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Sample records for agrobacterium tumefaciens-mediated transformation

  1. Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea.

    Science.gov (United States)

    Chen, Liang; Wang, Qun; Chen, Hua; Sun, Gengwu; Liu, Huixiang; Wang, Hongkai

    2016-07-01

    Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10(5) protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis. PMID:27263001

  2. Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper.

    Science.gov (United States)

    Karthikeyan, A S; Sarma, K S; Veluthambi, K

    1996-01-01

    Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.

  3. Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species).

    Science.gov (United States)

    Prasad, Binod; Vadakedath, Nithya; Jeong, Hyun-Jeong; General, Thiyam; Cho, Man-Gi; Lein, Wolfgang

    2014-10-01

    Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 μM of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 μM of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future.

  4. Agrobacterium tumefaciens-mediated transformation of CryⅠA(b) gene to Trichoderma harzianum

    Institute of Scientific and Technical Information of China (English)

    GAO Xingxi; YANG Qian

    2004-01-01

    In this study, CryⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 106 spores by using Agrobacterium tumefaciens-mediated trans- formation. Putative transformants were analyzed to test the presence of CryⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the CryⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.

  5. Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

    Institute of Scientific and Technical Information of China (English)

    Ji-ye WANG; Hong-ye LI

    2008-01-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

  6. Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

    NARCIS (Netherlands)

    Muniz, C.R.; Silva, da C.F.; Souza, M.T.; Freire, F.C.O.; Kema, G.H.J.; Guedes, M.I.F.

    2014-01-01

    Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp)

  7. Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii.

    Directory of Open Access Journals (Sweden)

    Sook-Young Park

    Full Text Available Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway.

  8. Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding.

    Science.gov (United States)

    Zhang, Tian; Qi, Zhen; Wang, Yueyue; Zhang, Fangyuan; Li, Renyong; Yu, Qingsheng; Chen, Xiangbin; Wang, Huojun; Xiong, Xin; Tang, Kexuan

    2013-03-30

    Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/10(5)condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future. PMID:23265791

  9. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish

    2010-07-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  10. Improved dominant selection markers and co-culturing conditions for efficient Agrobacterium tumefaciens-mediated transformation of Ustilago scitaminea.

    Science.gov (United States)

    Sun, Longhua; Yan, Meixin; Ding, Zhaojian; Liu, Yanbin; Du, Minge; Xi, Pinggen; Liao, Jinling; Ji, Lianghui; Jiang, Zide

    2014-06-01

    Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen. PMID:24563317

  11. Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans

    NARCIS (Netherlands)

    Vijn, I.; Govers, F.

    2003-01-01

    Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blig

  12. Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

    Science.gov (United States)

    Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F

    2014-01-01

    Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation. PMID:24634294

  13. Agrobacterium tumefaciens-mediated transformation of a taxol-producing endophytic fungus, Cladosporium cladosporioides MD2.

    Science.gov (United States)

    Zhang, Peng; Liu, Ting-Ting; Zhou, Peng-Peng; Li, Shu-Tao; Yu, Long-Jiang

    2011-04-01

    In this study, an Agrobacteriurn tumefaciens-mediated transformation (ATMT) protocol was successfully developed for the genetic transformation of a taxol-producing fungus, Cladosporium cladosporioides MD2, and the co-cultivation conditions affecting the transformation efficiency were optimized. The optimal transformation conditions were that 1 ml of C. cladosporioides MD2 spore suspension (10(8) spores/ml) was mixed with an equal volume of A. tumefaciens cultures, which contained 400 μl of A. tumefaciens LBA4404 (OD(660) ≈ 0.6) and 600 μl LB medium that were used to make up difference in volume, and the mix cultures were supplemented with 300 μM acetosyringone (AS) and co-cultivated at 26°C and 50 rpm for 48 h. Stable transformants were obtained through analysis of the mitotic stability of inserted T-DNA and the presence of hygromycin resistance gene (hpt II). This study laid a fine groundwork for development of transgenic C. cladosporioides MD2 strains.

  14. An Improved Agrobacterium tumefaciens Mediated Transformation of Artemisia annua L. by Using Stem Internodes as Explants

    NARCIS (Netherlands)

    Tian, N.A.; Liu, S.; Huang, J.; Krol, van der A.R.; Bouwmeester, H.J.; Liu, Z.

    2013-01-01

    Transformation of Artemisia annua, which produces the sesquiterpenoid endoperoxide artemisinin widely used for the treatment of malaria, has been hampered by the low efficiency of adventitious shoot and root formation on a selective medium containing additional compounds for Agrobacterium decontamin

  15. Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops.

    Science.gov (United States)

    Singh, Roshan Kumar; Prasad, Manoj

    2016-05-01

    Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement. PMID:26660352

  16. Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.

    Science.gov (United States)

    Zhang, Yan-Jun; Zhao, Jin-Jin; Xie, Ming; Peng, De-Liang

    2014-10-01

    Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain. PMID:25107375

  17. Efficient Agrobacterium tumefaciens-mediated transformation and regeneration of garlic (Allium sativum) immature leaf tissue.

    Science.gov (United States)

    Kenel, Fernand; Eady, Colin; Brinch, Sheree

    2010-03-01

    Transgenic garlic (Allium sativum) plants have been recovered directly from immature leaf material by selective culture following Agrobacterium-mediated transformation. This method involved the use of a binary vector containing the mgfp-ER reporter gene and hpt selectable marker, and followed a similar protocol developed previously for the transformation of immature onion embryos. The choice of tissue and post-transformation selection procedure resulted in a large increase in recovery of transgenic plants compared with previously confirmed allium transformation protocols. The presence of transgenes in the genome of the plants was confirmed using Southern analysis. This improvement in frequency and the use of clonal commercial "Printanor" germplasm now makes possible the integration of useful agronomic and quality traits into this crop. PMID:20099065

  18. Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

    Institute of Scientific and Technical Information of China (English)

    WU Guan-ting; CHEN Jin-qing; HU Zhang-hua; LANG Chun-xiu; CHEN Xiao-yun; WANG Fu-lin; JIN Wei; XIA Ying-wu

    2006-01-01

    In order to improve stress tolerances of turf-type tall fescue (Festuca arundinacea Schreb.), Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses,and that relative electronic conductivity of in vitro leaves treated with low and high temperatures, dehydration and high salinity stresses was 25-30% lower in transgenic plants than in control plants. In addition, it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

  19. First Report of Agrobacterium tumefaciens mediated genetic transformation of aquatic Rice paddy herb (Limnophila aromatica

    Directory of Open Access Journals (Sweden)

    Allah Bakhsh

    2016-08-01

    Full Text Available The study presents first report of Agrobacterium mediated genetic transformation in Rice paddy herb (Linmophila aromatica. A. tumefaciens strain C58C1 harboring pBin19 Plasmid containing β-glucuronidase (GUS and neomycin phosphotransferase II (nptII gene, under the control of 35S promoter and NOS terminator was used. Shoot tip explants were inoculated for 30 min followed by co-cultivation for 72 h and selected on agar semi solidified MS medium containing 100 mg/l Kanamycin and 1.0 mg/l BA; whereas total number of 78 putative transgenic shoots were obtained. The shoots were rooted on MS medium containing 1.0 mg/l IBA and 100 mg/l Kanamycin where 43 plants survived and rooted. Expression of GUS gene in the putative transgenics was confirmed by histochemical GUS assay. Visible localised gus expression was noted in a few cells and callus tissues of 4 plantlets that were photographed using compound light microscope.

