behind the attachment is to perform two simultaneous bombardments with control and test DNA preparations onto the same leaf. The control bombardment measures the efficiency of the transformation while the ratio of the test bombardment to the control bombardment measures the activity of the gene of interest. With care, the ratio between the pair of bombardments can be highly reproducible from bombardment to bombardment. The double-barreled attachment has been used to study plant resistance (R) gene-mediated responses to effectors, induction and suppression of cell death by a wide variety of pathogen and host molecules, and the role of oömycete effector RXLR motifs in cell reentry.

  11. "Agrolistic" transformation of plant cells: integration of T-strands generated in planta.

    PubMed

    Hansen, G; Chilton, M D

    1996-12-10

    We describe a novel plant transformation technique, termed "agrolistic," that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as "agrolistic" inserts, as distinguished from "biolistic" inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts. PMID:8962167

  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. PMID:21626148

  13. Genetic transformation of the model green alga Chlamydomonas reinhardtii.

    PubMed

    Neupert, Juliane; Shao, Ning; Lu, Yinghong; Bock, Ralph

    2012-01-01

    Over the past three decades, the single-celled green alga Chlamydomonas reinhardtii has become an invaluable model organism in plant biology and an attractive production host in biotechnology. The genetic transformation of Chlamydomonas is relatively simple and efficient, but achieving high expression levels of foreign genes has remained challenging. Here, we provide working protocols for algal cultivation and transformation as well as for selection and analysis of transgenic algal clones. We focus on two commonly used transformation methods for Chlamydomonas: glass bead-assisted transformation and particle gun-mediated (biolistic) transformation. In addition, we describe available tools for promoting efficient transgene expression and highlight important considerations for designing transformation vectors.

  14. 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.

  15. Chloroplast-Based Expression of Recombinant Proteins by Gateway® Cloning Technology.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas

    2016-01-01

    Plastid transformation for the expression of recombinant proteins and entire enzymatic pathways has become a promising tool for plant biotechnology in the past decade. Several improvements of the technology have turned plant plastids into robust and dependable expression platforms for multiple high value compounds. In this chapter, we describe our current methodology based on Gateway(®) recombinant cloning, which we have adapted for plastid transformation. We describe the steps required for cloning, biolistic transformation, identification, and regeneration of transplastomic plant lines and Western blot analysis. PMID:26614278

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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. PMID:26634291

  2. Transgenic oil palm: production and projection.

    PubMed

    Parveez, G K; Masri, M M; Zainal, A; Majid, N A; Yunus, A M; Fadilah, H H; Rasid, O; Cheah, S C

    2000-12-01

    Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.

  3. [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.

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

    PubMed Central

    Schwartz, Christine; Gao, Huirong; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    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. PMID:26269544

  5. The complete nucleotide sequence of a new bipartite begomovirus from Brazil infecting Abutilon.

    PubMed

    Paprotka, T; Metzler, V; Jeske, H

    2010-05-01

    The complete nucleotide sequence of Abutilon mosaic Brazil virus (AbMBV), a new bipartite begomovirus from Bahia, Brazil, is described and analyzed phylogenetically. Its DNA A is most closely related to those of Sida-infecting begomoviruses from Brazil and forms a phylogenetic cluster with pepper- and Euphorbia-infecting begomoviruses from Central America. The DNA B component forms a cluster with different Sida- and okra-infecting begomoviruses from Brazil. Both components are distinct from those of the classical Abutilon mosaic virus originating from the West Indies. AbMBV is transmissible to Nicotiana benthamiana and Malva parviflora by biolistics of rolling-circle amplification products and induces characteristic mosaic and vein-clearing symptoms in M. parviflora.

  6. Stable chloroplast transformation of the unicellular red alga Porphyridium species.

    PubMed

    Lapidot, Miri; Raveh, Dina; Sivan, Alex; Arad, Shoshana Malis; Shapira, Michal

    2002-05-01

    Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalga Porphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.

  7. 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.

  8. Transformation of the developing barley endosperm by particle bombardment.

    PubMed

    Knudsen, S; Müller, M

    1991-10-01

    Delivery of DNA into intact cells of the developing barley (Hordeum vulgare L.) endosperm was performed with the BIOLISTIC particle gun. It is shown that the proximal 532 base pairs (bp) of the upstream region of a B1-hordein gene drive the expression of the β-glucuronidase (GUS) gene (uidA) in sub-aleurone and starchy-endosperm cells but not in cells devoid of starch, i.e. developing aleurone cells. The 35S promoter from cauliflower mosaic virus was active in all three cell types. This cell-specific activity of the hordein promoter was verified by a detailed histological study of the regions of the extruded endosperms expressing the uidA gene. The analysis included a histological study of the developing endosperm as a base for classifying the different cell types in the developing endosperm.

  9. 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. PMID:23812656

  10. 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. PMID:22351008

  11. 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.

  12. 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. PMID:24599867

  13. Recent advances in plant transformation.

    PubMed

    Barampuram, Shyamkumar; Zhang, Zhanyuan J

    2011-01-01

    Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops. Over the last decade, significant progress has been made in the development of new and efficient transformation methods in plants. Despite a variety of available DNA delivery methods, Agrobacterium- and biolistic-mediated transformation remain the two predominantly employed approaches. In particular, progress in Agrobacterium-mediated transformation of cereals and other recalcitrant dicot species has been quite remarkable. In the meantime, other transgenic-enabling technologies have emerged, including generation of marker-free transgenics, gene targeting, and chromosomal engineering. Although transformation of some plant species or elite germplasm remains a challenge, further advancement in transformation technology is expected because the mechanisms of governing the regeneration and transformation processes are now better understood and are being creatively applied to designing improved transformation methods or to developing new enabling technologies. PMID:21181522

  14. 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.

