WorldWideScience

Sample records for biolistics

  1. Barley Transformation Using Biolistic Techniques

    Science.gov (United States)

    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. Biolistic transformation of Scoparia dulcis L.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. Targeted biolistics for improved transformation of Impatiens balsamina.

    Science.gov (United States)

    Wetten, Andy C; Thomas, Jean-Luc; Wagiran, Alina; Chiurugwi, Tinashe

    2012-01-01

    A transgenesis programme has been developed for Impatiens balsamina that will allow elucidation of the roles played by individual genes in the flower reversion phenomenon shown by this model species. The lack of explants exhibiting adventitious shooting in I. balsamina hinders Agrobacterium-based transformation, but the multiple shoots that arise from cotyledonary nodes present a suitable target for biolistics. These tissues can be disrupted by the helium blast effect associated with conventional biolistic devices, so we have utilised modifications to the PDS 1000/He equipment originally developed for transformation of fragile insect tissues. By loading microcarriers on to a rigid, rather than flexible, macrocarrier, the blast effect is largely eliminated, and the use of a focussing nozzle allows the bombardment to be concentrated on the target tissues. This approach reduces waste of plasmid DNA and gold microcarriers and achieves transfection at lower, less disruptive helium pressures than would otherwise be necessary to efficiently penetrate below the shoot epidermis and generate heritable transgenic lines. PMID:22351015

  6. Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

    Directory of Open Access Journals (Sweden)

    Lummis Sarah CR

    2011-06-01

    Full Text Available Abstract Background Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles. Results Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles. Conclusions We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.

  7. In situ DNA transfer to chicken embryos by biolistics

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    Luciana A. Ribeiro

    1999-12-01

    Full Text Available Fertilized chicken eggs were bombarded with a biolistic device. Transient expression of the lacZ gene under the control of a human cytomegalovirus (CMV promoter was assessed after in situ gene transfer using this approach. The influence of different pressures, vacuum levels and particles was tested. Survival rate improved as particle velocity decreased, but resulted in lower levels of expression. The best survival and expression were obtained with gold particles, a helium gas pressure of 600 psi and a vacuum of 600 mmHg. Under these conditions, all bombarded embryos showed b-galactosidase activity, indicating that this was an effective method for transformation of chicken embryos.Ovos fertilizados de galinha foram bombardeados através da técnica de biobalística. A expressão transiente do gene lacZ, sob o controle do promotor humano citomegalovírus, foi verificada após a transferência in situ. Diferentes níveis de pressão de gás hélio, vácuo e tipos de partículas foram testados. A taxa de sobrevivência aumentou à medida que a velocidade das partículas diminuíram, entretanto, o nível de expressão foi menor. Os melhores resultados, combinando taxa de sobrevivência e expressão, foram obtidos com partículas de ouro, 600 libras por polegada ao quadrado de hélio e 600 mmHg de vácuo. Nestas condições, todos os embriões bombardeados apresentaram atividade da b-galactosidase, indicando que esta técnica é eficiente para a transformação de embriões de galinhas.

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

    Directory of Open Access Journals (Sweden)

    Daniell Henry

    2011-06-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    Jean-Luc Thomas

    2003-01-01

    There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473–479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting th...

  11. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

    Science.gov (United States)

    Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V

    2015-11-01

    Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. PMID:26284695

  12. A transgenic wheat with a stilbene synthase gene resistant to powdery mildew obtained by biolistic method

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Stilbene, a kind of phytoalexin, plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spore. Stilbene synthase gene (Vst1) obtained from grapevine has been transferred into common spring wheat Jinghong 5 by using the biolistic transformation method. Five transgenic plants (T0) were obtained from the bombarded 2014 immature embryos. One immune plantlet and 3 plantlets with mid-resistance to powdery mildew were identified from the transgenic plants of T3 generation which came from 2 T0 transgenic plants.

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

    Science.gov (United States)

    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

  14. Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

    OpenAIRE

    Romulo Marino Llamoca-Zárate; Luiz Ferreira Aguiar Ponte; Joerg Landsmann; Francisco de Assis de Paiva Campos

    1999-01-01

    We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transf...

  15. A biolistic process for in vitro gene transfer into chicken embryos

    Directory of Open Access Journals (Sweden)

    L.A. Ribeiro

    2001-09-01

    Full Text Available Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

  16. Study on the Obtaining of Transgenic Wheat with GNA Alien Gene by Biolistic Particle

    Institute of Scientific and Technical Information of China (English)

    XU Qiong-fang; LI Lian-cheng; CHEN Xiao; TIAN Fang; MA You-zhi; YE Xing-guo; ZHANG Zeng-yan; XU Hui-jun; XIN Zhi-yong

    2002-01-01

    The immature embryos of wheat plants, cv. Jing 411, 12 - 14 days after pollination, were cultured on SD2 medium for callus induction. After 10 days culture, 800 wheat calli were bombarded by biolistic particle coated with theDNA of plasmid pBI121-2 harboring both Galanthus nivalis agglutinin gene and bar gene. 67 green plants were finally regenerated from the bombardment calli on selection medium containing 4mg/L Basta. The results of bioassay by both inoculating wheat aphids onto the plants and applying Basta solution of 50 mg/L and 75 mg/L onto the wheat leaves in the field, and the molecular analysis, such as PCR and Southern blotting, indicated that 8 T2 plants contaning the target genes were obtained.

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

    Science.gov (United States)

    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

  18. Studies of Improving the Frequency of Indica Rice Transformation by Biolistic Bombardment

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to improve the frequency of indica rice transformation by biolistic bombardment, suitable culture conditions for embryonic calli,an optimal selection scheme for resistant calli and seedlings, and optimum bombardment parameters a investigated by using 14 commercially important indica rice cultivars. The main results show that the CC medium with 36g/L mannitol is a scheme subculture medium in which the browning of indica rice calli can be mitigated significantly; The concentration of 30~40mg/L Hyg or 150~200mg/L G418 or 10~20 mg/L Basta is suitable for selection of resistant calli; The transformation parameters of 100μg gold powder absorbing 0.2μg DNA per shot and 900 psi helium pressure and 6 cm bombardment distance and bombarded twice for each plate give the best result; Keeping the target calli on osmotic medium containing 60g/L mannitol from 12 ~24h before bombardment to 24~48h after it can increase the efficiencies of transformation . Furthermore, some transgenic indica rice plants are obtained using this optimized transformation system.

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

    Science.gov (United States)

    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.

  20. Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica

    Directory of Open Access Journals (Sweden)

    Romulo Marino Llamoca-Zárate

    1999-01-01

    Full Text Available We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transformation system can be used to express a transgene in a member of the Cactaceae.Nós demonstramos a expressão transiente do gene GUS nas células do meristema apical de Opuntia ficus-indica. A introdução do DNA nas células foi realizada através de um sistema de biolística PDS-1000He da Bio-Rad Laboratories. Para transformação, partículas de tungstenio com diâmetro de 1,3 µm foram cobertas com DNA e a distância entre o disco das partículas e o tecido alvo foi de 7,5cm, a pressão de tiro foi 1200 psi. Esta é a primeira demonstração que o sistema de biolística de transformação pode ser usado para a expressão de transgenes nas cactáceas.

  1. Determination of the most efficient target tissue and helium pressure for biolistic transformation of oil palm (Elaeis guineensis Jacq.

    Directory of Open Access Journals (Sweden)

    Amornrat Phongdara

    2008-03-01

    Full Text Available An efficient genetic transformation system for oil palm using particle bombardment was established. The transformation was performed using the pCAMBIA 1302 DNA which contains the green fluorescent protein (mgfp5 reporter gene and the selectable marker hygromycin phosphotransferase (hph gene. Oil palm explants were bombarded under the following conditions: rupture disk to macrocarrier distance, 11 mm; macrocarrier to target tissue, 90 mm and using 1 μm gold particles as microcarrier. Four different pressures of helium were tested with three types of target tissues (mature embryo, embryogenic callus and young seedlings. From the transformation efficiency, calli were much more efficiently transformed in the biolistic process compared with mature embryos and seedlings. A 100% transformation efficiency for DNA delivery into callus oil palm explants was obtained at 850 psi helium pressures, for embryos a maximum 81.8% efficiency required 850 psi and for seedlings a maximum 75.9% efficiency required 1,550 psi. Using a confocal laser scanning microscope, and appropriate filters to block out the red fluorescence of chlorophyll, expression of the GFP gene was observed in all three bombarded explant types by a bright-green fluorescence. The mgfp5 gene was still present more than 8 months after bombardment, hence it indicated the stability of transgene in those transformants.

  2. A new biolistic intradermal injector

    Science.gov (United States)

    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.

  3. Improved biolistic transfection of hair cells.

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    Hongyu Zhao

    Full Text Available Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C and PMCA2 (ATP2B2; plasma-membrane Ca(2+-ATPase isoform 2 to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.

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

    Directory of Open Access Journals (Sweden)

    Jean-Luc Thomas

    2001-10-01

    Full Text Available To compensate for the extremely low penetration efficiency of the original PDS/1000-He Bio Rad biolisticreg; 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.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    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 beta...