  20. Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1IA

    Institute of Scientific and Technical Information of China (English)

    YANG Ying-qing; YANG Mei; Li Ming-hai; Li Yong; HE Xiao-xia; ZHOU Er-xun

    2011-01-01

    To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solaniAG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone (AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium (ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation (ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

  1. Production of herbicide-resistant coffee plants (Coffea canephora P. via Agrobacterium tumefaciens-mediated transformation

    Directory of Open Access Journals (Sweden)

    Alessandra Ferreira Ribas

    2006-01-01

    Full Text Available Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supplemented with 5µM N6 - (2-isopentenyl-adenine (2-iP. Ten µM ammonium glufosinate was used for putative transgenic somatic embryos selection. Presence and integration of the bar gene were confirmed by PCR and Southern blot analysis. Selected transgenic coffee plants sprayed with up to 1600 mg.L-1 of FinaleTM, a herbicide containing glufosinate as the active ingredient, retained their pigmentation and continued to grow normally during ex vitro acclimation.Plantas transgênicas de Coffea canephora P resistentes ao herbicida glufosinato de amônio foram regeneradas a partir de explantes foliares co-cultivados com Agrobacterium tumefaciens EHA105 contendo o plasmídio pCambia3301 que contém os genes bar e uidA ambos sob controle do promotor 35S. Embriogênese somática direta foi induzida no meio contendo ¼ da concentração de macro, metade da concentração de micronutrientes do meio MS, constituintes orgânicos do meio B5 e 30 g.L-1 de sacarose suplementado com 5µM N6 - (2-isopentenil-adenina (2-iP e 10 µM de glufosinato de amônio para seleção de embriões transgênicos putativos. A presença e a integração do gene bar foram confirmados pelas análises de PCR e Southern blot. As plantas transgênicas selecionadas de café, pulverizadas com 1600 mg.L-1 do herbicida FinaleTM que contém glufosinato como ingrediente ativo, mantiveram a coloração e continuaram crescendo normalmente na aclimatação ex vitro.

  2. Agrobacterium tumefaciens-mediated transformation of taro (Colocasia esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a fungal pathogen Sclerotium rolfsii.

    Science.gov (United States)

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Moore, Paul H; Zhu, Yun J

    2008-05-01

    Taro (Colocasia esculenta) is one of the most important crops in the Pacific Islands, however, taro yields have been declining in Hawaii over the past 30 years partly due to diseases caused by oomycete and fungal pathogens. In this study, an efficient Agrobacterium tumefaciens-mediated transformation method for taro is first reported. In total, approximately 200 pieces (8 g) of embryogenic calluses were infected with the super-virulent A. tumefaciens strain EHA105 harboring the plant transformation plasmid pBI121/ricchi11 that contains the rice chitinase gene ricchi11. The presence and expression of the transgene ricchi11 in six independent transgenic lines was confirmed using polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). Southern blot analysis of the six independent lines indicated that three out of six (50%) had integrated a single copy of the transgene, and the other three lines had two or three copies of the transgene. Compared to the particle bombardment transformation of taro method, which was used in the previous studies, the Agrobacterium-mediated transformation method obtained 43-fold higher transformation efficiency. In addition, these six transgenic lines via Agrobacterium may be more effective for transgene expression as a result of single-copy or low-copy insertion of the transgene than the single line with multiple copies of the transgene via particle bombardment. In a laboratory bioassay, all six transgenic lines exhibited increased tolerance to the fungal pathogen Sclerotium rolfsii, ranging from 42 to 63% reduction in lesion expansion.

  3. Effects of squirting cucumber (Ecballium elaterium) fruit juice onAgrobacterium tumefaciens-mediated transformation of plants

    OpenAIRE

    ÖZCAN, SANCAR FATİH; Yildiz, Mustafa; AHMED, HUSSEIN ABDULLAH AHMED; Aasim, Muhammad

    2015-01-01

    Abstract: Different concentrations of squirting cucumber (Ecballium elaterium (L.) A.Rich.) fruit juice were added to Agrobacterium tumefaciens growth, leaf disc inoculation, and cocultivation media, to investigate its effect on the transformation frequency of tobacco and potato. A. tumefaciens strain GV2260 harboring p35S GUS-INT and pAoPR1-GUS-INT plasmids were used separately in the transformation experiments. Neomycin phosphotransferase (NPT-II) gene was used as a plant selectable marker ...

  4. Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Immature embryos of rice varieties “Xiushui11” and “Chunjiang 11” precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7(containing the spider insecticidal gene).The resistant calli were transferred onto the differentiation medium and plants were regenerated.The transformation frequency reached 56%~72% measured as numbers of Geneticin(G418)-resistant calli produced and 36%~60% measured as numbers of transgenic plants regenerated,respectively.PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome.Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder(Cnaphalocrasis medinalis)after 7d of leaf feeding reached 38%~61% and the corrected mortality of the striped stem borer(Chilo suppressalis)after 7d of leaf feeding reached 16%~75%.The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

  5. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

    OpenAIRE

    Xu, Rongfang; Li, Hao; Qin, Ruiying; WANG, LU; LI Li; Wei, Pengcheng; Yang, Jianbo

    2014-01-01

    Background The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. Findings Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospa...

  6. A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand

    2011-01-01

    transformation tool for species in which it was previously impossible to conduct molecular genetics experiments. ATMT experiments can be divided into three groups: i) Forward genetics (i.e., random mutagenesis), ii) Reverse genetics (i.e., targeted genome modification and random integration) and iii......) the introduction of reporter genes (e.g., GFP, RFP and GUS) that allow in situ monitoring of the fungus. The use of ATMT for forward genetics experiments has primarily included classic random insertional inactivation strategies to obtain loss-of-function mutants. For reverse genetics experiments, ATMT has been...... used to introduce targeted genome modifications (e.g., disruptions, replacements, overexpression and complementation) and to generate random integrations for complementation, heterologous expression, expression of transcriptional and translational fusion reporters and RNAi-mediated down...

  7. Optimization ofAgrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize (Zea maysL.)

    Institute of Scientific and Technical Information of China (English)

    YU Gui-rong; LIU Yan; DU Wen-ping; SONG Jun; LIN Min; XU Li-yuan; XIAO Fang-ming; LIU Yong-sheng

    2013-01-01

    Since maize is one of the most important cereal crops in the world, establishment of an efifcient genetic transformation system is critical for its improvement. In the current study, several elite corn lines were tested for suitability ofAgrobacterium tumefaciens-mediated transformation by using immature embryos as explants. Infection ability and efficiency of transformation ofA. tumefacienssp. strains EHA105 and LBA4404, different heat treatment times of immature embryos before infection, inlfuence of L-cysteine addition in co-cultivation medium after transformation, and how different ways of selection and cultivation inlfuence the efifciency of transformation were compared. Glyphosate-resistant gene2mG2-EPSPS was transformed into several typical maize genotypes including 78599, Zong 31 and BA, under the optimum conditions. Results showed that the hypervirulentAgrobacterium tumefacienssp. strain EHA105 was more infectious than LBA4404. Inclusion of L-cysteine (100 mg L-1) in co-cultivation medium, and heating of the immature embryos for 3 min prior to infection led to a signiifcant increase in the transformation efifciency. Growth in resting medium for 4-10 d and delaying selection was beneifcial to the survival of resistant calli. During induction of germination, adding a high concentration of 6-BA (5 mg L-1) and a low concentration of 2,4-D (0.2 mg L-1) to regeneration medium signiifcantly enhanced germination percentage. Using the optimized transformation procedure, more than 800 transgenic plants were obtained from 78599, Zong 31 and BA. By spraying herbicide glyphosate on leaves of transgenic lines, we identiifed 66 primary glyphosate-resistant plants. The transformation efifciency was 8.2%. PCR and Southern-blot analyses conifrmed the integration of the transgenes in the maize genome.

  8. Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper, using Agrobacterium tumefaciens-mediated gene transfer to shoot apical meristem cultures.