  15. Genetic transformation: a tool to study protein targeting in diatoms.

    PubMed

    Kroth, Peter G

    2007-01-01

    Diatoms are unicellular photoautotrophic eukaryotes that play an important role in ecology by fixing large amounts of CO2 in the oceans. Because they evolved by secondary endocytobiosis-- a process of uptake of a eukaryotic alga into another eukaryotic cell--they have a rather unusual cell biology and genetic constitution. Because the preparation of organelles is rather difficult as a result of the cytosolic structures, genetic transformation and expression of preproteins fused to green fluorescent protein (GFP) became one of the major tools to analyze subcellular localization of proteins in diatoms. Meanwhile several groups successfully attempted to develop genetic transformation protocols for diatoms. These methods are based on "biolistic" DNA delivery via a particle gun and allow the introduction and expression of foreign genes in the algae. Here a protocol for the genetic transformation of the diatom Phaeodactylum tricornutum is described as well as the subsequent characterization of the transformants. PMID:17951693

  16. 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. PMID:27643854

  17. 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.

  18. 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

  19. 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.

  20. Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun

    PubMed Central

    2013-01-01

    Background Organotypic brain slices (OTBS) are an excellent experimental compromise between the facility of working with cell cultures and the biological relevance of using animal models where anatomical, morphological, and cellular function of specific brain regions can be maintained. The biological characteristics of OTBS can subsequently be examined under well-defined conditions. They do, however, have a number of limitations; most brain slices are derived from neonatal animals, as it is difficult to properly prepare and maintain adult OTBS. There are ample problems with tissue integrity as OTBS are delicate and frequently become damaged during the preparative stages. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells, and cells imbedded within tissues, have been consistently difficult to achieve. Results Following the ex vivo extraction of adult mouse brains, mounted inside a medium-agarose matrix, we have exploited a precise slicing procedure using a custom built vibroslicer. To transfect these slices we used an improved biolistic transfection method using a custom made low-pressure barrel and novel DNA-coated nanoparticles (40 nm), which are drastically smaller than traditional microparticles. These nanoparticles also minimize tissue damage as seen by a significant reduction in lactate dehydrogenase activity as well as propidium iodide (PI) and dUTP labelling compared to larger traditional gold particles used on these OTBS. Furthermore, following EYFP exogene delivery by gene gun, the 40 nm treated OTBS displayed a significantly larger number of viable NeuN and EYFP positive cells. These OTBS expressed the exogenous proteins for many weeks. Conclusions Our described methodology of producing OTBS, which results in better reproducibility with less tissue damage, permits the exploitation of mature fully formed adult brains for advanced neurobiological studies. The novel 40 nm particles are ideal for the viable

  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. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    PubMed

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

  3. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

    PubMed

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

    2004-04-01

    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 degrees 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 degrees 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

  4. Localized RNAi and ectopic gene expression in the medicinal leech.

    PubMed

    Shefi, Orit; Simonnet, Claire; Groisman, Alex; Macagno, Eduardo R

    2008-04-17

    In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls. The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of approximately 50-150 microm and extends over approximately 15 microm around the mean penetration depth, which is adjustable between 0 and 50 microm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches. For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the

  5. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    PubMed

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture. PMID:27259918

  6. Dendritic cell delivery of plasmid DNA. Applications for controlled genetic immunization.

    PubMed

    Mumper, R J; Ledebur, H C

    2001-09-01

    Positive human clinical data using biolistic-mediated gene transfer (i.e., gene gun) to administer a nucleic acid-based Hepatitis B vaccine has validated genetic immunization as an effective clinical vaccine modality. Although the precise mechanism of action has yet to be determined, preclinical studies using jet injection have indicated that direct targeting of resident antigen presenting cells (Langerhan's cells) in the skin as the primary immunological driving force for the potent and long-lived immune response. Moreover, positive results with topical delivery of genetic vaccines and ex vivo loading of dendritic cells with antigen has strengthened the movement toward directly targeting antigen presenting cells as a means to amplify, control, and mediate the immunological consequences of prophylactic and/or therapeutic genetic vaccines. Despite these encouraging results with the gene gun, it is unclear whether this technology will translate into commercially available vaccines due to potential product development barriers such as cost and convenience. It is clear that safety concerns in using genetic approaches to treat and prevent disease have highlighted the need for strict product requirements for genetic vaccines. A plausible strategy to meet these requirements is to combine controlled plasmid delivery systems with tissue-specific gene expression systems.

  7. Expression of Nav1.8 sodium channels perturbs the firing patterns of cerebellar Purkinje cells.

    PubMed

    Renganathan, M; Gelderblom, M; Black, J A; Waxman, S G

    2003-01-10

    The sensory neuron specific sodium channel Na(v)1.8/SNS exhibits depolarized voltage-dependence of inactivation, slow inactivation and rapid repriming, which differentiate it from other voltage-gated sodium channels. Na(v)1.8 is normally selectively expressed at high levels in sensory ganglion neurons, but not within the CNS. However, expression of Na(v)1.8 mRNA and protein are upregulated within cerebellar Purkinje cells in animal models of multiple sclerosis (MS), and in human MS. To examine the effect of expression of Na(v)1.8 on the activity pattern of Purkinje cells, we biolistically introduced Na(v)1.8 cDNA into these cells in vitro. We report here that Na(v)1.8 can be functionally expressed at physiological levels (similar to the levels in DRG neurons where Na(v)1.8 is normally expressed) within Purkinje cells, and that its expression alters the activity of these neurons in three ways: first, by increasing the amplitude and duration of action potentials; second, by decreasing the proportion of action potentials that are conglomerate and the number of spikes per conglomerate action potential; and third, by contributing to the production of sustained, pacemaker-like impulse trains in response to depolarization. These results provide support for the hypothesis that the expression of Na(v)1.8 channels within Purkinje cells, which occurs in MS, may perturb their function. PMID:12493611