  6. Wheat Transformation with Trichosantin Gene by Biolistic Particle%用天花粉蛋白基因转化小麦获得转基因植株

    Institute of Scientific and Technical Information of China (English)

    徐琼芳; 李连城; 马有志; 叶兴国; 杜丽璞; 辛志勇; 张增艳; 李晓兵; 邵鹏柱

    2001-01-01

    取普通小麦品种京411未成熟胚诱导愈伤组织,10天左右,对820个胚性愈伤组织用含有35S启动子的天花粉蛋白(trichosanthin, TCS)基因轰击。2周后,将这些被轰击的愈伤组织转到含潮霉素50mg/L的筛选培养基上,经分化和生根,获得了33棵再生植株,经接饲毒蚜虫抗病性鉴定和PCR、Southern杂交分析,从中筛选出4株含有编码TCS的转基因小麦植株。转化频率为0.49%。%Wheat immature embryo calli of 10 days culture from Jing 411,a common wheat variety, Triticum aestivum, were used as the receptor of Trichosanthin gene droven by 35S promotor to be bombardmented.Two weeks after bombardment, 820 embryonic calli were transferred onto selection medium containing 50mg/L hygromycin,and 33 plantlets with healthy roots were regenerated finaly from the calli. By the barley yellow dwarf virus resistance test of inoculating the wheat aphids carrying the virus on the plants, PCR analysis, and Southern blotting analysis, 4 plants were identified to be the transgenic wheats, containing the alien DNA that ecodes TCS gene. The transformed efficiency was around 0.12 percent.

  7. 基因枪法获得GNA转基因小麦植株的研究%Study on the Obtaining of Transgenic Wheats with GNA Alien Gene by Biolistic Particle

    Institute of Scientific and Technical Information of China (English)

    徐琼芳; 李连城; 陈孝; 马有志; 叶兴国; 张增艳; 徐惠君; 辛志勇

    2001-01-01

    以小麦品种京411作为基因枪转化的靶材料,取其未成熟胚诱导愈伤组织,经过10d左右培养后,用含有雪花莲凝集素(Galanthus nivalis agglutinin,GNA)和bar基因的质粒pBI121-2轰击800个胚性愈伤组织,在含有4mg/L Basta溶液的培养基上进行筛选,分化及生根培养,获得67棵再生植株.田间涂抹Basta溶液(50、75mg/L)检测和接麦蚜实验,提取转基因植株基因组DNA,用扩增GNA基因的引物经PCR扩增Southern杂交,结果表明利用基因枪转化已从T2代获得了8株含有编码bar/GNA基因的转基因植株.

  8. Introdução de genes em segmentos foliares de cupuaçu (Theobroma grandiflorum schumm.) usando biobalística Genes introduction in foliar segments of cupuassu (Theobroma grandiflorum schumm.) using biolistic

    OpenAIRE

    Maria das Graças Rodrigues Ferreira; Carlos Henrique Siqueira de Carvalho; Andréa Almeida Carneiro; Fernando Enrique Ninamango Cárdenas

    2004-01-01

    A técnica da biobalística foi utilizada com o objetivo de adaptar um protocolo para transformação de plantas de cupuaçu. Segmentos foliares de cupuaçu foram bombardeados com um plasmídio contendo o gene reportador da antocianina, utilizando-se as pressões de hélio de 650, 1000 e 1100psi. Após o bombardeamento, os explantes foram transferidos para meio MS por 24 horas e, após este período de incubação, pontos vermelhos foram detectados utilizando-se um estereomicroscópio Stemi SV11 Zeiss (Germ...

  9. Experiment list: SRX331210 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ne was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  10. Experiment list: SRX331129 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  11. Experiment list: SRX065683 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(...ansgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony

  12. Experiment list: SRX043974 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available il Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipel...ine. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  13. Experiment list: SRX151400 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ne was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  14. Experiment list: SRX277042 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  15. Experiment list: SRX043973 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available l Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  16. Experiment list: SRX065638 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipel...ine. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  17. Experiment list: SRX277082 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available nsgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Dresden using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  18. Experiment list: SRX331069 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ne was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  19. Experiment list: SRX043849 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  20. Experiment list: SRX065639 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ne was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  1. Experiment list: SRX277083 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available nsgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Dresden using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  2. Experiment list: SRX043971 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available il Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipel...ine. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  3. Experiment list: SRX331127 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  4. Experiment list: SRX331216 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ne was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeli...ne. The resulting plasmid was used for biolistic transformation of an unc-119(ed3)

  5. Experiment list: SRX277041 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available gene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipe...line. The resulting plasmid was used for biolistic transformation of an unc-119(ed3

  6. Micro-shock Wave Assisted Plant Transformation

    Science.gov (United States)

    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.

  7. Transformace ptDNA \\kur{Chlamydomonas reinhardtii}

    OpenAIRE

    Husáková, Jana

    2011-01-01

    The aim of this master thesis was to test and compare two available methods of genetic transformation (biolistics, electroporation) of the plastid genome of green algae Chlamydomonas reinhardtii. For biolistic transformation a wide range of experimental parameters which generally influence ptDNA transformation efficiency was optimized: physiological condition of acceptor cells, type and size of microparticles, pressure of propulsion gas (helium), length of projectile trajectory, transformatio...

  8. UCE: A uracil excision (USERTM)-based toolbox for transformation of cereals

    DEFF Research Database (Denmark)

    Hebelstrup, Kim H; Christiansen, Michael W; Carciofi, Massimiliano;

    2010-01-01

    Background Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs...... (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient...

  9. Main Achievements of Cotton Large-scale Transformation System

    Institute of Scientific and Technical Information of China (English)

    LI Fu-guang; LIU Chuan-liang; WU Zhi-xia; ZHANG Chao-jun; ZHANG Xue-yan

    2008-01-01

    @@ Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacteriurn turnefaciens-mediated,pollen-tube pathway and biolistic methods together efficiently.More than 1000 transgenie lines are selected from the transgenic plants with molecular assistant breeding and conventional breeding methods.

  10. Some factors involved in microprojectile-mediated indica rice transformation

    Institute of Scientific and Technical Information of China (English)

    XUERui; QIUXiaohui; YANMeixian; HUANGDanian; CAOShouyun; TIANWenzhong

    1997-01-01

    Immature embryos and calli derived from immature or mature embryos of the 16 indica rice varieties have been transformed by using particle bombardment. To improve growth state of subcultured calli, we set up a variety of parameters to optimize the important tactors involved in Biolistic system, and added various hormones, mannitol, and MES to CC or NB medium.

  11. Development of transgenic papayas expressing the coat protein gene from a Brazilian isolate of Papaya ringspot virus (PRSV) = Desenvolvimento de mamoeiros transgênicos resistentes a vírus expressando o gene da capa protéica de um isolado brasileiro de Papaya ringspot virus

    NARCIS (Netherlands)

    Souza, M.T.; Níckel, O.; Gonsalves, D.

    2005-01-01

    Translatable and nontranslatable versions of the coat protein (cp) gene of a Papaya ringspot virus (PRSV) isolate collected in the state of Bahia, Brazil, were engineered for expression in Sunrise and Sunset Solo varieties of papaya (Carica papaya). The biolistic system was used to transform seconda

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Wu, Huixia; Doherty, Angela; 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.

  14. Main Achievements of Cotton Large-scale Transformation System

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacterium tumefaciens-mediated,pollen-tube pathway and biolistic methods together efficiently.More than

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

    Science.gov (United States)

    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.

  16. Scientific Opinion on applications (EFSA-GMO-UK-2008-57 and EFSA-GMO-RX-MON15985) for the placing on the market of insect-resistant genetically modified cotton MON 15985 for food and feed uses, import and processing, and for the renewal of authorisation of existing products produced from cotton MON 15985, both under Regulation (EC) No 1829/2003 from Monsanto

    OpenAIRE

    EFSA Panel on Genetically Modified Organisms (GMO)

    2014-01-01

    Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 and GUS in addition to the Cry1Ac and NPTII proteins. Cry proteins in MON 15985 confer resistance to major lepidopteran cotton pests, whereas the GUS and NPTII proteins were used as markers during product development. Molecular characterisation of MON 15985 did not give rise to safety issues. The EFSA GMO Panel could not co...

  17. Efficient transient expression of the $\\beta$-glucuronidase reporter gene in garlic (Allium sativum L.)

    OpenAIRE

    Ferrer, Esther; Linares, Concha; González, Juan,

    2000-01-01

    International audience A biolistic particle delivery system was used to introduce DNA containing a $\\beta$-glucuronidase (gus) reporter gene under the control of the CaMV35S promoter in three different garlic (Allium sativum L.) tissues: embryogenic calli, leaves and basal plate discs. Expression of the reporter gene was assayed histochemically and fluorimetrically when the tissues were bombarded with 1 $\\mu$m diameter gold particles coated with DNA, at a distance of 3 cm from the stopping...