    Science.gov (United States)

    Saini, Raman; Jaiwal, Pawan K

    2005-06-01

    The efficiency of Vigna mungo L. Hepper transformation was significantly increased from an average of 1% to 6.5% by using shoot apices excised from embryonic axes precultured on 10 microM benzyl-6-aminopurine (BAP) for 3 days and wounded prior to inoculation in Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a beta-glucuronidase (GUS) gene (gusA) interrupted by an intron. The transformed green shoots that were selected and rooted on medium containing kanamycin, and which tested positive for nptII gene by polymerase chain reaction, were established in soil to collect seeds. GUS activity was detected in whole T(0) shoots and T(1) seedlings. All T(0) plants were morphologically normal, fertile and the majority of them transmitted transgenes in a 3:1 ratio to their progenies. Southern analysis of T(1) plants showed integration of nptII into the plant genome.

  9. Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation.

    Science.gov (United States)

    Boyko, Alex; Matsuoka, Aki; Kovalchuk, Igor

    2011-04-01

    Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations.

  10. Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

    Institute of Scientific and Technical Information of China (English)

    Zhang Xiao-ying; Wang Hua-fang; Yin Wei-lun; Zhu Zhen

    2006-01-01

    Agrobacterium-mediated genetic transformation of Sophorajaponica was standardized using the Agrobacterium tumefaciens strain LBA4404 that harbored the binary vector pBI121 containing genes for β-glucuronidase (GUS) and neomycin phosphotransterase (npt Ⅱ). S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets from the explants. The transformed plants resembled their parents in morphology.

  11. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    Science.gov (United States)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  12. Agrobacterium-mediated transformation: rice transformation.

    Science.gov (United States)

    Slamet-Loedin, Inez H; Chadha-Mohanty, Prabhjit; Torrizo, Lina

    2014-01-01

    Agrobacterium is a common soil bacterium with natural capacity for trans-kingdom transfer of genetic information by transferring its T-DNA into the eukaryotic genome. In agricultural plant biotechnology, combination of non-phytopathogenic strain of Agrobacterium tumefaciens with modified T-DNA and vir-genes in a binary vector system is the most widely utilized system for genetic improvement in diverse plant species and for gene function validation. Here we have described a highly efficient A. tumefaciens-mediated transformation system for indica and japonica rice cultivars based on an immature embryo system.

  13. Agrobacterium tumefaciens-mediated transformation (ATMT) for the screening for genes involved in laccase glucose repression in the pathogenic yeast Cryptococcus neoformans%利用根癌农杆菌T-DNA插入突变寻找参与漆酶葡萄糖阻遏的关键基因

    Institute of Scientific and Technical Information of China (English)

    李中明; 潘皎; 朱旭东

    2012-01-01

    [目的]新型隐球酵母(Cryptococcus neoformans)是人类重要致病真菌,主要毒性因子之一漆酶的表达受葡糖糖阻遏,机制未知.本文拟寻找参与葡萄糖阻遏的关键基因.[方法]建立根癌农杆菌介导的转化方法(Agrobacterium tumefanciens-mediated transformation,ATMT)建立一个容量约200000的随即插入突变文库,在高浓度葡萄糖条件下从中筛选葡萄糖去阻遏的突变株.通过Southern确定突变株中T-DNA的拷贝数,利用反向PCR获得依赖葡萄糖的漆酶阻遏基因序列.[结果]筛选到了30株葡萄糖去阻遏突变株,Southern blot发现83%的葡萄糖去抑制突变株含有单个T-DNA拷贝.初步鉴定了可能参与漆酶阻遏的10个不同生物学功能基因,如参与碳水化合物的代谢,固醇的合成,几丁质的合成,GPI脂锚钩的合成等等.[结论]ATMT突变策略可以找到一些参与漆酶葡萄糖阻遏的关键基因,为理解漆酶在致病过程中的作用机制和工业改进漆酶活性提供参考.%[Objective] To identify genes in glucose repression of laccase in the human pathogen Cryptococcus neoformans. [Methods] We created a random insertional mutagenesis library containing over 200000 transformants by Agrobacterium tumefaciens-mediated transformation ( ATMT). We screened the glucose derepression mutants under high-glucose condition and obtained the genes for glucose repression of laccase via inverse polymerase chains reaction ( PCR). [Results] Totally, we isolted 30 glucose derepression mutants from the library. We found that that 83% of the mutants contain a single T-DNA via Southern blot. We preliminarily identified 10 genes, which fall into a broad range of biological processes including: carbohydrate metabolism, sterol biosynthesis, chitin biosynthesis and glycosylphatidylinositol (GPI) anchor biosynthesis. Additionally, we found that three glucose derepression mutants have a single T-DNA insertion in the promoter region of LAC1, which

  14. Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum)

    Energy Technology Data Exchange (ETDEWEB)

    VanderGheynst, J.S.; Guo, H.-Y.; Simmons, C.W. [Department of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616 (United States)

    2008-04-15

    Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) experiments were performed in harvested switchgrass (Panicum virgatum) leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta {beta}-glucuronidase expression and leaf decay. Expression was scored based on a consistent pattern of visual observations of histochemical staining over the leaf surface as might be observed in stable gene expression in switchgrass leaves. Assays on extracts from leaves were also performed to measure expression levels; however, these assays showed low expression levels, which may have been due to low recombinant protein recovery and decomposition in the leaf. Bead beating was successful for wounding the plant surface, but did not improve the consistency of expression based on histochemical staining observations. Surfactant was necessary for improving contact between the leaf surface and Agrobacterium suspension and consistently improved expression when vacuum application level was low (25 kPa). Increasing vacuum application from 25 to 5 kPa improved expression only when surfactant concentration was low. When a suspension of A. tumefaciens containing 1000 ppm Break-Thru surfactant was added to harvested leaves and 25 kPa vacuum applied, a fairly uniform expression was visualized across the leaf surface within 2-3 days of incubation, suggesting that agroinfiltration is a rapid tool for examining expression of transgenes in switchgrass leaves. (author)

  15. Agrobacterium-mediated transformation of Fusarium proliferatum.

    Science.gov (United States)

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-01-01

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process. PMID:27323127

  16. Agrobacterium-mediated transformation of Fusarium proliferatum.

    Science.gov (United States)

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-06-03

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

  17. Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.

    Science.gov (United States)

    Lee, Hyeyoung; Zhang, Zhanyuan J

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds.

  18. 高渗处理对根癌农杆菌介导转化平菇菌丝体的影响%Effect of Hyper-osmosis and Co-cultivation Treatments on Agrobacterium tumefaciens-Mediated Transformation of Pleurotus ostreatus

    Institute of Scientific and Technical Information of China (English)

    任艳; 黄玉杰; 张新建; 杨合同

    2010-01-01

    采用根癌农杆菌(Agrobacterium tumefaciens ,EHA105菌株)介导法,对平菇(Pleurotus ostreatus)菌丝体遗传转化的最佳试验条件进行研究,结果表明:当平菇菌丝体与甘露醇和山梨醇的混合液预处理20 min,且与农杆菌共培养60 min时,转化效率高且用时较少.经PCR和Southern Blotting检测表明,转移DNA (transferred DNA,T-DNA)中的潮霉素抗性基因hph已整合到转化子基因组中.

  19. Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Frandsen, Mette; Giese, Nanna Henriette

    2012-01-01

    Genome sequence data on fungal pathogens provide the opportunity to carry out a reverse genetics approach to uncover gene function. Efficient methods for targeted genome modifications such as knockout and in locus over-expression are in high demand. Here we describe two efficient single-step clon...

  20. Review of Factors Affecting Organogenesis, Somatic Embryogenesis and Agrobacterium tumefaciens-Mediated Transformation of Strawberry

    NARCIS (Netherlands)

    Husaini, A.M.; Mercado, J.A.; Abdin, M.Z.; Teixeira da Silva, J.A.; Schaart, J.

    2011-01-01

    Invited Review: Standardization of an efficient regeneration system for each strawberry genotype is generally an indispensible pre-requisite for the successful development of transgenic plants. In this paper, we review some key factors affecting the regeneration of strawberry plants via adventitious

  1. Review of Factors Affecting Organogenesis,Somatic Embryogenesis and Agrobacterium tumefaciens-Mediated Transformation of Strawberry

    OpenAIRE

    Masood Husaini, Amjad; Mercado, José A; JAIME A. TEIXEIRA DA SILVA; Schaart, Jan G.