  8. In vivo dissection of cis-acting determinants for plastid RNA editing.

    PubMed

    Bock, R; Hermann, M; Kössel, H

    1996-09-16

    Substitutional RNA editing changes single C nucleotides in higher plant chloroplast transcripts into U residues. To determine the cis-acting sequence elements involved in plastid RNA editing, we constructed a series of chloroplast transformation vectors harboring selected editing sites of the tobacco ndhB transcript in a chimeric context. The constructs were inserted into the tobacco plastid genome by biolistic transformation leading to the production of stable chimeric RNAs. Analysis of RNA editing revealed unexpected differences in the size of the essential cis elements or in their distance from the editing site. Flanking sequences of identical size direct virtually complete editing for one pair of editing sites, partial editing for a second and no editing at all for a third pair of sites. Serial 5' and 3' deletions allowed us to define the cis-acting elements more precisely and to identify a sequence element essential for editing site recognition. In addition, a single nucleotide substitution immediately upstream of an editing position was introduced. This mutation was found drastically and selectively to reduce the editing efficiency of the downstream editing site, demonstrating that position -1 is important for either site recognition or catalysis. Our results indicate that the editing of adjacent sites is likely to be mechanistically coupled. In no case did the presence in the plastome of the additional editing sites have any effect on the editing efficiency of the endogenous ndhB sites, indicating that the availability of site-specific trans-acting factors is not rate limiting.

  9. 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.

  10. 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.

  11. Characterization of Rhynchosia yellow mosaic Yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico.

    PubMed

    Hernández-Zepeda, C; Brown, J K; Moreno-Valenzuela, O A; Argüello-Astorga, G; Idris, A M; Carnevali, G; Rivera-Bustamante, R F

    2010-10-01

    Rhynchosia minima (L.) DC. (Fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, México [1]. To characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. Sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic R. minima, and the name Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) is proposed. Pairwise comparisons indicated that RhYMYuV DNA-A [2,597 nt, (EU021216)] and DNA-B [2,542 nt, (FJ792608)] components shared the highest nt sequence identity with Cabbage leaf curl virus (CaLCuV), 87% for component A and 71% for component B. Phylogenetic analysis indicated that both components of RhYMYuV are most closely related to other New World begomoviruses, having as closest relatives immediate outliers to the major Squash leaf curl virus (SLCV) clade. Recombination analysis of the RhYMYuV genome indicated that the DNA-A component has arisen through intermolecular recombination. R. minima plants inoculated with the monomeric clones developed a bright yellow mosaic similar to symptoms observed in naturally infected plants, confirming that the clones were infectious. Nicotiana benthamiana plants biolistically inoculated with monomeric clones developed curling and chlorosis in the newly emerging leaves. RhYMYuV was also detected in symptomatic Desmodium sect. Scorpiurus Benth. (Fabaceae) that were collected near the RhYMYuV-infected plants.

  12. 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

  13. Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

    PubMed Central

    Staub, J M; Maliga, P

    1992-01-01

    We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector. PMID:1356049

  14. 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). PMID:17273915

  15. 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. PMID:18551377

  16. Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects.

    PubMed

    Manimaran, P; Ramkumar, G; Sakthivel, K; Sundaram, R M; Madhav, M S; Balachandran, S M

    2011-01-01

    Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program. PMID:21672619

  17. [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.

  18. A simple method for chloroplast transformation in Chlamydomonas reinhardtii.

    PubMed

    Ramesh, Vellupillai M; Bingham, Scott E; Webber, Andrew N

    2011-01-01

    Photosystem I (PSI) is a multisubunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photosynthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of Photosystem I. C. reinhardtii is also a particularly favorable organism for chloroplast transformation because it contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This article describes the procedures for chloroplast transformation.

  19. [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.

  20. Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome.

    PubMed

    Jinkerson, Robert E; Jonikas, Martin C

    2015-05-01

    The success of the green alga Chlamydomonas reinhardtii as a model organism is to a large extent due to the wide range of molecular techniques that are available for its characterization. Here, we review some of the techniques currently used to modify and interrogate the C. reinhardtii nuclear genome and explore several technologies under development. Nuclear mutants can be generated with ultraviolet (UV) light and chemical mutagens, or by insertional mutagenesis. Nuclear transformation methods include biolistic delivery, agitation with glass beads, and electroporation. Transforming DNA integrates into the genome at random sites, and multiple strategies exist for mapping insertion sites. A limited number of studies have demonstrated targeted modification of the nuclear genome by approaches such as zinc-finger nucleases and homologous recombination. RNA interference is widely used to knock down expression levels of nuclear genes. A wide assortment of transgenes has been successfully expressed in the Chlamydomonas nuclear genome, including transformation markers, fluorescent proteins, reporter genes, epitope tagged proteins, and even therapeutic proteins. Optimized expression constructs and strains help transgene expression. Emerging technologies such as the CRISPR/Cas9 system, high-throughput mutant identification, and a whole-genome knockout library are being developed for this organism. We discuss how these advances will propel future investigations.

  1. Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii.

    PubMed

    Hu, Zhangli; Fan, Zhun; Zhao, Zhonglin; Chen, Jun; Li, Jiancheng

    2012-01-01

    The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii. PMID:22530046

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

    PubMed Central

    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. PMID:24322586

  3. 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.

  4. 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.

  5. Overexpression of Arabidopsis and rice stress genes' inducible transcription factor confers drought and salinity tolerance to rice.