  18. Scientific Opinion on an application (EFSA-GMO-BE-2011-98) for the placing on the market of herbicide-tolerant genetically modified soybean FG72 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Bayer CropScience

    OpenAIRE

    Arpaia, Salvatore; Birch, Andrew Nicholas Edmund; Chesson, Andrew; du Jardin, Patrick; Gathmann, Achim; Gropp, Jürgen; Herman, Lieve; Hoen-Sorteberg, Hilde-Gunn; Jones, Huw; Kiss, József; Kleter, Gijs; Løvik, Martinus; Messéan, Antoine; Naegeli, Hanspeter; Kaare Magne Nielsen, Kaare Magne

    2015-01-01

    Soybean FG72 was developed by biolistic transformation to express the HPPD W336 and 2mEPSPS proteins, which confer tolerance to isoxaflutole- and glyphosate-based herbicides. The molecular characterisation of soybean FG72 did not give rise to safety issues. The agronomic and phenotypic characteristics of soybean FG72 tested under field conditions revealed no biologically relevant differences between soybean FG72 and its conventional counterpart that would give rise to any food and feed or env...

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

    Science.gov (United States)

    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

  20. A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

    OpenAIRE

    Choi, Sangdun; Begum, Dilara; Koshinsky, Heather; Ow, David W; Wing, Rod A

    2000-01-01

    With current plant transformation methods (Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombinati...

  1. Optimización de las condiciones de inoculación por biobalística de un Begomovirus en tomate y tabaco

    Directory of Open Access Journals (Sweden)

    Karina López-López

    2013-12-01

    Full Text Available Optimizing conditions for biolistic inoculation of Begomovirus in tomato and tobaccoResumen: La transmisión experimental de Begomovirus es problemática. La mayoría de estos virus se pueden transmitir de planta a planta por su vector biológico, Bemisia tabaci. Las inoculaciones experimentales con mosca blanca son problemáticas debido a sus hábitos de alimentación, una planta viva infectada e instalaciones de contención para el vector. Por su parte la inoculación mecánica de Begomovirus es posible, pero generalmente a tasas bajas y no en todos los casos. Por esta razón el bombardeo de partículas (biobalística de DNA viral como una estrategia de inoculación fue desarrollada. La posibilidad de utilizar el dispositivo de mano Helios Gen System Gun (Biorad®, un equipo de biobalística, para la transmisión de un Begomovirus bipartita a plantas de tomate y tabaco fue ensayado y optimizado. Los parámetros evaluados fueron: número de disparos (1-2, presión de helio (220 y 320 psi y diámetro de las partículas de oro (0.6 y 1.6µm. Los síntomas característicos de la enfermedad viral (deformación clorosis, mosaico y de la hoja aparecieron 3 semanas después del bombardeo en las hojas jóvenes no inoculadas. La replicación del DNA viral en las plantas se confirmó por Reacción en cadena de la polimerasa. Plantas infectadas en un 100% se obtuvieron cuando en el bombardeo se emplearon partículas de oro de 1.6 µm recubiertas con DNA viral a una presión de 320psi. A nuestro entender este es el primer reporte en Colombia de la inoculación directa de plantas de tomate y tabaco con un Begomovirus bipartita usando un dispositivo portátil de biobalística.Palabras clave: geminivirus, Solanum lycopersicum, Nicotiana tabacum,  N. benthamiana, Bemisia tabaciAbstract: Experimental transmission of Begomovirus is problematic. Most Begomoviruses can be transmitted readily from plant to plant by the whitefly vector, but this also requires a

  2. Methods for transient assay of gene function in floral tissues

    Directory of Open Access Journals (Sweden)

    Pathirana Nilangani N

    2007-01-01

    Full Text Available Abstract Background There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue. Results Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum chalcone synthase gene (CHS and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression. Conclusion We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to

  3. Expression of a methionine-rich storage albumin from the Brazil nut (Bertholletia excelsa H.B.K., Lecythidaceae in transgenic bean plants (Phaseolus vulgaris L., Fabaceae

    Directory of Open Access Journals (Sweden)

    Aragão F.J.L.

    1999-01-01

    Full Text Available Bean (Phaseolus vulgaris, an important component in the diet of people in developing countries, has low levels of the essential amino acid, methionine. We have attempted to correct this deficiency by introducing a transgene coding for a methionine-rich storage albumin from the Brazil nut via biolistic methods. The transgene's coding sequence was driven by a doubled 35S CaMV promoter and AMV enhancer sequences. The transgene was stable and correctly expressed in homozygous R2 to R5 seeds. In two of the five transgenic lines the methionine content was significantly increased (14 and 23% over the values found in untransformed plants.

  4. Transgenic Plants as Expression Factories for Bio Pharmaceuticals

    Directory of Open Access Journals (Sweden)

    Shabir H Wani

    2015-06-01

    Full Text Available At present agriculture not only provides food, but is also used for the production of pharmaceuticals or industrial compounds such as pharmaceutical drugs, vaccines along with biodegradable plastic and industrial chemicals. Since last three decades, plant genetic engineering has played a vital role in the production of bio-pharmaceutical products from crops. Due to technological advancement of genetic engineering, biotechnologist are able to engineer plants by using living organism with the help of different transformation techniques like Agrobacterium mediated transformation, biolistic gene gun, and so on to produce biopharmaceuticals products for diagnostic purposes as well as nutritional supplements.

  5. Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:27130499

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

    Science.gov (United States)

    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

  8. Transgenic oil palm: production and projection.

    Science.gov (United States)

    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. PMID:11171275

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Transgenic oil palm: production and projection.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2005-10-01

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

  12. DNA vaccines for viral diseases

    Directory of Open Access Journals (Sweden)

    J.J. Donnelly

    1999-02-01

    Full Text Available DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

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

    Science.gov (United States)

    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.

  14. Transformation of TrxS Gene into Barley by Particle Bombardment

    Institute of Scientific and Technical Information of China (English)

    WEI Li; YIN Jun; KONG Wei-wei; REN Jiang-ping; LI Lei; LIU Lei

    2005-01-01

    The production of malting barley in China can't meet the demand of beer industries because of poor quality and it becomes a bottleneck problem in beer manufacture industry. In this paper, TrxS gene cloned from Phalaris coerulescens was transferred into barley cultivar Yupi 1 (YP1)via biolistic bombardment. 1206 immature embryos were bombarded and seven transgenic plants carrying TrxS gene were confirmed by PCR and PCR-Southern blotting analysis. TrxS gene was expressed in transgenic plants by RT-PCR analysis. The activity of Trxh and αt-amylase of transgenic line were higher than that of non-transgenic line, which is helpful to improve malting quality of barley.

  15. Differential Cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds

    KAUST Repository

    Idris, Ali

    2010-12-01

    A Cotton leaf crumple virus (CLCrV)-based gene silencing vector containing a fragment of the Gossypium hirsutum Magnesium chelatase subunit I was used to establish endogenous gene silencing in cotton of varied genetic backgrounds. Biolistic inoculation resulted in systemic and persistent photo-bleaching of the leaves and bolls of the seven cultivars tested, however, the intensity of silencing was variable. CLCrV-VIGS-mediated expression of green fluorescent protein was used to monitor the in planta distribution of the vector, indicating successful phloem invasion in all cultivars tested. Acala SJ-1, one of the cotton cultivars, was identified as a particularly optimal candidate for CLCrV-VIGS-based cotton reverse-genetics. © 2010 Elsevier Ltd.

  16. Plastid transformation in sugar beet: Beta vulgaris.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele

    2014-01-01

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

  17. Plastid transformation in sugar beet: Beta vulgaris.

    Science.gov (United States)

    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

  18. The application of PCR for the isolation of a lipase gene from the genomic DNA of an Antarctic microfungus.

    Science.gov (United States)

    Bradner, J Ron; Bell, Philip J L; Te'o, V S Junior; Nevalainen, K M Helena

    2003-12-01

    We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes. We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium. The recombinant lipase had a temperature optimum of 25 degrees C at pH 7.9 and retained greater than 50% of the maximum activity from 10 degrees C to 35 degrees C and over a pH range from 4.0 to 8.5. PMID:13680154

  19. A novel approach to the generation of seamless constructs for plant transformation

    DEFF Research Database (Denmark)

    Kronbak, Remy; Ingvardsen, Christina R.; Madsen, Claus K.;

    2014-01-01

    sequences from the vector leaving no undesired, short sequences behind.Results: We designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent......Background: When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone....... Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  3. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants.

    Directory of Open Access Journals (Sweden)

    Dipak Kumar Sahoo

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2012-12-01

    The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03 mg/g FW), sucrose content (ca 60 mg/g FW) was not affected, while starch content (<0.4 mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01 mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate. PMID:22903192

  8. 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.

    Science.gov (United States)

    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.

  9. 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

    Science.gov (United States)

    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

  10. Developing nanotechnology for biofuel and plant science applications

    Energy Technology Data Exchange (ETDEWEB)

    Valenstein, Justin [Iowa State Univ., Ames, IA (United States)

    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.