    2010-01-01

    Standardization of an efficient regeneration system for each strawberry genotype is generally an indispensible pre-requisite for the successful development of transgenic plants. In this paper, we review some key factors affecting the regeneration of strawberry plants via adventitious organogenesis or somatic embryogenesis, such as type of explant, growth regulators or dark/light treatments

  2. Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.

    Science.gov (United States)

    Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

    2014-05-01

    Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling. PMID:24154495

  3. Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.

    Science.gov (United States)

    Khan, Shamshad Ahmad; Ur Rahman, Laiq; Shanker, Karuna; Singh, Manju

    2014-05-01

    Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.

  4. One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE

    OpenAIRE

    Untergasser, A.; Bijl, G.J.M.; W. Liu; Bisseling, T.; Schaart, J.G.; Geurts, R.

    2012-01-01

    Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination...

  5. An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

    Science.gov (United States)

    Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2013-04-01

    Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

  6. Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of b-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic em-bryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of b-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zy-gotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transfor-mation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transfor-mation system could be useful for the future studies on transferring economically important genes to loblolly pine.

  7. An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

    Science.gov (United States)

    Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

    2014-01-01

    Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.

  8. Agrobacterium tumefaciens – Mediated transformation of Woodfordia fruticosa (L. Kurz

    Directory of Open Access Journals (Sweden)

    Mallesham Bulle

    2015-12-01

    Full Text Available In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L. Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA containing intron as the reporter gene and hygromycin phosphotransferase (hpt as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM and Indole-3-acetic acid IAA (1.14 μM + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1 for 4 weeks (includes a single subculture onto the same medium at a 2 week interval. They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM and Indole-3-acetic acid IAA (1.14 μM + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2 medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

  9. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    Science.gov (United States)

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  10. Agrobacterium-mediated transformation of cotton.

    Science.gov (United States)

    Zhang, Baohong

    2013-01-01

    There are many methods and techniques that can be used to transfer foreign genes into cells. In plant biotechnology, Agrobacterium-mediated transformation is a widely used traditional method for inserting foreign genes into plant genome and obtaining transgenic plants, particularly for dicot plant species. Agrobacterium-mediated transformation of cotton involves several important and also critical steps, which includes coculture of cotton explants with Agrobacterium, induction and selection of stable transgenic cell lines, recovery of plants from transgenic cells majorly through somatic embryogenesis, and detection and expression analysis of transgenic plants. In this chapter, we describe a detailed step-by-step protocol for obtaining transgenic cotton plants via Agrobacterium-mediated transformation.

  11. 根癌农杆菌介导灭蚊卵菌贵阳腐霉的遗传转化%The genetic transformation of Pythium guiyangense mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    赵竟男; 苏晓庆

    2008-01-01

    Pythium guiyangense is a mosquito pathogen,and has been proved to be a promising agent for biological control of mosquitoes.In order to develop the strains adaptable to different ecological environment having stable virulence to mosquito larvae,and being able to prolong the shelf life,an effort was made on transforming the fungus by using homologous or heterologous virulence genes.In this paper,a genetic transformation experiment of P.guiyangense mediated by Agrobacterium tumefaciens is reported.As a result,an A.tumefaciens mediated genetic transformation system was established successfully.

  12. Optimization of genetic transformation with Agrobacterium rhizogenes

    International Nuclear Information System (INIS)

    To optimize the genetic transformation efficiency using Agrobacterium rhizogenes, carrot sections inoculated with the Agrobacterium strain A4TC were co-cultivated with acetosyringone, phloroglucinol, and a mix of both. Acetosyringone is one of the phenolic compounds produced by plant tissues in response to wounding, which induces the transfer of T-DNA from the agrobacteria to the plant. Phloroglucinol is also a phenolic compound; however, it has a synergistic action with auxins by partially inhibiting cytokinin activity. The highest transformation efficiency (75%) was obtained with acetosyringone (100 mM) in combination with phloroglucinol (25 mg l-1). In general, a 6-day co-cultivation, independently of treatments, induced the best transformation rate. Inclusion of 100 mg l-1 kanamycin efficiently discriminated transformed roots from non-transgenic ones. This paper also presents a novel bacterial elimination method, by which Agrobacterium can be completely eliminated in 48 h with Cefotaxime at a dosage of 500 mg l-1. Author

  13. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell.

    Science.gov (United States)

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

  14. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C; knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

  15. 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

  16. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    Science.gov (United States)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  17. Agrobacterium-mediated transformation of Petunia leaf discs

    NARCIS (Netherlands)

    Meer, van der I.M.

    2006-01-01

    Many dicotyledonous and also several monocotyledonous plant species are susceptible to Agrobacterium-mediated transformation. This current and well-established method has been used successfully with a large number of plant species to mediate gene transfer. This chapter describes an Agrobacterium-med

  18. Agrobacterium-mediated genetic transformation of Coffea arabica (L. is greatly enhanced by using established embryogenic callus cultures

    Directory of Open Access Journals (Sweden)

    Lashermes Philippe

    2011-05-01

    Full Text Available Abstract Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%. At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our

  19. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

    DEFF Research Database (Denmark)

    Sorensen, Lisette Quaade; Lysøe, Erik; Larsen, Jesper Erup;

    2014-01-01

    The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infe...

  20. Agrobacterium-mediated transformation of Brachypodium distachyon.

    Science.gov (United States)

    Thole, Vera; Vain, Philippe

    2012-01-01

    Brachypodium distachyon is an attractive genomics and biological model system for grass research. Recently, the complete annotated genome sequence of the diploid line Bd21 has been released. Genetic transformation technologies are critical for the discovery and validation of gene function in Brachypodium. Here, we describe an efficient procedure enabling the Agrobacterium-mediated transformation of a range of diploid and polyploid genotypes of Brachypodium. The procedure relies on the transformation of compact embryogenic calli derived from immature embryos using either chemical selection alone or a combination of chemical and visual screening of transformed tissues and plants. Transformation efficiencies of around 20% can routinely be achieved using this protocol. In the context of the BrachyTAG programme (BrachyTAG.org), this procedure made possible the mass production of Bd21T-DNA mutant plant lines.

  1. Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

    Science.gov (United States)

    2012-01-01

    overexpression of DAT increased the yield of vindoline in transgenic plants. Conclusions In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants. PMID:22748182

  2. Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

    Directory of Open Access Journals (Sweden)

    Wang Quan

    2012-06-01

    showed that overexpression of DAT increased the yield of vindoline in transgenic plants. Conclusions In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11% of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene DAT involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.

  3. VIP1: linking Agrobacterium-mediated transformation to plant immunity?

    Science.gov (United States)

    Liu, Yukun; Kong, Xiangpei; Pan, Jiaowen; Li, Dequan

    2010-08-01

    Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved in plant immunity responses. Agrobacterium is able to activate and abuse VIP1 for transformation. These findings highlight Agrobacterium-host interaction and unveil how Agrobacterium hijacks host cellular mechanism for its own benefit. This review focuses on the roles played by VIP1 in Agrobacterium-mediated transformation and plant immunity. PMID:20473505

  4. VIP1: linking Agrobacterium-mediated transformation to plant immunity?

    Science.gov (United States)

    Liu, Yukun; Kong, Xiangpei; Pan, Jiaowen; Li, Dequan

    2010-08-01

    Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved in plant immunity responses. Agrobacterium is able to activate and abuse VIP1 for transformation. These findings highlight Agrobacterium-host interaction and unveil how Agrobacterium hijacks host cellular mechanism for its own benefit. This review focuses on the roles played by VIP1 in Agrobacterium-mediated transformation and plant immunity.