    PubMed

    Datta, Karabi; Baisakh, Niranjan; Ganguly, Moumita; Krishnan, Sellapan; Yamaguchi Shinozaki, Kazuko; Datta, Swapan K

    2012-06-01

    Rice yield is greatly affected by environmental stresses such as drought and salinity. In response to the challenge of producing rice plants tolerant to these stresses, we introduced cDNA encoding the transcription factors DREB1A and DREB1B under the control of the stress inducible rd29 promoter. Two different indica rice cultivars were used, BR29, an improved commercially cultivated variety from Bangladesh and IR68899B, an IRRI bred maintainer line for hybrid rice. Agrobacterium mediated transformation of BR29 was done independently with DREB1A isolated from rice and Arabidopsis and DREB1B isolated from rice, whereas biolistic transformation was done with rice- DREB1B in the case of IR68899B. Initial genetic integration was confirmed by PCR and Southern blot analysis. Salinity tolerance was assayed in very young seedlings. Drought stress tests were found to be more reliable when they were carried out at the pre-flowering booting stage. RNA gel blot analysis as well as quantitative PCR analysis was performed to estimate the transcription level under stressed and unstressed conditions. Agronomic performance studies were done with stressed and unstressed plants to compare the yield losses due to dehydration and salt loading stresses. Noticeably enhanced tolerance to dehydration was observed in the plants transformed with DREB1A isolated from Arabidopsis while DREB1B was found to be more effective for salt tolerance.

  6. 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. PMID:27446112

  7. 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. PMID:27480687

  8. 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

  9. Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans.

    PubMed

    Sloan, Megan A; Reaves, Barbara J; Maclean, Mary J; Storey, Bob E; Wolstenholme, Adrian J

    2015-11-01

    The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing.

  10. 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.

  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. 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. PMID:27306679

  13. [Obtainment of transgenic wheat with the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) gene and analysis of resistance to aphid].

    PubMed

    Liang, Hui; Zhu, Yin-Feng; Zhu, Zhen; Sun, Dong-Fa; Jia, Xu

    2004-02-01

    Snowdrop lectin (Galanthus nivalis agglutinin; GNA) is toxic to sap sucking injurious insects of Homopteran. A new gna gene has been transferred into common spring wheat Zhong60634 and winter wheat Yumai66 with high yield by using the biolistic transformation method. Transgenic wheat plants have been obtained in both of the two varieties. Two transgenic plants (T0) have been obtained from the bombarded 535 immature embryos of Zhong60634. Bioassay results show that the development of aphid could be slowed down and the survival rate of young aphid could be reduced by gna gene. Seventeen transgenic plants (T0) were obtained from the bombarded 4636 immature embryos of Yumai66. Twenty plantlets with good resistance to Rhopalosiphum padi and Macrosiphum avenae, which are mainly aphid in north wheat area, were identified from the transgenic plants of T1 generation that came from 8 T0 transgenic plants with good resistance to aphid. The anti-aphid bioassay shows that resistance to the different grain aphid is not the same in transgenic wheat plants. To Rhopalosiphum padi, the rate of survival aphid 8 days after exposing transgenic plants to aphids is significantly lower than that of nontransgenic plants. To Macrosiphum avenae, growth speed of aphids is slowed down but not killed. At the same time, the death rate of young aphids is increased. Anyway, feeding of the two kinds of aphids has been controlled in a certain degree by gna gene when aphids can free to move in plants.

  14. A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane1[W][OPEN

    PubMed Central

    Chevalier, Adrien S.; Bienert, Gerd Patrick; Chaumont, François

    2014-01-01

    Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER. PMID:24989232

  15. A new LxxxA motif in the transmembrane Helix3 of maize aquaporins belonging to the plasma membrane intrinsic protein PIP2 group is required for their trafficking to the plasma membrane.

    PubMed

    Chevalier, Adrien S; Bienert, Gerd Patrick; Chaumont, François

    2014-09-01

    Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER.

  16. Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration

    PubMed Central

    Engels, Cibelle; Fuganti-Pagliarini, Renata; Marin, Silvana Regina Rockenbach; Marcelino-Guimarães, Francismar Corrêa; Oliveira, Maria Cristina Neves; Kanamori, Norihito; Mizoi, Junya; Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Nepomuceno, Alexandre Lima

    2013-01-01

    The loss of soybean yield to Brazilian producers because of a water deficit in the 2011–2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2–17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress. PMID:24385860

  17. 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.

  18. 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.

  19. 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

  20. Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development.

    PubMed

    Liu, Hui; Hedley, Peter; Cardle, Linda; Wright, Kathryn M; Hein, Ingo; Marshall, David; Waugh, Robbie

    2005-06-01

    Two full-length cDNA sequences homologous to caleosin, a seed-storage oil-body protein from sesame, were identified from a series of barley grain development cDNA libraries and further characterised. The cDNAs, subsequently termed HvClo1 and HvClo2, encode proteins of 34 kDa and 28 kDa, respectively. Real-time RT-PCR indicated that HvClo1 is expressed abundantly during the later stages of embryogenesis and is seed-specific, accumulating in the scutellum of mature embryos. HvClo2 is expressed mainly in the endosperm tissues of the developing grain. We show that HvClo1 and HvClo2 are paralogs that co-segregate on barley chromosome 2HL. Transient expression of HvClo1 in lipid storage and non-storage cells of barley using biolistic particle bombardment indicates that caleosins have different subcellular locations from the structural oil-body protein oleosin, and by inference participate in different sorting pathways. We observe that caleosin sorts via small vesicles, suggesting a likely association with lipid trafficking, membrane expansion and oil-body biogenesis. PMID:15702354

  1. 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.

  2. Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

    PubMed Central

    Foehring, Robert C.; Guan, Dongxu; Toleman, Tara; Cantrell, Angela R.