  11. Dual Targeting of a Mitochondrial Protein: The Case Study of Cytochrome C1

    Institute of Scientific and Technical Information of China (English)

    Anja R(o)diger; Bianca Baudisch; Uwe Langner; Ralf Bernd Kl(o)sgen

    2011-01-01

    As a result of the endosymbiotic gene transfer, the majority of proteins of mitochondria and chloroplasts is encoded in the nucleus and synthesized in the cytosol as precursor molecules carrying N-terminal transit peptides for the transport into the respective target organelle. In most instances, transport takes place into either mitochondria or chlor-oplasts, although a few examples of dual targeting into both organelles have been described. Here, we show by a com-bination of three different experimental strategies that also cytochrome c of potato, a component of the respiratory electron transport chain, is imported not only into mitochondria, but also into plastids. In organello import experiments with isolated mitochondria and chloroplasts, which were analyzed in both single and mixed organelle assays, demonstrate that the processing products accumulating after import within the two endosymbiotic organelles are different in size. Dual targeting of cytochrome c is observed also in vivo, after biolistic transformation of leaf epidermal cells with suitable reporter constructions. Finally, Western analyses employing cytochrome c-specific antiserum provide evidence that the protein accumulates in significant amounts in mitochondria and chloroplasts of both pea and spinach. The possible consequences of our findings on the relevance of the dual targeting phenomenon are discussed.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants

    Institute of Scientific and Technical Information of China (English)

    LI Yi-nü; SUN Bing-yao; SU Ning; MENG Xiang-xun; ZHANG Zhi-fang; SHEN Gui-fang

    2009-01-01

    In contrast to the situation of random integration of foreign genes in nuclear transformation,the introduction of genes via chloroplast genetic engineering is characterized by site-specific pattern via homologous recombination.To establish an expression system for alien genes in rice chloroplast,the intergenic region of ndhF and trnL was selected as target for sitespecific integration of PPT-resistant bar gene in this study.Two DNA fragments suitable for homologous recombination were cloned from rice chloroplast genome DNA using PCR technique,and the chloroplast-specific expression vector pRB was constructed by fusing a modified 16S rRNA gene promoter to bar gene together with terminator of psbA gene 3'sequence.Chloroplast transformation was carried out by biolistic bombardment of sterile rice calli with the pRB construct.Subsequently,the regenerated plantlets and seeds of progeny arising from reciprocal cross to the wild-type lines were obtained.Molecular analysis suggested that the bar gene has been integrated into rice chloroplast genome.Genetic analysis revealed that bar gene could be transmitted and expressed normally in chloroplast genome.Thus,the bar gene conferred not only selection pressure for the transformation of rice chloroplast genome,but PPT-resistant trait for rice plants as well.It is suggested that an efficient gene expression system in the rice chloroplast has been established by chloroplast transformation technique.

  15. Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

    Institute of Scientific and Technical Information of China (English)

    YANG Zongqi; LI yinü; CHEN Feng; LI Dong; ZHANG Zhifang; LIU Yanxin; ZHENG Dexian; WANG Yong; SHEN Guifang

    2006-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expression vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and cultivation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%-0.67% of the total soluble proteins.These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.

  16. Efficient and rapid C. elegans transgenesis by bombardment and hygromycin B selection.

    Directory of Open Access Journals (Sweden)

    Inja Radman

    Full Text Available We report a simple, cost-effective, scalable and efficient method for creating transgenic Caenorhabditis elegans that requires minimal hands-on time. The method combines biolistic bombardment with selection for transgenics that bear a hygromycin B resistance gene on agar plates supplemented with hygromycin B, taking advantage of our observation that hygromycin B is sufficient to kill wild-type C. elegans at very low concentrations. Crucially, the method provides substantial improvements in the success of bombardments for isolating transmitting strains, the isolation of multiple independent strains, and the isolation of integrated strains: 100% of bombardments in a large data set yielded transgenics; 10 or more independent strains were isolated from 84% of bombardments, and up to 28 independent strains were isolated from a single bombardment; 82% of bombardments yielded stably transmitting integrated lines with most yielding multiple integrated lines. We anticipate that the selection will be widely adopted for C. elegans transgenesis via bombardment, and that hygromycin B resistance will be adopted as a marker in other approaches for manipulating, introducing or deleting DNA in C. elegans.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Plant regeneration and genetic transformation in switchgrass-A review

    Institute of Scientific and Technical Information of China (English)

    Paul Merrick; Shuizhang Fei

    2015-01-01

    Switchgrass is native to the tal grass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid (2x) to dodecaploid (12x) with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow wel in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocel ulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We ifrst reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efifciency. Final y we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.

  2. Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

    Institute of Scientific and Technical Information of China (English)

    ZHU HuaChen; XU XinPing; XIAO GuoYing; YUAN LongPing; LI BaoJian

    2007-01-01

    A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of '9311', an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four antifungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of '9311', co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R1 and R2 progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F1 plants, resulting from a cross of R2 homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).

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

    Science.gov (United States)

    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.

  4. Integration of C4-specific PPDK gene of maize to C3 rice and its characteristics in relation to photosynthesis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jianfu; Swapan K.Datta; WANG Guoying; XIE Huaan

    2007-01-01

    Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in photosynthesis in some plants that exploit the C4 photosynthetic pathway for the fixation of CO2.The C4-specific PPDK encoding pyruvate orthophosphate dikinase was introduced into C3 plant,a rice (Oryza sativa L.cv.indica IR64) mediated by biolistic and Agrobacterium transformation.The C4-PPDK gene of maize was integrated to indica IR64 with polymerase chain reaction (PCR)-Southern blotting.The total nitrogen of flag leaves of transgenic IR64 was analyzed with Kj eldahl method for quantitative determination of nitrogen,indicating that the total nitrogen of flag leaves of most transgenic IR64 was higher than that of non-transgenic control IR64 formants in the greenhouse.The maximum value of total nitrogen of flag leaves was 3.61% among transgenie IR64 plants,1.07% higher than that of non-transgenic control IR64 formants.The total nitrogen of flag leaves of transgenic IR64 was increased by 42.1%.The factors for yield of transgenic IR64 plants were analyzed,indicating there was a greater difference in yield-forming factors among transgenic IR64 plants in the greenhouse,i.e.dried plant weight,harvested index and so on.Thus,it could help rice breeders select different materials for breeding.

  5. Investigations into the structure of the genomes of thermophilic Methanobacterium strains. Untersuchungen zur Genomstruktur von thermophilen Methanobacterium Arten

    Energy Technology Data Exchange (ETDEWEB)

    Stettler, R.

    1994-01-01

    In the present work, attempts were made to develop a gene transfer system for Methanobacterium thermoautotrophicum Marburg, and the genomes of thermophilic Methanobacterium strains were analyzed. Chapter 1 describes studies for developing a transformation system for M. thermoautotrophicum Marburg. Elements for establishing a cloning system in this strain, such as auxotrophic and resistance markers as well as plasmids are available, but methods for plasmid transformation are still lacking. A variety of techniques for introducing DNA into strain Marburg were investigated, including electroporation, chemical transformation, protoplast transformation, and biolistic transformation. However, no transformants were detected in any of these experiments. In order to find related Methanobacterium strains which may be transformable, new thermophilic methanogens were isolated from sewage sludge. One of these isolates, Methanobacterium sp. ZH3, contained the cryptic plasmid pME2200. The characterization of strain ZH3 and the analysis of plasmid pME2200 are described in Chapter 2. With respect to its physiological characteristics Methanobacterium sp. ZH3 closely resembled M. thermoautotrophicum Marburg. Analysis of its 16S rRNA sequence confirmed the close relationship of Methanobacterium sp. ZH3 with M. thermoautotrophicum Marburg at the molecular level. A single nucleotide difference was found between the 16S rRNA sequences of these two strains. Strain ZH3 thus is the first M. thermoautotrophicum strain which is closely related to strain Marburg. The construction of a physical map of the M. thermoautotrophicum Marburg chromosome is reported in Chapter 3. (author) figs., tabs., refs.

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

    Directory of Open Access Journals (Sweden)

    Cibelle Engels

    2013-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2010-11-01

    Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance. PMID:20653413

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

    Science.gov (United States)

    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

  9. Plant-Based Vaccines: Production and Challenges

    Directory of Open Access Journals (Sweden)

    Erna Laere

    2016-01-01

    Full Text Available Plant-based vaccine technologies involve the integration of the desired genes encoding the antigen protein for specific disease into the genome of plant tissues by various methods. Agrobacterium-mediated gene transfer and transformation via genetically modified plant virus are the common methods that have been used to produce effective vaccines. Nevertheless, with the advancement of science and technology, new approaches have been developed to increase the efficiency of former methods such as biolistic, electroporation, agroinfiltration, sonication, and polyethylene glycol treatment. Even though plant-based vaccines provide many benefits to the vaccine industry, there are still challenges that limit the rate of successful production of these third-generation vaccines. Even with all the limitations, continuous efforts are still ongoing in order to produce efficient vaccine for many human and animals related diseases owing to its great potentials. This paper reviews the existing conventional methods as well as the development efforts by researchers in order to improve the production of plant-based vaccines. Several challenges encountered during and after the production process were also discussed.

  10. Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four anti-fungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R1 and R2 progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F1 plants, resulting from a cross of R2 homo-zygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).

  11. Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar-dependent and gibberellin action involves SLENDER1 but not GAMYB.