  5. AGROBACTERIUM-MEDIATED TRANSFORMATION OF WHEAT

    Directory of Open Access Journals (Sweden)

    K. Mészáros

    2008-09-01

    Full Text Available Transformation of cereals is one of the emerging areas for plant genomic and biotechnology research. Wheat was among the last major crops to be transformed by particle bombardment about 10 years ago. However, Agrobacterium-mediated transformation has several advantages over bombardment, including a reduction in copy number, fewer rearrangements and preferential integration into transcriptionally active chromosome regions. As a first step, we started to adapt an immature embryo-based transformation method for the model variety ‘Cadenza’. The regeneration of this variety was low and especially the cost of generating donor plants was high. Therefore, we decided (i to test regeneration capacity of winter and spring wheats using four different explants, (ii to determine the optimal genotype-regeneration system combinations, and (iii to work out the details of mature embryo transformation with Agrobacterium. The experiment was carried out with 16 cultivated winter wheat and 2 model spring wheat varieties. Four different explants: anther, immature embryo, mature embryo and dry seed were tested for callus induction and plant regeneration. The regeneration capacity was the lowest in the case of anther culture and ranged from 20% (‘Mv Béres’ to 0.1% (‘Mv Magvas’ with four varieties exerting significantly higher regeneration than ‘Cadenza’. Plant regeneration from immature embryos ranged between 59% (‘Mv Regiment’ and 0.1% (‘Mv Toborzó’. Again, four varieties produced significantly more plants than the control ‘Cadenza’. We tested two systems for the plant regeneration from mature embryos. First, mature embryos were isolated from seeds, which resulted in an average of 17% plant regeneration (from 63% in ‘Fatima’ to zero in ‘Mv Palotás’. ‘Cadenza’ was one of the worse regenerating genotype (7%. The highest plant regeneration (average 54% was in the case of seed explants. There were no significant differences

  6. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.

    Science.gov (United States)

    Paz, Margie M; Martinez, Juan Carlos; Kalvig, Andrea B; Fonger, Tina M; Wang, Kan

    2006-03-01

    The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term "half-seed" to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5-7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T1 generation by phenotypic assay using the herbicide Liberty (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method. PMID:16249869

  7. [Agrobacterium-mediated transformation of Cymbidium sinensis].

    Science.gov (United States)

    Xie, Li; Wang, Fen; Zeng, Ruizhen; Guo, Herong; Zhou, Yuliang; Zhang, Zhisheng

    2015-04-01

    Genetic transformation is an effective method to improve breeding objective traits of orchids. However, there is little information about genetic transformation of Cymbidium sinensis. Rhizomes from shoot-tip culture of C. sinensis cv. 'Qijianbaimo' were used to establish a practical transformation protocol of C. sinensis. Pre-culture time, concentration and treating methods of acetosyringone, concentration of infection bacteria fluid (OD600), infection time, and co-culture time had significant effects on β-glucuronidase (GUS) transient expression rate of C. sinensis cv. 'Qijianbaimo' rhizome. The GUS transient expression rate of rhizome was the highest (11.67%) when rhizomes pre-cultured for 39 d were soaked in bacterium suspension (OD600 = 0.9) supplemented with 200 μmol/L acetosyringone for 35 min, followed by culturing on co-culture medium supplemented with 200 μmol/L acetosyringone for 7 d. Under this transformation conditions, 3 transgenic plantlets, confirmed by GUS histochemical assay and PCR, were obtained from 400 regenerated plantlets, and the genetic transformation rate was 0.75%. This proved that it was feasible to create new cultivars by the use of Agrobacterium-mediated genetic transformation in C. sinense.

  8. The Efficient Transformation of Wheat in Planta by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    HE Dao-yi; LI Zhong-cun; WANG Hong-gang

    2003-01-01

    Transformation of wheat was performed by pipetting spikelets with Agrobacterium tumefaciens which contained expression vectors using Npt Ⅱ as reporter gene. Transformants were identified through kanamycin resistance, PCR and Southern blot. The results showed that transformation efficiency was within 2.0 to 3. 2 % in all tested varieties of wheat. Then the simple and efficient protocol of wheat transformation by Agrobacterium tumefaciens in planta was primarily established.

  9. Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

    Institute of Scientific and Technical Information of China (English)

    Lei Zhou; Jun Zhao; Wangzhen Guo; Tianzhen Zhang

    2013-01-01

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi.However,the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood.Here,we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes,VdATG8 and VdATG12,by means of targeted gene replacement and complementadon.Transformation of a cotton-infecting Verticilliun dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 106 conidia.V.dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production.Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants,compared with the wildtype and gene complemented strains.Surprisingly,in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants.These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V.dahliae.

  10. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    Science.gov (United States)

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  11. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

    Science.gov (United States)

    Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

    2015-08-01

    The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi.

  12. Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

    Institute of Scientific and Technical Information of China (English)

    LI Xiu-ying; LANG Zhi-hong; ZHANG Jie; HE Kang-lai; ZHU Li; HUANG Da-fang

    2014-01-01

    A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efifciency of 5%around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further conifrmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

  13. Agrobacterium tumefaciens-mediated Transformation of Trichoderma Harzianum%根癌农杆菌介导的木霉菌遗传转化方法

    Institute of Scientific and Technical Information of China (English)

    高兴喜; 杨谦; 宋金柱; 郭兆奎

    2004-01-01

    利用根癌农杆菌介导转化系统,成功实现了丝状真菌哈茨木霉(Trichoderma harzianum)的遗传转化,转化率约为60~190个转化子/106个孢子.PCR检测和Southern 杂交分析表明,T-DNA已整合进木霉菌基因组中,而且大多都是以单拷贝的形式整合,转化子都能够稳定遗传.农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具.

  14. Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange Transformação genética de laranja 'Hamlin' via Agrobacterium

    Directory of Open Access Journals (Sweden)

    Beatriz Madalena Januzzi Mendes

    2002-07-01

    Full Text Available The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L. Osbeck plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS assays and, subsequently, by PCR amplification for the nptII and GUS genes.O desenvolvimento e otimização de protocolos eficientes de transformação genética é essencial nos programas atuais de melhoramento de citros, tanto para porta-enxertos, como para copas de valor comercial. Plantas transgênicas de laranja 'Hamlin' (Citrus sinensis (L. Osbeck foram obtidas pela transformação genética de segmentos de epicótilo, coletados de plântulas germinadas in vitro, com Agrobacterium tumefaciens. Foram avaliados fatores que influenciam a eficiência da transformação genética, como: condições de incubação das plântulas utilizadas para coleta de explantes, tempo de inoculação com Agrobacterium e condições de co-cultivo. A regeneração de plantas a partir de segmentos de epicótilo ocorreu em alta freqüência, por organogênese direta. A maior

  15. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    Science.gov (United States)

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-04-26

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

  16. Genetic Transformation of Switchgrass

    Science.gov (United States)

    Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

    Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

  17. Study on Transformation of 2mG2-epsps Gene into Immature Embryos of Maize Inbred-line 18-599R by Agtobactetium tumefaciens Mediated%农杆菌介导的2mG2-epsps 基因转化玉米自交系18-599R 幼胚的研究

    Institute of Scientific and Technical Information of China (English)