    2011-01-01

    We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice. PMID:21673642

  3. Use of Tomato yellow leaf curl virus (TYLCV) Rep Gene Sequences to Engineer TYLCV Resistance in Tomato.

    PubMed

    Yang, Y; Sherwood, T A; Patte, C P; Hiebert, E; Polston, J E

    2004-05-01

    ABSTRACT Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus (family Geminiviridae), causes severe losses in tomato production in the tropics and subtropics. In order to generate engineered resistance, eight different constructs of the TYLCV replication-associated protein (Rep) and C4 gene sequences were tested in transformed tomato inbred lines. Transgenic plants were screened for resistance to TYLCV using viruliferous whiteflies. No symptoms were observed and no TYLCV genomic DNA was detected by both hybridization and polymerase chain reaction in progenies of plants transformed with three constructs. This resistance was observed in plants that contained one of the following transgenes: 2/5Rep (81 nucleotides [nt] of the intergenic region [IR] plus 426 nt of the 5' end of the TYLCV Rep gene), Delta2/5Rep (85 nt of the IR plus 595 nt of the 5' end of the TYLCV Rep gene in the antisense orientation), and RepDelta2/5Rep (81 nt of the IR, the entire Rep gene, and 41 nt 3' to the end of the Rep gene fused to Delta2/5Rep). Our study differs from other transgenic Geminivirus resistance reports involving the Rep gene in that viruliferous whiteflies were used for challenge inoculation instead of agroinoculation or biolistic inoculation, and TYLCV resistance was evaluated under field conditions. PMID:18943768

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

    PubMed

    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

  5. 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.

  6. 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

  7. 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. PMID:23821951

  8. 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.

  9. Development of an edible rabies vaccine in maize using the Vnukovo strain.

    PubMed

    Loza-Rubio, E; Rojas, E; Gómez, L; Olivera, M T J; Gómez-Lim, M A

    2008-01-01

    The objective of this study was to obtain transgenic maize expressing the rabies virus glycoprotein (G) of the Vnukovo strain and to evaluate its immunogenicity in mice, by the oral route. The ubiquitin maize promoter fused to the whole coding region of the rabies virus G gene, and a constitutive promoter from cauliflowermosaic virus (CaMV)were used. Maize embryogenic callus were transformed with the above construct by biolistics. Regenerated maize plants were recovered and grown in a greenhouse. The presence of the G gene and its product was detected by PCR and western blot, respectively. The amount of G protein detected in the grains was approximately 1% of the total soluble plant protein. Transformed kernels containing 50 microg of G protein were given once by the oral route in adult mice (BALB-C strain). Challenge was undertaken at 90-days post-vaccination using a lethal dose of a vampire bat rabies virus (100 LD 50% in mice); vampire bats are one of the main reservoirs in Latin America. The edible vaccine induced viral neutralizing antibodies (VNA) which, protected mice 100% against challenge. The control group did not survive. The G protein of the Vnukovo strain expressed in transgenic maize may be considered as an oral immunogen against rabies, conferring cross-protection. PMID:18634510

  10. Cell-free construction of disarmed Abutilon mosaic virus-based gene silencing vectors.

    PubMed

    Krenz, Björn; Wege, Christina; Jeske, Holger

    2010-10-01

    The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics. PMID:20638413

  11. Genetic transformation with untranslatable coat protein gene of sugarcane yellow leaf virus reduces virus titers in sugarcane.

    PubMed

    Zhu, Yun J; McCafferty, Heather; Osterman, Greg; Lim, Steven; Agbayani, Ricelle; Lehrer, Axel; Schenck, Susan; Komor, Ewald

    2011-06-01

    Sugarcane yellow leaf syndrome, characterized by a yellowing of the leaf midrib followed by leaf necrosis and growth suppression, is caused by sugarcane yellow leaf virus (SCYLV). We produced SCYLV-resistant transgenic sugarcane from a susceptible cultivar (H62-4671) and determined the amount of virus present following inoculation. The transgenic plants were produced through biolistic bombardment of cell cultures with an untranslatable coat protein gene. Presence of the transgene in regenerated plants was confirmed using PCR and Southern blot analysis. The transgenic lines were inoculated by viruliferous aphids and the level of SCYLV in the plants was determined. Six out of nine transgenic lines had at least 10(3)-fold lower virus titer than the non-transformed, susceptible parent line. This resistance level, as measured by virus titer and symptom development, was similar to that of a resistant cultivar (H78-4153). The selected SCYLV-resistant transgenic sugarcane lines will be available for integration of the resistance gene into other commercial cultivars and for quantification of viral effects on yield. PMID:20661641

  12. Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine.

    PubMed

    Hassan, Syed Waqas; Waheed, Mohammad Tahir; Müller, Martin; Clarke, Jihong Liu; Shinwari, Zabta Khan; Lössl, Andreas Günter

    2014-01-01

    Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV. PMID:25483463

  13. Tobacco (Nicotiana tobaccum) nuclear transgenics with high copy number can express NPTII driven by the chloroplast psbA promoter.

    PubMed

    Ye, G N; Pang, S Z; Sanford, J C

    1996-03-01

    A chloroplast expression vector containing the NPTII gene under the control of apsbA promoter (psbA-NPTII) was constructed, and was biolistically delivered into both suspension cells and leaf strips of tobacco (Nicotiana tabaccum). Analyses of subsequently recovered kanamycin-resistant transgenic plants indicate that the psbA-NPTII gene was not located in the chloroplast, but was in the nucleus in very high copy number. This conclusion was based upon results from: (1) Southern hybridization analyses of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes; (2) pulse-field gel electrophoresis; and (3) kanamycin screening of sexual progenies. This study suggests that the nuclear expression of the NPTII gene may have been associated with many copies of the psbA-NPTII construction. Very high copy number in the nucleus might either allow NPTII expression from the otherwise inadequate psbA promoter, or might increase the chance of recombining with upstream tobacco regulatory sequences. PMID:24178457

  14. 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.