    Science.gov (United States)

    Eastmond, Peter J; Jones, Russell L

    2005-11-01

    Storage oil is a major constituent in the cereal aleurone layer. The aim of this study was to investigate how gibberellin (GA) and abscisic acid (ABA) regulate conversion of oil to sugar in barley aleurone. The activity of the glyoxylate cycle enzyme isocitrate lyase (ICL) was surveyed in eight barley cultivars. Surprisingly, some cultivars do not require GA for the induction of ICL (e.g. Himalaya), whereas some do (e.g. Golden Promise). Furthermore, in Golden Promise, GA also stimulates triacylglycerol breakdown and enhances the net flux of carbon from acetate to sugar. In contrast, ABA strongly represses ICL activity and the flux of carbon from oil to sugar in both Golden Promise and Himalaya. Biolistics using a promoter reporter showed that GA and ABA regulate ICL at the level of transcription. Studies using barley and rice mutants and pharmacological agents show that GA-dependent induction of ICL activity is mediated by SLENDER1 and requires cGMP, but does not involve the transcription factor GAMYB. Gibberellin and ABA therefore act antagonistically to regulate gluconeogenesis in the aleurone layer as well as controlling the production and secretion of hydrolases into the starchy endosperm. We suggest that the variation between different barley cultivars might be a result of selective breeding to alter seed dormancy. PMID:16236157

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

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  14. Genetic transformation of major cereal crops.

    Science.gov (United States)

    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.

  15. A ENGENHARIA GENÉTICA APLICADA NO MELHORAMENTO DA CANA-DE-AÇÚCAR: UMA NOVA ALTERNATIVA PARA A PRODUÇÃO DE BIODIESEL

    Directory of Open Access Journals (Sweden)

    Elisângela Távora da Silva

    2011-01-01

    Full Text Available The biotechnology have made great advances in genetic improvement. In this sense, aimed to address currentcontexts of dynamic institutional, organizational and technological in which genetically modified organisms are included.The initial concern was to describe the technologies for sustainable production and genetic modification of sugar cane forthe production of biodiesel. For this, we used recombinant DNA technology, which allows you to isolate, prepare,manipulate and study small segments of DNA, and have been widely used to become resistant to herbicides, somecommercially important crops. The gene transformation allows you to transfer the gene of interest through naturalmethods such as Agrobacterium tumefaciens, and direct physical methods such as electroporation and biolistic. Theproduction of biodiesel is an alternative option that would circumvent the shortage of oil, because it is made carbonneutral. However, using biotechnology to improve the production process of biodiesel, requires attention for the controlof possible risks to the environment and ecological balance. Based on this literature review, we conclude that there arestill few conclusive studies on the genetic improvement of sugar cane in the production of biodiesel, and future studiesare needed to validate the process.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Zhangli Hu

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Kedong Xu

    Full Text Available 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. Development of a gene silencing DNA vector derived from a broad host range geminivirus

    Directory of Open Access Journals (Sweden)

    Hancock Leandria C

    2009-07-01

    Full Text Available Abstract Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV, named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS, transketolase, the sulfur allele of magnesium chelatase (ChlI, and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

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

    Science.gov (United States)

    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

  6. Expression of the intact C4 type pepc gene cloned from maize in transgenic winter wheat

    Institute of Scientific and Technical Information of China (English)

    CHEN Xuqing; ZHANG Xiaodong; LIANG Rongqi; ZHANG Liquan; YANG Fengping; CAO Mingqing

    2004-01-01

    Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as Pbac214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of Mrna sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two Mrna, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector Pbac214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4-pepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  9. Comparison of the activity of salt and 35S promoters in transgenic calli of sorghum (Sorghum bicolor L. (Moench))

    International Nuclear Information System (INIS)

    Use of specific promoters under clearly defined conditions in transgenic plants is valuable for determining gene expression. In this study, we used the Salt promoter derived from the rice variety Taichung native-1 and linked to the uidA gene in plasmid pGVB310, and compared its activity with that of the 35S promoter in the transformed calli of sorghum. The 35S promoter, as the constitutive promoter, is also linked to the uidA gene in plasmid p35SGus. Plasmid pGVB310 was transferred to sorghum cell suspension using biolistic bombardment in the same way as co-transformation was carried out for plasmid p35SGus with pROB5 containing the hpt gene as the selectable marker. A transformed callus was selected using 50 mg/L of hygromycin in callus medium for 3 weeks. The resistant colonies obtained during selection were then transferred to a fresh callus medium containint 50 mg/L of hygromycin. A histochemical Gus assay was performed on those calli that were resistant to 50 mg/L of hygromycin; the results showed that a blue colour appeared in the transformed calli after 5 hours of incubation at 37 deg C. A fluorimetric Gus assay was performed to detect quantitative Gus expression in the resistant calli. Resistant calli were grown under various conditions, e.g. 0.1% NaCl, or 2 mg/L of ABA in callus medium, or without stress (normal conditions). The level of promoter activity for both the 35S and Salt promoters was measured on the basis of Gus expression detected with fluorimetric assay. The results showed that the transformed calli (varieties Arval and White Martin) with plasmid pGVB310 showed Gus expression that was about 8-12 times higher than that of the transformed calli with plasmid p35SGus. 2 refs, 1 fig., 1 tab

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  12. The sense and antisense expression of gibberellin 20-oxidase gene (rga5) in rice and its effects on GA1 level and agronomic traits

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A gibberellin 20-oxidase gene rga5 was isolated by PCR from genomic DNA of rice (Oryza sativa ssp indica) cultivars 'Aizizhan' and 'Nante'. Compared with the reported OsGA20ox, the rga5 was partial-frame-shifted with 11 different amino acids. Then the rga5 with CaMV 35S promotor and NOS terminator was inserted into the polylinker site of pCambia1301 to construct sense and antisense gene expressing vectors pSrga5 and pArga5. The transgenic plants were obtained by biolistic bombardment with pSrga5 or pArga5. The transgenic rice plants showed that the over- expression and antisense-expression of rga5 have remarkable effects on the biological characters of rice. The sense transgenic plants showed heightening with longer spike, more seed-bearing and unaffected flowering, whereas antisense transgenic plants showed dwarfing, early-flowering with slender stem, dark leaf color, shorter leaf and shorter spike. The PCR amplification and Southern blot hybridization showed that the rga5 has been integrated into the transgenic rice genome and the transcription of rga5 was identified by Northern blot hybridization. In the sense transgenic plants the GA1 content increased of about 50%, however, the antisense transgenic rice decreased of 90% approximately compared with control plant 'Zhonghua 8'. These results demonstrated that the rga5 is a functional gene encoding gibberellin 20-oxidase in rice. Over-expressing rga5 significantly increases the endogenous GA1 level and plant height of rice, whereas the expression of antisense rga5 decreases the GA1 level and plant height of rice dramatically.

  13. Cell surface area regulation in neurons in hippocampal slice cultures is resistant to oxygen-glucose deprivation

    Directory of Open Access Journals (Sweden)

    Natalya Shulyakova

    2010-09-01

    Full Text Available Natalya Shulyakova1,2, Jamie Fong2, Diana Diec2, Adrian Nahirny1,2, Linda R Mills1,21Department of Physiology, University of Toronto, Toronto, ON, Canada, M5T 2S8; 2Toronto Western Hospital Research Institute, University Health Network, 11-430, 399 Bathurst St, Toronto, ON, Canada, M5T 2S8Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs, to accommodate the decrease in the bilayer.Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP targeted to the cytoplasm; and a membrane targeted GFP (lckGFP, targeted to the plasma membrane.Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normo-osmotic media changes. Oxygen-glucose deprivation (OGD caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge.Conclusion: These results suggest cell surface area regulation (SAR is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.Keywords: neurons, swelling, ischemia, cell surface area, hippocampal slice culture

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

    Science.gov (United States)

    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. PMID:20574644

  15. The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

    Directory of Open Access Journals (Sweden)

    von Arnim Albrecht G

    2009-05-01

    Full Text Available Abstract Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially

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

    Science.gov (United States)

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

    2003-03-01

    Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene beta-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition. PMID:17147745

  17. Spatial relationships between GABAergic and glutamatergic synapses on the dendrites of distinct types of mouse retinal ganglion cells across development.

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    Adam Bleckert

    Full Text Available Neuronal output requires a concerted balance between excitatory and inhibitory (I/E input. Like other circuits, inhibitory synaptogenesis in the retina precedes excitatory synaptogenesis. How then do neurons attain their mature balance of I/E ratios despite temporal offset in synaptogenesis? To directly compare the development of glutamatergic and GABAergic synapses onto the same cell, we biolistically transfected retinal ganglion cells (RGCs with PSD95CFP, a marker of glutamatergic postsynaptic sites, in transgenic Thy1-YFPγ2 mice in which GABAA receptors are fluorescently tagged. We mapped YFPγ2 and PSD95CFP puncta distributions on three RGC types at postnatal day P12, shortly before eye opening, and at P21 when robust light responses in RGCs are present. The mature IGABA/E ratios varied among ON-Sustained (S A-type, OFF-S A-type, and bistratified direction selective (DS RGCs. These ratios were attained at different rates, before eye-opening for ON-S and OFF-S A-type, and after eye-opening for DS RGCs. At both ages examined, the IGABA/E ratio was uniform across the arbors of the three RGC types. Furthermore, measurements of the distances between neighboring PSD95CFP and YFPγ2 puncta on RGC dendrites indicate that their local relationship is established early in development, and cannot be predicted by random organization. These close spatial associations between glutamatergic and GABAergic postsynaptic sites appear to represent local synaptic arrangements revealed by correlative light and EM reconstructions of a single RGC's dendrites. Thus, although RGC types have different IGABA/E ratios and establish these ratios at separate rates, the local relationship between excitatory and inhibitory inputs appear similarly constrained across the RGC types studied.