    贾艾敏; 张玲; 蒋琴; 胡冲; 刘坚

    2015-01-01

    [Objective]In this study,a new glyphosate-resistant gene 2mG2-epsps was transformed into immature embryos of maize inbred line 18-599R by Agrobacterium tumefaciens mediated to obtain transgenic plants with significant resistance to glyphosate.[Method]2mG2-epsps gene was introduced into 18-599R immature corn embryos by Agrobacterium mediated,transformation system was optimized by control the pretreatment conditions,bacterial concentration × immer-sion time and concentration of glyphosate filter setting process.The 2mG2-epsps gene had been expressed in transgenic plant by PCR,real time PCR and spraying glyphosate exogenous gene i-dentification.[Results]Heat shock treatment didn't improve the confrontational callus rate,and centrifugation reduced the rate of resistance callus.When the concentration of Agrobacterium in the inoculation medium was adjusted to OD600 =0.6,and the infection time was 10 min,the aver-age rate of resistance callus was up to 37.33%.The ideal concentration of Glyphosate was 2.0 mmol/L.Methods using PCR of 46 transgenic plants,5 positive transgenic individuals were ob-tained,with a positive frequency of 10.87%.Real-time PCR showed that the foreign gene was ex-pressed in roots,stems and leaves and inherited to next generation stably.And the T2 generation transgenic plants showed tolerance to glyphosate.[Conclusion]2mG2-epsps gene was successful-ly imported into 18-599R genome,and gained significant glyphosate resistance.%【目的】对农杆菌介导的抗草甘膦新基因2mG2-epsps 转化玉米18-599R 幼胚的体系进行优化,以获得具有明显草甘膦抗性的转基因植株。【方法】以玉米18-599R 幼胚为受体,采用农杆菌介导法,对转化时的预处理条件、菌液浓度×浸染时间、草甘膦筛选浓度设置处理,以优化转化体系;并用 PCR、实时荧光定量 PCR 和喷洒草甘膦鉴定外源基因在转基因植株中的表达情况。【结果】热激处理对抗性愈伤率没有明显

  18. An efficient Agrobacterium-mediated transformation system for poplar.

    Science.gov (United States)

    Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

    2014-06-13

    Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides×P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.

  19. An Efficient Agrobacterium-Mediated Transformation System for Poplar

    Directory of Open Access Journals (Sweden)

    Ali Movahedi

    2014-06-01

    Full Text Available Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.

  20. Agrobacterium-mediated genetic transformation of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    WANG Hui-mei; ZU Yuan-gang

    2007-01-01

    UGPase gene related with wood cellulose synthesis was transferred into C. Acuminata using the method of Agrobacterium-mediated genetic transformation, and an efficient transformation system was developed for C. Acuminata on the basis of evaluations of several factors affecting Agrobacterium-mediated DNA transfer rate. The highest transformation rate was achieved when pre-cultured leaf explants were infected with an Agrobacterium culture corresponding to OD600 (0.5) for 10 min, and cultured on explant regeneration medium for three days. The results of Southern hybridization showed that genomic DNA of the kanamycin-resistant shoots to an UGPase gene probe substantiated the integration of the transgene. Transformation efficiency (6%) was achieved under the optimized transformation procedure. This system should facilitate the introduction of important useful genes into C. Acuminata.

  1. Strain specific Agrobacterium-mediated genetic transformation of Bacopa monnieri

    Directory of Open Access Journals (Sweden)

    Sheetal Yadav

    2014-12-01

    Full Text Available Agrobacterium-mediated genetic transformation is the most preferred strategy utilized for plant genetic transformation. The present study was carried out to analyze the influence of three different strains of Agrobacterium tumefaciens on genetic transformation of Bacopa monnieri (L. Pennell. In the present study, B. monnieri was genetically transformed with three different strains of A. tumefaciens viz. LBA4404, EHA105 and GV3101 harbouring expression vector pCAMBIA2301 containing β-glucuronidase (GUS as a reporter gene. The putative transformants were analyzed by PCR method using transgene specific primers. Expression and presence of GUS reporter protein were analyzed by histochemical staining assay and quantitative analysis of GUS enzyme was done using fluorometric assay. No statistically significant difference in transformation efficiency was found for all the three strains. Interestingly, Gus expression was variable with LBA4404 plants showing highest GUS activity.

  2. Transformation Fava Beans by Agrobacterium using Chitinase, Glucanase and CryIA (b genes

    Directory of Open Access Journals (Sweden)

    A.H Gorji

    2014-01-01

    Full Text Available In this study two plasmid vectors that are appropriate for plant transformation were made by preparation of gene cassettes for β-1, 3-glucanase from barley, chitinase from bean and cryIA (b from Bacillus thuringiensis (BT. Each of these genes were cloned under the control of the CaMV35S  promoter and the Nos terminator in pBI121 binary vector. pBI-Chi  and pBI-Glu and recombinant plasmid vectors were constructed via cloning of chitinase , β-1,3- glucanase and cryIA (b genes, respectively, instead of the gus gene in T-DNA region of pBI121 vector. Construction of pBI-ChiGlu recombinant plasmid vector was performed by means of cloning both of the complete chitinase and glucanase gene cassettes in pBI121 vector, with the intention of production synergistic effects against fungal infection.pBI-ChiBt  recombinant plasmid vector containing both of the complete chitinase and Bt gene cassettes was also constructed in order to contemporaneous plants resistance to pest pathogens and fungal in a single transformation event. pBI-ChiGlu and pBI-ChiBt that have been  introduced into the A. tumefaciens strain LBA4404 that was subsequently used for  transformation. Results indicate that embryogenic calli are well appropriate as objective material for Agrobacterium tumefaciens-mediated transformation in Faba bean.Seventeen well established shoots  were transferred to new MLS medium including suitable  antibiotics.Finally six independent transgenic plants were successfully rooted on kanamycin-containing  selection media and then transferred to soil after 20 days .Four plants out of six putative transgenic plants displaied to contain the end part of the chit transgene and nos terminator.The corresponding  piece, 700 bp of the chit gene, was amplified using specific primer.These putative transgenic plants were also be measured for the presence of the bgn13.1 and cryIA (b genes by PCR using specific primers.Two  pieces with expected sizes (1221 bp and 640

  3. Is VIP1 important for Agrobacterium-mediated transformation?

    Science.gov (United States)

    Shi, Yong; Lee, Lan-Ying; Gelvin, Stanton B

    2014-09-01

    Agrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1-Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium-mediated transformation or VirE2 subcellular localization.

  4. Agrobacterium-mediated transformation of three freshwater microalgal strains.

    Science.gov (United States)

    Sanitha, Mary; Radha, Sudhakar; Fatima, Anwar Aliya; Devi, Selvaraju Gayathri; Ramya, Mohandass

    2014-01-01

    Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

  5. Agrobacterium-mediated transformation of barley (Hordeum vulgare L.).

    Science.gov (United States)

    Ismagul, Ainur; Mazonka, Iryna; Callegari, Corinne; Eliby, Serik

    2014-01-01

    Barley biotechnology requires efficient genetic engineering tools for producing transgenic plants necessary for conducting reverse genetics analyses in breeding and functional genomics research. Agrobacterium-mediated genetic transformation is an important technique for producing barley transgenics with simple low-copy number transgenes. This chapter reports a refined protocol for the systematic high-throughput transformation of the advanced Australian spring barley breeding line WI4330.

  6. One-step Agrobacterium mediated transformation of eight genes essential for rhizobium symbiotic signaling using the novel binary vector system pHUGE.

    Directory of Open Access Journals (Sweden)

    Andreas Untergasser

    Full Text Available Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.

  7. Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea.

    Science.gov (United States)

    Bhalla, Prem L; Singh, Mohan B

    2008-01-01

    Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.