  15. 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.

  16. 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.

  17. Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects.

    PubMed

    Manimaran, P; Ramkumar, G; Sakthivel, K; Sundaram, R M; Madhav, M S; Balachandran, S M

    2011-01-01

    Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program.

  18. Genetic transformation of sweet potato by particle bombardment.

    PubMed

    Prakash, C S; Varadarajan, U

    1992-03-01

    Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.

  19. 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. PMID:15585287

  20. Transformation of the US bread wheat 'Butte 86' and silencing of omega-5 gliadin genes.

    PubMed

    Altenbach, Susan B; Allen, Paul V

    2011-01-01

    Complex groups of proteins determine the unique functional properties of wheat flour and are sometimes responsible for food intolerances and allergies in individuals that consume wheat products. Transgenic approaches can be used to explore the functions of different flour proteins, but are limited to the few wheat cultivars that can be transformed and also by the lack of detailed information about genes and proteins expressed in grain from those cultivars. The US bread wheat Butte 86 has been extensively characterized and a comprehensive proteome map was developed in which flour proteins were distinguished by mass spectrometry and associated with specific gene sequences. Here, this information has been used to design an RNA interference construct to silence the expression of genes encoding omega gliadins that trigger the food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA). The construct was introduced into immature embryos from Butte 86 using biolistics and bialaphos-resistant plants were regenerated. Stable transformation and inheritance of the transgene were confirmed by PCR. Analysis of proteins in grain from transgenic plants demonstrated that the omega-5 gliadins were either absent or substantially reduced relative to non-transformed controls. The ability to genetically transform Butte 86 makes it possible to alter flour composition in a targeted manner in a commercial US wheat cultivar and should accelerate future research on flour quality and immunogenic potential. PMID:21844700

  1. Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine.

    PubMed

    Hassan, Syed Waqas; Waheed, Mohammad Tahir; Müller, Martin; Clarke, Jihong Liu; Shinwari, Zabta Khan; Lössl, Andreas Günter

    2014-01-01

    Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV.

  2. Gene transfer and expression in plants.

    PubMed

    Lorence, Argelia; Verpoorte, Robert

    2004-01-01

    Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

  3. Overexpression of TaPIEP1, a pathogen-induced ERF gene of wheat, confers host-enhanced resistance to fungal pathogen Bipolaris sorokiniana.

    PubMed

    Dong, Na; Liu, Xin; Lu, Yan; Du, Lipu; Xu, Huijun; Liu, Hongxia; Xin, Zhiyong; Zhang, Zengyan

    2010-05-01

    Bipolaris sorokiniana is an economically important phytopathogen of wheat and other cereal species. In this paper, a novel pathogen-induced ethylene-responsive factor (ERF) gene of wheat, TaPIEP1, was isolated and characterized. The transcript of TaPIEP1 was significantly and rapidly induced by treatments with B. sorokiniana, and with ethylene (ET), jasmonate (JA), and abscisic acid. Molecular and biochemical assays demonstrated that TaPIEP1 is a new ERF transcription activator belonging to B-3c subgroup of the ERF family. Transgenic wheat lines overexpressing TaPIEP1 were generated by biolistic bombardment and molecular screening. Compared with the host wheat Yangmai12, six stable transgenic wheat lines overexpressing TaPIEP1 that exhibited significantly increased resistance to B. sorokiniana were identified by molecular detection in the T(0)-T(4) generations and by disease resistance tests. The degree of the enhanced resistance was correlated with an accumulation of the transcript level of TaPIEP1. Furthermore, the transcript levels of certain defense-related genes in the ET/JA pathways were markedly increased in the transgenic wheat plants with enhanced resistance. These results reveal that TaPIEP1 overexpression in wheat could obviously improve resistance to B. sorokiniana via activation of some defense genes, and TaPIEP1 gene may be useful in improving crop resistance to the pathogen.

  4. 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

  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. 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.

  7. 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. PMID:24416168

  8. 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.

  9. 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. PMID:23797042

  10. Experimental Transmission of Pospiviroid Populations to Weed Species Characteristic of Potato and Hop Fields▿

    PubMed Central

    Matoušek, J.; Orctová, L.; Ptáček, J.; Patzak, J.; Dědič, P.; Steger, G.; Riesner, D.

    2007-01-01

    Weed plants characteristic for potato and hop fields have not been considered in the past as potential hosts that could transmit and lead to spreading of potato spindle tuber (PSTVd) and hop stunt (HSVd) viroids, respectively. To gain insight into this problem, we biolistically inoculated these weed plants with viroid populations either as RNA or as cDNA. New potential viroid host species, collected in central Europe, were discovered. From 12 weed species characteristic for potato fields, high viroid levels, detectable by molecular hybridization, were maintained after both RNA and DNA transfers in Chamomilla reculita and Anthemis arvensis. Low viroid levels, detectable by reverse transcription-PCR (RT-PCR) only, were maintained after plant inoculations with cDNA in Veronica argensis and Amaranthus retroflexus. In these two species PSTVd concentrations were 105 and 103 times, respectively, lower than in tomato as estimated by real-time PCR. From 14 weeds characteristic for hop fields, high HSVd levels were detected in Galinsoga ciliata after both RNA and DNA transfers. HSVd was found, however, not to be transmissible by seeds of this weed species. Traces of HSVd were detectable by RT-PCR in HSVd-cDNA-inoculated Amaranthus retroflexus. Characteristic monomeric (+)-circular and linear viroid RNAs were present in extracts from weed species propagating viroids to high levels, indicating regular replication, processing, and circularization of viroid RNA in these weed species. Sequence analyses of PSTVd progenies propagated in C. reculita and A. arvensis showed a wide spectrum of variants related to various strains, from mild to lethal variants; the sequence variants isolated from A. retroflexus and V. argensis exhibited similarity or identity to the superlethal AS1 viroid variant. All HSVd clones from G. ciliata corresponded to a HSVdg variant, which is strongly pathogenic for European hops. PMID:17715233