  18. Spatial Relationships between GABAergic and Glutamatergic Synapses on the Dendrites of Distinct Types of Mouse Retinal Ganglion Cells across Development

    Science.gov (United States)

    Bleckert, Adam; Parker, Edward D.; Kang, YunHee; Pancaroglu, Raika; Soto, Florentina; Lewis, Renate; Craig, Ann Marie; Wong, Rachel O. L.

    2013-01-01

    Neuronal output requires a concerted balance between excitatory and inhibitory (I/E) input. Like other circuits, inhibitory synaptogenesis in the retina precedes excitatory synaptogenesis. How then do neurons attain their mature balance of I/E ratios despite temporal offset in synaptogenesis? To directly compare the development of glutamatergic and GABAergic synapses onto the same cell, we biolistically transfected retinal ganglion cells (RGCs) with PSD95CFP, a marker of glutamatergic postsynaptic sites, in transgenic Thy1­YFPγ2 mice in which GABAA receptors are fluorescently tagged. We mapped YFPγ2 and PSD95CFP puncta distributions on three RGC types at postnatal day P12, shortly before eye opening, and at P21 when robust light responses in RGCs are present. The mature IGABA/E ratios varied among ON-Sustained (S) A-type, OFF-S A-type, and bistratified direction selective (DS) RGCs. These ratios were attained at different rates, before eye-opening for ON-S and OFF-S A-type, and after eye-opening for DS RGCs. At both ages examined, the IGABA/E ratio was uniform across the arbors of the three RGC types. Furthermore, measurements of the distances between neighboring PSD95CFP and YFPγ2 puncta on RGC dendrites indicate that their local relationship is established early in development, and cannot be predicted by random organization. These close spatial associations between glutamatergic and GABAergic postsynaptic sites appear to represent local synaptic arrangements revealed by correlative light and EM reconstructions of a single RGC's dendrites. Thus, although RGC types have different IGABA/E ratios and establish these ratios at separate rates, the local relationship between excitatory and inhibitory inputs appear similarly constrained across the RGC types studied. PMID:23922756

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

    Science.gov (United States)

    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.

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

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    Li Li

    2010-01-01

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

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

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    Masci Stefania

    2010-07-01

    Full Text Available Abstract 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.

  2. Scientific Opinion on applications (EFSA-GMO-UK-2008-57 and EFSA-GMO-RX-MON15985 for the placing on the market of insect-resistant genetically modified cotton MON 15985 for food and feed uses, import and processing, and for the renewal of authorisation of existing products produced from cotton MON 15985, both under Regulation (EC No 1829/2003 from Monsanto

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Genetically Modified Organisms (GMO

    2014-07-01

    Full Text Available Cotton MON 15985 was developed by biolistic transformation of cotton MON 531 to express Cry2Ab2 and GUS in addition to the Cry1Ac and NPTII proteins. Cry proteins in MON 15985 confer resistance to major lepidopteran cotton pests, whereas the GUS and NPTII proteins were used as markers during product development. Molecular characterisation of MON 15985 did not give rise to safety issues. The EFSA GMO Panel could not conclude on the potential occurrence of unintended effects for agronomic and phenotypic characteristics owing to data limitations. Compositional data gave no indication of unintended effects for which further assessment was needed. The Panel concludes that cotton MON 15985, as described in these applications, is as safe and nutritious as its conventional counterpart and other non-genetically modified varieties, and considers it unlikely that the overall allergenicity of the whole plant is changed. Environmental risk assessment was restricted to the exposure through faecal material from animals fed with cotton products of MON 15985 and its accidental spillage. Following a weight of evidence approach and considering the poor ability of cotton to survive outside cultivated land, despite the agronomic and phenotypic data limitations, the Panel concludes that there is very low likelihood of any adverse environmental impacts. The aadA and oriV sequences in MON 15985 may facilitate the stabilisation of nptII through double homologous recombination. However, considering the limited presence of intact DNA from MON 15985 in feed and the limited occurrence of horizontal transfer of DNA from plant material to bacteria, the Panel concludes that it is highly unlikely that nptII from MON 15985 will be transferred to bacteria.

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

    Science.gov (United States)

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

    2015-11-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Pamela Lachance-Touchette

    2014-10-01

    Full Text Available 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 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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2003-09-01

    Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCl. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography-mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state (13)C cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactogluco-mannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression. PMID:12811558

  9. 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

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    Mayra Janeth Esparza-Araiza

    2015-12-01

    Full Text Available 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 resistant S. peruvianum compared to susceptible S. lycopersicum following infection by Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of the gene 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, which resulted in leaf bleaching. The ToMoV_SCEI construct resulted in approx. 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. VIGS of SCEI in S. peruvianum resulted in unilateral wilting (15 dpi and subsequent death (20 dpi of the entire plant after Cmm inoculation, whereas empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. SCEI-silenced plants also showed higher Cmm colonization with 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 WRKY transcription factors, which may lead to expression of proteins involved in salicylic acid-dependent defense responses.

  10. 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.

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    Science.gov (United States)

    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. PMID:25920485

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

    Directory of Open Access Journals (Sweden)

    Khurana Paramjit

    2003-09-01

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

  14. Description and evaluation of transformation approaches used in wheat%小麦转基因方法及其评述

    Institute of Scientific and Technical Information of China (English)

    叶兴国; 陈明; 杜丽璞; 徐惠君

    2011-01-01

    小麦是遗传转化比较困难的作物之一.为了克服小麦基因工程育种和功能基因组学研究的障碍,人们分别尝试利用基因枪、花粉管通道、超声波、离子束注入,激光微柬穿刺、PEG(Polyethylene glycol)、电击和农杆菌等方法转化小麦.涉及的受体材料包括幼胚、成熟胚、花药愈伤组织、幼穗,芽尖和花器官.文章对小麦主要遗传转化方法及其应用进行了介绍、回顾和评述,分析、比较了获得安全型转基因小麦的几种策略,以期增强读者对小麦转基因技术和进展的了解,促进小麦转化技术的持续改进和提高.%Genetic transformation is a valuable tool for direct crop improvement and functional genomics study.Unfortunately, wheat is considered as a recalcitrant plant to genetic transformation due to its low efficiency and genotype dependency.To overcome these problems, various transformation methods such as biolistic bombardment, Agrobacterium tumefaciens, pollen-tube pathway, ion implantation, laser microbeams puncture, treatment with polyethylene glycol and ultrasonic wave, and electroporation have been reported in wheat using various types of explants including immature embryos, mature embryos, anthers derived calluses, inflorescences, apical meristems, and other floral organs.In this review, several major transformation approaches and their applications in wheat are reviewed, and potential strategies for the development of safe transgenic wheat plants are discussed.The objective of this review is to provide an update on current status of wheat transformation, and to stimulate further research for improving transformation efficiency in wheat.

  15. 绿色荧光蛋白表达载体pXS75-GFP的构建及在嗜热四膜虫中的应用%Construction of Green Fluorescent Protein Expression Vector pXS75-GFP and Application in Tetrahymena thermophila

    Institute of Scientific and Technical Information of China (English)

    梁海霞; 王伟

    2012-01-01

    To construct a vector for studying the localization of protein in Tetrahymena thermophila, GFP expression vector pXS75-GFP was constructed by ligating GFP with Cd2+-inducible metallothionein (MTT1) promoter and terminator sequences. The target gene—GFP fusion gene can integrate into the MTT1 locus through homologous recombination and resistance screening. Expression of the target protein in-fusion with the C-terminal GFP tag was controllable by Cd2+. The recombinant plasmid pXS75-GFP-ATUl was constructed and biolistically transformed into Tetrahymena. The expression of a-tubulin-GFP was analyzed by Western blot. Confocal microscopy showed that a-tubulin-GFP localized at cortex in living and fixed Tetrahymena cells. The results revealed that pXS75-GFP can be used for studying the subcellular localization of proteins in Tetrahymena thermophila.%为获得能够用于构建嗜热四膜虫蛋白定位的载体,该研究将GFP基因与镉(Cd2+)诱导的四膜虫金属硫蛋白基因(MTT1)启动子序列和终止子序列融合,获得表达载体pXS75-GFP.通过同源重组和抗性筛选,pXS75-GFP载体携带的目的基因整合入四膜虫MTT1位点,在Cd2+诱导下实现GFP融合蛋白的可控表达.将α-tubulin基因ATU1克隆入pXS75-GFP中,重组质粒pXS75-GFP-ATU1通过基因枪转化入四膜虫细胞,在巴龙霉素筛选下获得稳定的α-tubulin-GFP过表达细胞株.激光共聚焦显微镜观察α-tubulin-GFP的定位,结果显示,α-tubulin-GFP融合蛋白在四膜虫细胞中表达并分布于皮层上,表明pXS75-GFP载体可用于嗜热四膜虫功能蛋白的定位分析.