  8. EXPRESIÓN GUS EN EXPLANTES DE Solanum phureja (Juz. et. Buk Var. Criolla Colombia , TRANSFORMADOS CON Agrobacterium tumefaciens Gus Expression in Solanum phureja Explants (Juz. et. Buk Cultivar Criolla Colombia , Transformed with Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    IVÁN DARÍO BARRERO-FARFÁN

    Full Text Available La expresión transitoria y estable del gen gusA-intron en explantes internodales de papa criolla variedad Criolla Colombia cocultivados con Agrobacterium tumefaciens es reportada. Con el fin de determinar la susceptibilidad de esta variedad a la transformación mediada por A. tumefaciens, explantes internodales de Solanum phureja fueron infectados con la cepa LBA4404 de A. tumefaciens que contiene el plásmido pCAMBIA2301. Este plásmido contiene el gen ntpII que confiere resistencia a kanamicina y el gen reportero gusA-intron. La selección de los explantes potencialmente transgénicos fue realizada en medios con kanamicina. La eficiencia de transformación estable y transitoria fue calculada con base en la actividad GUS (ß-glucuronidasa, detectada por el ensayo histoquímico X-gluc. La expresión transitoria y estable del gen gusA-intron fue observada en células del explante más bien que en tejidos completos. Estos resultados demuestran que la papa criolla (S. phureja Juz. et. Buk variedad Criolla Colombia es susceptible a la infección por A. tumefaciens.The stable and transient expression of the gusA-intron reporter gene in internodal explants of "Papa Criolla" cultivar Criolla Colombia co-cultivated with Agrobacterium tumefaciens is reported. In order to determine the susceptibility of this cultivar to the A. tumefaciens-mediated transformation, internodal explants of Solanum phureja were infected by A. tumefaciens containing the vector pCAMBIA2301. This vector contains the kanamycin resistance gene ntpII and the reporter gene gusA-intron. The selection of potential transgenic explants was performed on kanamycin-containing media. The stable and transient transformation efficiency was calculated on the basis of the GUS (ß-glucuronidase activity, detected by the histochemical X-Gluc essay. Transient and stable expression of the gusA-intron gene is observed in explants cells rather than in whole tissues. Nonetheless, these results

  9. Agrobacterium-mediated transformation of ornamental species: A review

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    Milošević Snežana

    2015-01-01

    Full Text Available Integration of desirable traits into commercial ornamentals using genetic engineering techniques is a powerful tool in contemporary biotechnology. However, these techniques have had a limited impact in the domain of ornamental horticulture, particularly floriculture. Modifications of the color, architecture or fragrance of the flowers as well as an improvement of the plant tolerance/resistance against abiotic and biotic stresses using plant transformation techniques, is still in its infancy. This review focuses on the application of Agrobacterium-mediated transformation, a major plant genetic engineering approach to ornamental plant breeding and the impact it has had to date. [Projekat Ministarstva nauke Republike Srbije, br. TR 31019

  10. The effect of hygromycin on regeneration in different Alstroemeria explant types after Agrobacterium-mediated transformation

    OpenAIRE

    Kim, J. B.; Jeu, de, M.J.; Raemakers, C.J.J.M.; Jacobsen, E.; Visser, R. G. F.

    2002-01-01

    This is the first successful report of Agrobacterium-mediated transformation in Alstroemeria by infection of FEC (Friable Embryogenic Callus) lines and leaves with axil tissues. Of the transformation methods, particle bombardment and Agrobacterium-mediated transformation have been widely used to transfer foreign DNA into the plant genome. Especially, Agrobacterium tumefaciens efficiently infects most plants. Most monocotyledonous plants, including Alstroemeria, are recalcitrant to A. tumefaci...

  11. Efficient production of transgenic melon via Agrobacterium-mediated transformation.

    Science.gov (United States)

    Bezirganoglu, I; Hwang, S Y; Shaw, J F; Fang, T J

    2014-04-25

    Oriental melon (Cucumis melo L. var. makuwa) is an important fruit for human consumption. However, this plant species is one of the most recalcitrant to genetic transformation. The lack of an efficient in vitro system limits the development of a reproducible genetic transformation protocol for Oriental melon. In this study, an efficient transgenic production method for Agrobacterium-mediated transformation using cotyledon explants of Oriental melon was developed. Cotyledon explants were pre-cultivated for two days in the dark, and the optimal conditions for transformation of melon were determined to be a bacteria concentration of OD600 0.6, inoculation for 30 min, and two days of co-cultivation. Transgenic melon plants were produced from kanamycin-resistant shoots. A total of 11 independent transgenic plants were regenerated with a transformation efficiency of 0.8% of the inoculated explants. The transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the co-cultivation time.

  12. The effect of hygromycin on regeneration in different Alstroemeria explant types after Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Kim, J.B.; Jeu, de M.J.; Raemakers, C.J.J.M.; Jacobsen, E.; Visser, R.G.F.

    2002-01-01

    This is the first successful report of Agrobacterium-mediated transformation in Alstroemeria by infection of FEC (Friable Embryogenic Callus) lines and leaves with axil tissues. Of the transformation methods, particle bombardment and Agrobacterium-mediated transformation have been widely used to tra

  13. Transformation of Mortierella alpina (fatty acid supplier myceliums via AMT system (Agrobacterium Mediated Transformation

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    Aida Javanmard

    2016-09-01

    Full Text Available Introduction: Mortierella alpina is one of the most important fungi in food industry because of having ability of synthesizing unsaturated fatty acids, particularly Arashidonic Acid. This is a precursor of Eicosanoidregulate-lipoprotein metabolism which is involved in blood rheology, platelet activation and leukocyte-function, and the functional characteristics of the cell membrane. Materials and methods: In this study genetic transformation of M. alpina CBS754.68 fungus was evaluated via Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacteriums containing pBI121 vector were used for transformation of three days of old mycelia. Three days old hyphae were exposed to the bacteria with three level of time (one, two and three hours in the present of acetosyringone. Mitotic stability of the third generation of transgenic (T2 was confirmed by GUS assay and amplification of CaMV 35S promoter by polymerase chain reaction. Results: The highest percentage of transformation and mitotic stability were obtained by using A. tumefaciens and A. rhizogenese, respectively. Discussion and conclusion: The results showed that to obtain more efficient and more stable transformation, the fundamental factor is the use of suitable species of Agrobacterium. It is the first report for transformation of autothroph strain of M. alpine via Agrobacterium.

  14. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

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    Yukoh eHiei

    2014-11-01

    Full Text Available Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites, which are the basis of tissue culture and transformation in dicotyledons, in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was determined that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

  15. 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.

  16. Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants.

    Science.gov (United States)

    Anand, Ajith; Krichevsky, Alexander; Schornack, Sebastian; Lahaye, Thomas; Tzfira, Tzvi; Tang, Yuhong; Citovsky, Vitaly; Mysore, Kirankumar S

    2007-05-01

    Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore, we provide evidence supporting VIP2 interaction with VIP1, a basic domain/leucine zipper motif-containing protein required for nuclear import and integration of T-DNA. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and characterization of the Arabidopsis vip2 mutant (At vip2) demonstrate that VIP2 is required for Agrobacterium-mediated stable transformation but not for transient transformation. Assays based upon a promoter-trap vector and quantification of T-DNA integration further confirmed VIP2 involvement in T-DNA integration. Interestingly, VIP2 transcripts were induced to a greater extent over prolonged periods after infection with a T-DNA transfer-competent Agrobacterium strain compared with the transfer-deficient Agrobacterium strain. Transcriptome analyses of At vip2 suggest that VIP2 is likely a transcriptional regulator, and the recalcitrancy to transformation in At vip2 is probably due to the combination of muted gene expression response upon Agrobacterium infection and repression of histone genes resulting in decreased T-DNA integration events. PMID:17496122

  17. Development of Transgenic Papaya through Agrobacterium-Mediated Transformation

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    Md. Abul Kalam Azad

    2013-01-01

    Full Text Available Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII gene as the selectable marker and β-glucuronidase (GUS as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

  18. Factors Affecting Agrobacterium-Mediated Transformation Efficiency in Rice

    Institute of Scientific and Technical Information of China (English)

    CHEN En-hui; ZHANG Ping; ZUO Shi-min; LI Ai-hong; ZHANG Ya-fang; CHEN Zong-xiang; PAN Xue-biao

    2004-01-01

    Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was concerned, CC medium was the best and the quality of callus was improved with the addition of 1.0 to 2.0 mg/L ABA. It decreased the percentage of browning calli and improved the callus growing state by addition of a certain amount of sorbitol to the subculture medium. NB medium was the best for callus initiation of japonica rice, but the improvement in the quality of callus of japonica subspecies was not obvious by adding ABA. During the period of subculture, to a certain degree, increasing the sucrose concentration could improve the proportion of hygromycin resistant calli. Furthermore, the transformation efficiency would be higher by applying selection pressure in the selection stage, removing selection pressure during the plantlet differentiation period and applying selection pressure again during seedling hardening period. Besides, suitable combination of plant hormones was beneficial for callus differentiation. An efficient Agrobacterium-mediated rice transformation system had been established for several rice cultivars and a lot of transgenic rice plants had been obtained.