  11. Skin Immunization Obviates Alcohol-Related Immune Dysfunction.

    PubMed

    Brand, Rhonda M; Stottlemyer, John Mark; Cline, Rachel A; Donahue, Cara; Behari, Jaideep; Falo, Louis D

    2015-01-01

    Alcoholics suffer from immune dysfunction that can impede vaccine efficacy. If ethanol (EtOH)-induced immune impairment is in part a result of direct exposure of immune cells to EtOH, then reduced levels of exposure could result in less immune dysfunction. As alcohol ingestion results in lower alcohol levels in skin than blood, we hypothesized that the skin immune network may be relatively preserved, enabling skin-targeted immunizations to obviate the immune inhibitory effects of alcohol consumption on conventional vaccines. We employed the two most common chronic EtOH mouse feeding models, the liver-damaging Lieber-DeCarli (LD) and liver-sparing Meadows-Cook (MC) diets, to examine the roles of EtOH and/or EtOH-induced liver dysfunction on alcohol related immunosuppression. Pair-fed mice were immunized against the model antigen ovalbumin (OVA) by DNA immunization or against flu by administering the protein-based influenza vaccine either systemically (IV, IM), directly to liver (hydrodynamic), or cutaneously (biolistic, ID). We measured resulting tissue EtOH levels, liver stress, regulatory T cell (Treg), and myeloid-derived suppressor cell (MDSC) populations. We compared immune responsiveness by measuring delayed-type hypersensitivity (DTH), antigen-specific cytotoxic T lymphocyte (CTL), and antibody induction as a function of delivery route and feeding model. We found that, as expected, and independent of the feeding model, EtOH ingestion inhibits DTH, CTL lysis, and antigen-specific total IgG induced by traditional systemic vaccines. On the other hand, skin-targeted vaccines were equally immunogenic in alcohol-exposed and non-exposed subjects, suggesting that cutaneous immunization may result in more efficacious vaccination in alcohol-ingesting subjects. PMID:26561838

  12. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat

    PubMed Central

    Chen, Liang; Zhang, ZengYan; Liang, HongXia; Liu, HongXia; Du, LiPu; Xu, Huijun; Xin, Zhiyong

    2008-01-01

    Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a β-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway. PMID:18953072

  13. Microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system.

    PubMed

    Mitrasinovic, Olivera M; Grattan, Alicia; Robinson, Christopher C; Lapustea, Nicolae B; Poon, Clara; Ryan, Heather; Phong, Connie; Murphy, Greer M

    2005-04-27

    Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimer's disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury. Increased expression of M-CSFR causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. To determine whether M-CSFR-induced microglial activation affects neuronal survival, we assembled a coculture system consisting of BV-2 microglia transfected to overexpress the M-CSFR and hippocampal organotypic slices treated with NMDA. Twenty-four hours after assembly of the coculture, microglia overexpressing M-CSFR proliferated at a higher rate than nontransfected control cells and exhibited enhanced migration toward NMDA-injured hippocampal cultures. Surprisingly, coculture with c-fms-transfected microglia resulted in a dramatic reduction in NMDA-induced neurotoxicity. Similar results were observed when cocultures were treated with the teratogen cyclophosphamide. Biolistic overexpression of M-CSFR on microglia endogenous to the organotypic culture also rescued neurons from excitotoxicity. Furthermore, c-fms-transfected microglia increased neuronal expression of macrophage colony-stimulating factor (M-CSF), the M-CSFR, and neurotrophin receptors in the NMDA-treated slices, as determined with laser capture microdissection. In the coculture system, direct contact between the exogenous microglia and the slice was necessary for neuroprotection. Finally, blocking expression of the M-CSF ligand by exogenous c-fms-transfected microglia with a hammerhead ribozyme compromised their neuroprotective properties. These results demonstrate a protective role for microglia overexpressing M-CSFR in our coculture system and suggest under certain circumstances, activated microglia can help rather than harm neurons subjected to excitotoxic and teratogen-induced injury.

  14. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    PubMed

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts.

  15. 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. PMID:26734014

  16. 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

  17. Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

    PubMed Central

    Stone, Brad C.; Kas, Arnold; Billman, Zachary P.; Fuller, Deborah H.; Fuller, James T.; Shendure, Jay; Murphy, Sean C.

    2016-01-01

    Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens. PMID:27070430

  18. 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.

  19. Biallelic Gene Targeting in Rice1[OPEN

    PubMed Central

    Mikami, Masafumi

    2016-01-01

    Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained. PMID:26668334

  20. 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.

  1. Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

    PubMed Central

    Manning, Viola A.; Ciuffetti, Lynda M.

    2005-01-01

    The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen. PMID:16199615

  2. 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

  3. 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.

  4. In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase.

    PubMed

    van der Vyver, Christell; Conradie, Tobie; Kossmann, Jens; Lloyd, James

    2013-04-01

    Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide. PMID:23543883

  5. 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. PMID:26896301

  6. 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.

  7. Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

    PubMed

    Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

    2011-08-01

    The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

  8. 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.

  9. 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. PMID:26887319

  10. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    PubMed

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. PMID:25554634

  11. 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.

  12. Ecological Studies of Transformed Trichoderma harzianum Strain 1295-22 in the Rhizosphere and on the Phylloplane of Creeping Bentgrass.