  16. 优良籼型恢复系转基因植株的获得及其遗传稳定性%TRANSGENIC ELITE INDICA RESTORER LINE AND GENETIC STABILITY OF TRANSGENES

    Institute of Scientific and Technical Information of China (English)

    马炳田; 王玲霞; 李平; 朱祯; 周开达

    2004-01-01

    This paper reports that the transgenic elite indica restorer line containing snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene gna was obtained by biolistics and identified by PCR, PCR-Southern blotting and Southern blotting that the transgenes were integrated with estimated multi-copy at the same location of the genome of SH527 and transmitted stably from the first generation (T0) of transgenic plants to the third generation (T2). The plant height and seed-setting percentage of the first generation (T0) of transgenic plants were reduced apparently as compared with the corresponding plants derived from seeds and untransformed calli. However, this kind of variation could not be inherited stably. The protein analysis showed that the insect-resistant gene expressed in T0, T1 and T2 plants. The expressed protein had the biological activity and the ratio of GNA from transgenic plants to their total soluble protein was about 0.1%. The usage of genetic engineering in the breeding programs of hybrid rice was also discussed. Fig 4, Tab 2, Ref 18%报道了用基因枪转化法将雪花莲凝集素基因(gna)转移到优良籼型杂交稻恢复系蜀恢527中.PCR、PCR-Southern blotting和Southern blotting等分子检测证明,外源基因以多拷贝单位点方式整合到受体基因组中并稳定遗传到转基因第三代(T2).转基因第一代植株(T0)在株高和结实率上与相应的组培、种子实生苗植株相比,发生明显的不可遗传的变异,随着繁殖代数的增加,转基因植株恢复到与对照植株一致.Western blotting表明,目的基因在转基因植株中正确表达.蛋白活性分析显示,外源基因在转基因植株中合成的蛋白具有凝血生物活性,含量占可溶性总蛋白的0.1%左右.本文还对转基因技术在杂交稻育种中的应用进行了讨论. 图4 表2 参18

  17. Tendencias históricas de la educación general y de la sexualidad del escolar sordo / Trends of general and sexual education of death students

    Directory of Open Access Journals (Sweden)

    García, Mirna Maura

    2016-04-01

    Full Text Available La sistematización de las experiencias en el proceso educativo de los escolares sordos, permite proponer el análisis histórico dela educación de su sexualidad como componente de la personalidad. El objetivo del artículo es valorar la evolución histórica del proceso educativo de los escolares sordos y las tendencias en la educación de la sexualidad de estas personas en el contexto educativo de Cuba. El análisis tendencial fue realizado tomando como indicadores a los referentes relacionados con la evolución teórica respecto al proceso educativo de las personas sordas en general; los criterios relativos a la educación de la sexualidad de las personas con necesidades educativas especiales, particularizando en los escolares sordos; la incorporación de los contenidos relativos al tema de la sexualidad a los planes de estudio y la adecuación de estos objetivos y contenidos a las particularidades de los escolares sordos. Se constató que se ha transitado desde una etapa donde no se abordaba el tema, pasando por una fase biologicista hasta concebirla como un proceso educativo esencial en la formación de la personalidad en su condición de sujetos bilingües y biculturales. The study of the evolution of deaf students’ sexuality as a component of the individual personality is possible by systematizing experiences in its corresponding educative process. This paper is aimed at evaluating the historical evolution of the education of deaf students and the main trends in their sexuality education. This study was carried on the basis of the following indicators: theoretical framework underlying the educative process of deaf people; criteria related to sexual education of handicaps, particular deaf people, and inclusion of sexuality related topics in the curriculum. The findings lead to clear up that the main trends formerly ignore such contents, some years later it takes a biolistic approach and lately assumes them as component of individual

  18. Developing molecular tools for Chlamydomonas reinhardtii

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Harris Nilangani N

    2012-03-01

    Full Text Available Abstract 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

  20. 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

    Directory of Open Access Journals (Sweden)

    Argüello-Astorga Gerardo R

    2010-10-01

    Full Text Available Abstract 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

  1. A high-throughput transient gene expression system for switchgrass (Panicum virgatum L. seedlings

    Directory of Open Access Journals (Sweden)

    Agarwal Sujata

    2010-05-01

    Full Text Available Abstract Background Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L. has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency. Results A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS gene. The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG assays showed a peak of β-glucuronidase (GUS enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1. Conclusion Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock

  2. phbA基因对拟南芥质体转化及花粉表型分析%Genetic Transformation of phbA Gene into Arabidopsis thaliana Plastid and Pollen Phenotype of Transgenic Plants

    Institute of Scientific and Technical Information of China (English)

    杨正安; 丁玉梅; 吴兴恩; 迟淑娟; 何雍琴; 张应华

    2011-01-01

    采用来源于产碱杆菌(Alcaligenes eutrophus)的PHB合成酶基因phbA,经PCR扩增后,将验证正确的phbA插入到烟草质体表达载体pBio3-GFP中,取代载体中的gfp,形成prrn-phbA-aadA-TpsbA-ter表达盒,得到质体表达载体pCTHBA,通过基因枪介导,用包裹有质粒pCTHBA的金粉子弹轰击拟南芥无菌苗叶片,经壮观霉素筛选后获得拟南芥抗性植株12株;PCR验证初步表明,phbA已整合进拟南芥的质体基因组中;对转基因拟南芥植株的花器官表型和花粉显微观察表明,phbA基因在拟南芥中得到表达,显现出雄性不育的性状.%The most effective method in hybrid breeding is that many male sterility lines have been exploited for the production of F1 hybrid seeds.Based on the previously reported sequences of phbA gene, cloned from (Alcaligenes eutrophus) coding β-ketothiolase, we amplified phbA gene from plastid pBHR68 by PCR.After sequencing, digesting and ligasing, we substituted phbA gene for gfp gene in plastid vector pBio3-GFP,thus constructed plastid transformation vector pCTHBA that carries the expression cassette of prrn-phbA-aadA-TpsbA-ter.Then the vector was transformed into plastid of Arabidopsis thaliana using PDS-1000/He biolistic particle delivering system.We totally obtained 12 transgenic plants.The further PCR analysis fundamentally indicated that the phbA gene was intergrated into the plastid genome of A.thaliana.All 12 transgenic plants performed the male-sterile phenotype with 11 plants were lacking stamens and 1 plant failed to produce viable pollen by phenotype examination and microscopy scanning.

  3. A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

    Institute of Scientific and Technical Information of China (English)

    Yuan-Xiang Zhou; Maggie Yuk-Ting Lee; James Ming-Him Ng; Mee-Len Chye; Wing-Kin Yip; Sze-Yong Zee; Eric Lam

    2006-01-01

    AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids,and to evaluate the antigenic effect of the plastid-derived pE2 in mice.METHODS: Plastid-targeting vector pRB94-E2containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids,this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration .of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA.RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies.CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

  4. 猪传染性胃肠炎病毒纤突糖蛋白在转基因玉米中的表达%Expression of Spike Glycoprotein Gene of Porcine Transmissible Gastroenteritis Virus in Transgenic Maize

    Institute of Scientific and Technical Information of China (English)

    曾作财; 许承扬; 张晓东; 王金洛; 杨兵; 李国平

    2012-01-01

    The purpose of this study is constructing and measuring the expression of spike glycoprotein of porcine transmissible gastroenteritis virus in transgenic maize plant. To obtain transgenic maize expressing gene the spike glycoprotein of porcine transmissible gastroenteritis virus (TGEV-S), the 2.2 kb TGEV-S gene fragment was successfully amplified from TGEV-S carrying recombinant plasmid by PCR and it was inserted into the expression vector pBAC176. The recombinant plasmid pBAC176 uses the glyphosate-resistant gene EPSP as the selective marker and E35S was used as the promoter for the target gene. The pBAC176 was transformed into immature embryos of maize by biolistic bombardment transformation. Following callus induction, glyphosate resistance selection and plant regeneration, 74 plants of transgenic maize were obtained. TGEV-S gene was tested positive by PCR and Southern blot in 9 of the T0 generation transgenic plants. Further detection in T1 Transgenic maize showed that 14 transgenic lines were positive by PCR. The results showed preliminarily that the target gene displayed stable integration into the transgenic maize plants. The expression TGEV-S genes were detected in 7 lines of transgenic maize plants of T1 generation by indirect ELISA using the specific anti-TGEV-S antibody. The results from this study will provide basis for further studies on the immunogenicity of TGEV-S protein from transgenic maize, and its possibility as oral immunization.%本研究拟构建携带猪传染性胃肠炎病毒纤突糖蛋白基因(TGEV-S)的转基因玉米植株并检测其表达情况.首先利用PCR方法自携带TGEV-S基因的重组质粒中扩增2.2 kb的S基因片段,然后插入植物表达载体pBAC176中,该表达载体以编码除草剂草甘膦抗性的EPSP基因作为选择标记,目的基因以双增强子的CaMV 35S (E35S)及其玉米Hsp70第一内含子为驱动.以授粉后10~12 d约0.5~1 mm大小的玉米幼胚为受体,用基因枪进行转化,经过

  5. GENE TRANSFER IN TOBACCO MITOCHONDRIA IN VITRO AND IN VIVO

    Directory of Open Access Journals (Sweden)

    Katyshev A.I.