    PubMed

    Lo, C T; Nelson, E B; Hayes, C K; Harman, G E

    1998-02-01

    ABSTRACT A beta-glucuronidase (GUS) reporter gene and a hygromycin B (hygB) phosphotransferase gene were integrated separately into the Trichoderma harzianum strain 1295-22 genome, using biolistic transformation. The mycelial growth and biocontrol ability of the transformed strains did not differ from that of the original strain. The transformed Gus(+)-kanamycin-resistant (Gus(+)Kan(R)) strains were used to monitor growth and interactions with Rhizoctonia solani on creeping bentgrass plants. The hygB-resistant (hygB(R)) strains were used to selectively recover strain 1295-22 from the rhizosphere soil and phylloplane of creeping bentgrass after spray applications. The population levels of two hygB(R) strains and the original strain were very similar for all treatments. All three strains persisted for the duration of the experiment (28 days) in both the rhizosphere soil and on leaves, although population levels declined somewhat over the course of the experiment in unautoclaved soils. In this study, the results demonstrated that hygB(R) strains remained dominant over time when assayed on Trichoderma-selective medium containing hygB. The hygB(R) strains were not displaced by strains that colonized untreated plants. Microscopic observation showed that the Gus(+)Kan(R) strains colonized the rhizoplane, seed coat, and phylloplane of creeping bentgrass. These results supported our earlier observation that strain 1295-22 was rhizosphere and phyllo-plane competent. Interactions between T. harzianum and R. solani were readily observed in situ and changed over time. Two types of reactions were found in these experiments. In the first type, sections of hyphae of R. solani near the hyphae of T. harzianum appeared damaged, and the pathogen appeared necrotic when viewed with a microscope. The second type, observed less frequently than the first type, was typical of myco-parasitism. The findings in this study provide new insight into the interactions between R. solani and T

  13. Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis▿

    PubMed Central

    Steinbrenner, Jens; Sandmann, Gerhard

    2006-01-01

    Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis. PMID:17012596

  14. 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

  15. 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

  16. 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

  17. Role of Ca2+/calmodulin-dependent protein kinase II in dendritic spine remodeling during epileptiform activity in vitro.

    PubMed

    Zha, Xiang-ming; Dailey, Michael E; Green, Steven H

    2009-07-01

    Epileptiform activity (EA) in vivo and in vitro induces a loss of dendritic spines and synapses. Because CaMKII has been implicated in synaptogenesis and synaptic plasticity, we investigated the role of CaMKII in the effects of EA on spines, using rat hippocampal slice cultures. To visualize dendrites and postsynaptic densities (PSDs) in pyramidal neurons in the slices, we used biolistic transfection to express either free GFP or a PSD95-YFP construct that specifically labels PSDs. This allowed us to distinguish two classes of dendritic protrusions: spines that contain PSDs, and filopodia that lack PSDs and that are, on average, longer than spines. By these criteria, 48 hr of EA caused a decrease specifically in the number of spines. Immunoblots showed that EA increased CaMKII activity in the slices. Inhibition of CaMKII by expression of AIP, a specific peptide inhibitor of CaMKII, reduced spine number under basal conditions and failed to prevent EA-induced spine loss. However, under EA conditions, AIP increased the number of filopodia and the number of PSDs on the dendritic shaft. These data show at least two roles for CaMKII activity in maintenance and remodeling of dendritic spines under basal or EA conditions. First, CaMKII activity promotes the maintenance of spines and spine PSDs. Second, CaMKII activity suppresses EA-induced formation of filopodia and suppresses an increase in shaft PSDs, apparently by promoting translocation of PSDs from dendritic shafts to spines and/or selectively stabilizing spine rather than shaft PSDs.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. [Primary targeting of functional regions involved in transcriptional regulation on watermelon fruit-specific promoter WSP].

    PubMed

    Wu, Han-Ying; Liu, Jing-Mei; Yang, Xin-Ting; Zhu, Zhu-Jun; Shou, Sen-Yan

    2003-03-01

    cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-16. The same insert was then cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by HindIII + Bgl II to produce pISPA-16. Three 5'-end deletions of the promoter were obtained and fused to GUS gene in plant transient expression vector pBI426: the 1201bp fragment (from 551bp to 1752bp) was generated by digestion of pBSPA-16 with BamH I + SnaB I, the 898bp fragment (from 854bp to 1752bp) by BamH I + EcoRV. Both fragments were ligated with pBluescript SK(-) digested by BamH I + Sma I, to produce pBSPA-12 and pBS-PA-9. The inserts were cut out with HindmIII + BamH I and ligated with pBI426 digested by Hind III + Bgl II, to produce pISPA-12 and pISPA-9. The 795bp fragment (from 957bp to 1752bp) was generated by digestion of pSPA with Hinc II + EcoR I, promoter fragment was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-8. The same insert were cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by Hind III + Bgl II. The 1573bp fragment and three 5'-end deletions were delivered into watermelon leaf, stem, flower and fruit of different development stages (5, 10, 20 days after pollination) via particle bombardment using a biolistic PDS-1000/He particle gun. Bombardment parameters were as follows: a helium pressure of 1200 psi, vacuum of 91432.23Pa, 7 cm between the stopping screen and the plate. Histochemical assay were done on all the tissues bombarded after incubation for 2 days. The 1573bp fragment had the strongest promoter activity, and can induce GUS expression in fruits of 5 and 20 days after anthesis and flowers, but not in fruits of 10 days after anthesis, leaves and stems. Fragments of 1201bp and 898bp can induce GUS expression only in fruits of 20 days after anthesis, and with lower expression levels than 1573bp. Fragment of 795bp was not able to direct GUS expression in any of the tissues bombarded