    2012-08-01

    -cob* genetic construct with integrative properties. It contains the selective gene and the gene of interest under control of the 5'-regulatory regions of Arabidopsis orf262 gene and the tobacco cob gene. We used modified variant of the tobacco apocytochrome b gene as a gene for selection with the nucleotide substitution G128T (G43V which results in antimycin A resistance. The maize sod3.1 gene was used as a gene of interest. The construct was delivered into tobacco callus cells and leaf disks by biolistic method. The callus lines demonstrating the high growth rates in the presence of antimycin A in comparison with the non-transformed control lines were selected. PCR analysis of transformed callus lines revealed the presence of heterologous maize sod3.1 sequence and the integration of the construct elements in tobacco mitochondrial genome.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (Scion Research.)

    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.

  7. 花青素合成转录因子基因在玉米中的表达研究:一种新型基因可视化跟踪表达系统%Expression of foreign transcription genes Bi and Cl on anthocyanin synthesis pathway in maize: A novel expression system of visual tracking for transgene

    Institute of Scientific and Technical Information of China (English)

    宫硖; 杨凤萍; 薛静; 陈绪清; 张立全; 李向龙; 张晓东

    2012-01-01

    Anthocyanin is a kind of water-soluble flavonoids, which make the plants showing red, blue, purple, red purple or other colors. Expression of the metabolic regulation gene causes the accumulation of anthocyanins in plant cells, and makes the plant organs change color, easy to observe. In this paper, using Bi and Cl gene which involved in anthocyanin synthesis pathway as a visual reporter, and glyphosate resistant gene epsp, encoding 5-enolpyruvylshikimate-3 phosphate synthase, as a selection marker, plant expression vector pBAC9009 was constructed. Immature embryos and embryogenic callus of several excellent maize inbred lines were transformed with biolistic bombardment. Undertaking in vitro culture steps of callus induction, glyphosate resistance selection, differentiation and plant regeneration, 75 transgenic plants were regenerated, and 43 ears of corn were harvested. There are 18 T0 lines expressed parts or whole purple on the seedling or the growth stage, 8 ears of these lines with scattered purple seeds. Analysis of PCR, RT-PCR and anthocyanin content showed that exogenous genes were well integrated in the genome of maize and efficiently expressed in the purple leaves and seeds. In conclusion, we successfully constructed a visual tracking system for transgene which make the transformed plants can be easily distinguished from their appearance, only by its color appearance at the early stage. Application of the system can greatly improve efficiency of transgene, and save costs. It is very important for plant transgenic research.%花青素是一种水溶性的黄酮类物质,可使植物呈现出红、蓝、紫和红紫等颜色.外源花青素代谢调控基因的表达可使花青素在植物细胞内积累,使植物体外观上表现出色彩的变化,易于观察,因此可作为报告基因用于植物转基因研究,快速报告细胞、组织、器官或植株是否被转化.本研究用花青素代谢调控基因Bi和Cl作为报告基因,以epsp作为筛

  8. 玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证%Cloning and functional validation of promoter of mo-molybdopterin cofactor sulfurase gene in maize

    Institute of Scientific and Technical Information of China (English)

    高学焕; 付凤玲; 牟巍; 周树峰; 张素芝; 李晚忱

    2014-01-01

    为克服组成型启动子启动外源基因过量表达引起的诸多问题,同源克隆(Mo-molybdopterin cofactor sulfurase)基因(ABA3)的启动子(ABA3s)序列,并用PlantCARE软件分析其非生物逆境应答元件,实时定量PCR检测 ABA3基因在非生物逆境诱导下的差异表达后。然后,用该启动子构建启动 GUS(β-glucuronidase)基因的表达载体,基因枪法转化玉米愈伤组织。经组织化学染色法检测其表达后,在高渗、高盐、低温胁迫处理及ABA诱导下检测 GUS酶荧光值与荧光素酶(内参)发光值的比值(GUS/LUC),以此评价 ABA3s启动子在非生物逆境胁迫下的启动活性。结果表明, ABA3基因在模拟干旱、低温、高温、高盐胁迫及ABA、乙稀诱导下差异表达,说明该基因的启动子(ABA3s)具有非生物逆境诱导活性。序列分析表明, ABA3s启动子全长777 bp,含有ARE、HSE、MBS、TGA、Circadian等多种非生物逆境胁迫应答元件。用ABA3s启动GUS基因构建的表达载体转化的玉米愈伤组织,响应干旱、低温、高温、高盐胁迫等多种非生物逆境胁迫,及ABA和乙稀诱导, GUS检测呈阳性。在8%甘露醇高渗条件下, GUS/LUC比值比空白对照高6倍。上述结果表明, ABA3s启动子具有非生物逆境诱导特性,经进一步验证其功能后,可用于玉米抗逆转基因研究。%To overcome the problems caused by the over-expression of exogenous genes under the control of constitutive promoters, the promoter (ABA3s) sequence of maize (Zea mays) mo-molybdopterin cofactor sulfurase gene (ABA3) was cloned homologously, analyzed for its abiotic stress-responsive elements by the PlantCARE software, and detected for dif-ferential expression of the ABA3 gene under the abiotic stresses by real-time quantitative PCR. Then, this promoter was used to construct expression vector to start GUS (β-glucuronidase) gene, and transform maize calli by biolistics. After iden-tification by

  9. 盐地碱蓬2个DREB1/CBF基因的克隆与表达调控分析%Cloning and Expression Analysis of TwoDREB1/CBF Genes inSuaeda salsaL

    Institute of Scientific and Technical Information of China (English)

    孙晓波; 苏家乐; 贾新平; 梁丽建; 肖政; 邓衍明

    2016-01-01

    DREB1/CBFgenes were cloned fromSuaeda salsa L. At the same time, the sequence characteristics, subcellular localization and transcriptional activities of the two predicted DREB1/CBF proteins and their expression alteration in response to abiotic stress were analyzed.[Method]The fragments of twoDREB1/CBF genes were obtained by using the technique of homologous cloning. The full-length of cDNA sequences of the twoDREB1/CBFgenes were isolated by the method of rapid-amplification of cDNA ends (RACE), and they were named asSsCBF1 andSsCBF2, respectively. The structures and functions of the two proteins encoded by SsCBFs were predicted by the bioinformatics software. In order to detect the subcellular localization of the two proteins, the coding sequences of the twoSsCBF genes were fused, respectively, downstream to theGFP sequence to obtain two expression vectors and they were separately transferred into onion epidermal cells by the biolistic method. The binding specificity of SsCBF1 and SsCBF2 to DRE/CRT cis-acting element and their transcriptional activities were investigated by using a yeast one-hybrid system. The expression of the twoSsCBFgenesin response to low temperature, NaCl,PEG and ABA were assessed by the real-time quantitative PCR.[Result]SsCBF1 encoded a peptide of 225 amino acid residues with a predicted molecular mass of 25.4 kD and apI of 4.84. SsCBF2encoded a predicted protein of 260 amino acid residues with a predicted molecular mass of 28.6 kD and a calculatedpI of 5.05. SsCBF1and SsCBF2both contained a typical AP2/ ERF domain andshared 53.5% and 45.4% identity at the levels of coding nucleotides and amino acids. However, the AP2/ ERF domains of the twoSsCBFgenes were highly similar and shared 76.2% and 87.3% identity at the levels of nucleotides and amino acids, respectively. SsCBF1 and SsCBF2 were classified into A-1 subgroup of the DREB subfamily and both localized to the nucleus. The two proteins were able to specifically bind to the DRE/CRT sequence

  10. Establishment and Application of Large-Scale Transformation Systems in Wheat%小麦规模化转基因技术体系构建及其应用

    Institute of Scientific and Technical Information of China (English)

    叶兴国; 徐惠君; 杜丽璞; 何光源; 王轲; 林志珊

    2014-01-01

    Wheat is a plant that is relatively difficult to be genetically modified through traditional transgenic approaches among several major field crops. Factors restricting the transformation of wheat include small scale and low efficiency of transformation, poor repeatability, and fewer availability of transgenic materials with satisfied appearances, and thus leading to the lagged-behind of wheat in transgenic breeding compared to soybean, maize, cotton, and rice. Presently, transgenic techniques mainly used in wheat transformation are biolistic particle andAgrobacterium-mediated transformation based onin vitro tissue culture, although methods of pollen tube pathway,ion beam implantation, laser microbeam puncture, polyethylene glycol (PEG) medium, pollen-mediated, andAgrobacterium dipping floral have also been employed in some laboratories. In the aspect of recipient tissues, wheat immature embryo and its derived callus were mostly used as initiating materials for the transformation of target genes, but transgenic wheat plants were also reported to be obtained by using mature embryo, inflorescence, and anther derived callus, which still need to be investigated and confirmed. As for the transformation efficiency reported in wheat, 0.1%-16.7% is for particle bombardment and 0.7%-44.8% forAgrobacterium. A big variation still existed. In the matter of alien transferring genes for wheat, the target genes are mainly involved in wheat enhancement of flour quality, disease resistance, drought tolerance, aphids resistance, and herbicide tolerance except some selection or reporter genes such asnptⅡ,bar,hpt,GUS, GOX,pmi, andALS. The transformation efficiency of wheat immature embryos mediated byAgrobacterium and particle gun is dramatically affected not only by genotypes, but also by the physiological status of mother plants which is determined by temperature, light, nutrition, and water supply during the growth period. Especially, the suitable day/night temperature during the