  19. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori

    NARCIS (Netherlands)

    Michielse, C.B.; Ram, A.F.J.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins

  20. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    NARCIS (Netherlands)

    Michielse, C.B.; Arentshorst, M.; Ram, A.F.; Hondel, C.A. van den

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated tra

  1. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

    Science.gov (United States)

    Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

    2015-01-01

    The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

  2. Agrobacterium-mediated transformation of Ruta graveolens L.

    Science.gov (United States)

    Lièvre, Karine; Tran, Thi Lê Minh; Doerper, Sébastien; Hehn, Alain; Lacoste, Paul; Thomasset, Brigitte; Bourgaud, Frédéric; Gontier, Eric

    2009-01-01

    Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

  3. Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Sharma Krishna K

    2010-09-01

    Full Text Available Abstract Background White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA. The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi. Results This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA, green fluorescent protein (gfp and hygromycin phosphotransferase (hpt to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls

  4. Leaf Disk Transformation of Lactuca sativa Using Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Sergiu VALIMAREANU

    2010-12-01

    Full Text Available Reliable methods of transferring cloned genes into plants are essential for engineering crops with desired traits. In order to induce sap-sucking insect resistance Agrobacterium tumefaciens LBA 4404 (1065 strain containing the binary vector pMOG 23 and hypervirulent pTOK 47 plasmid was used. pta (Pinellia ternata agglutinin, salmon ct (calcitonin and cgrp (calcitonin gene related protein genes were successfully integrated into Lactuca sativa plants. This genetic modification conferred lettuce resistance to orthopteran and homopteran insects like Nilaparvata lugens Stl or Myzus persicae Sulzer. Lactuca sativa could be routinely transformed using Ti plasmids of A. tumefaciens containing a chimeric kanamycin resistance gene (nos nptII. nos.

  5. A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus

    OpenAIRE

    Chen, Xi; Stone, Michelle; Schlagnhaufer, Carl; Romaine, C. Peter

    2000-01-01

    We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

  6. Effect of Agrobacterium Induced Necrosis, Antibiotic Induced Phytotoxicity and Other Factors in Successful Plant Transformation

    Directory of Open Access Journals (Sweden)

    Sandip S. Magdum

    2013-08-01

    Full Text Available Agrobacterium tumefaciens infection and antibiotic wash are the critical steps of Agrobacterium mediated plant transformation procedure, most time responsible for lower transformation efficiency due to necrosis and phytotoxicity caused by biotic stress of Agrobacterium and abiotic stress by antibiotics respectively. Ammi majus Egyptian origin medicinal plant and Pearl millet cereal grain crop were studied for their stress responses to Agrobacterium mediated transformation (AMT. Agrobacterium strains LBA4404 (O.D.=0.6-0.8 and EHA105 (O.D.=0.2-0.4 were used for transformation experiments to infect calli of Ammi majus and embryogenic calli of Pearl millet respectively. Incase of antibiotic wash, Cefotaxime 500 mg L-1 was used for LBA4404 infected Ammi majus calli and Timentin 300 mg L-1 was used for EHA105 infected embryogenic calli of Pearl millet. Effects of Agrobacterium infection, antibiotic and NaOCl washes on Agrobacterium removal and both explants physiological changes during transformation experimental procedures were studied. At the end of the experiments explants survival efficiency of Ammi majus and pearl millet were 8% and 5% respectively. Biotic and abiotic stress factors responsible for lower efficiency were investigated with various other factors and strategies were discussed which are need to be considered for higher transformation events and target tissue survival.

  7. Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens.

    Science.gov (United States)

    Akutsu, M; Ishizaki, T; Sato, H

    2004-03-01

    An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration. Calli derived from ovules were co-cultivated with A. tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively. These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers. Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin. A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks. The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis. Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture. The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR. PMID:14615906

  8. Host genes involved in Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Soltani, Jalal

    2009-01-01

    Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the w

  9. Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

    OpenAIRE

    TOPRAK, Umut; COUTU, Cathy; BALDWIN, Doug; Erlandson, Martin; Hegedus, Dwayne

    2014-01-01

    Plant-mediated RNA interference (RNAi) has shown a great potential in pest control and requires i) subcloning of sense/antisense regions in compatible vectors, ii) transfer of the silencing cassette into a binary vector, iii) transformation of Agrobacterium tumefaciens with desired binary plasmids, and iv) transformation of plants with Agrobacterium. The procedure is long and should ensure plasmid backbone stability; however, plasmid recombination due to antibiotic selection is a common probl...

  10. Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

    Science.gov (United States)

    Pelczar, Pawel; Kalck, Véronique; Gomez, Divina; Hohn, Barbara

    2004-06-01

    Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis. PMID:15153934

  11. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

    Science.gov (United States)

    Zhang, Wan-Jun; Dewey, Ralph E; Boss, Wendy; Phillippy, Brian Q; Qu, Rongda

    2013-02-01

    Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

  12. 农杆菌介导甘蔗梢腐病病原菌YN41的遗传转化%Agrobacterium tumefaciens-Mediated Transformation of Sugarcane Pokkah Boeng Pathogen YN41

    Institute of Scientific and Technical Information of China (English)

    郭强; 王鑫; 徐世强; 王继华; 崔一平; 张木清

    2016-01-01

    甘蔗梢腐病是甘蔗生长中期的主要真菌病害.目前,研究甘蔗梢腐病病原菌和甘蔗品种的互作机制是防治梢腐病害的重点.本研究运用农杆菌介导的转化方法将含有绿色荧光蛋白(GFP)的双元载体PCA-MBgfp转化到甘蔗梢腐病野生菌株(YN41)中,获得41个转化子.随机挑取7个转化子进行PCR扩增,均可扩增出潮霉素基因目标条带;且转化菌株的菌落形态、生长速度和致病力与野生型菌株相比没有显著差异;单孢继代培养后仍能发出稳定的绿色荧光,并且能够稳定遗传.同时发现60 μg/mL潮霉素B能够完全抑制甘蔗梢腐病菌株YN41的生长.以上结果表明GFP基因已成功转化到甘蔗梢腐病菌株YN41中,可为后续甘蔗梢腐病病原菌和甘蔗品种的互作机制研究提供技术支撑.

  13. Agrobacterium tumefaciens-mediated transformation of the lichen forming fungus Cladonia metacorallifera%根癌农杆菌介导的地衣型真菌Cladonia metacorallifera的转化

    Institute of Scientific and Technical Information of China (English)

    王毅; 王晨晨; 周旭; 许宰铣; 王娟

    2015-01-01

    以潮霉素抗性和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为筛选标记,利用地衣型真菌Cladonia metacorallifera的菌丝,成功实现了根癌农杆菌介导的遗传转化,PCR检测证明转化子中存在潮霉素抗性基因,共聚焦显微镜检测到转化子菌丝能够产生绿色荧光,证明EGFP能够在trpC启动子控制下在地衣型真菌中表达.

  14. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

    Science.gov (United States)

    Michielse, C B; Ram, A F J; Hooykaas, P J J; Hondel, C A M J J van den

    2004-05-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. PMID:15050546

  15. A high-efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    Science.gov (United States)

    Ozawa, Kenjirou

    2012-01-01

    Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

  16. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

    Science.gov (United States)

    Srinivasan, Ramachandran

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer. PMID:27351975

  17. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

    Directory of Open Access Journals (Sweden)

    Ramachandran Srinivasan

    Full Text Available An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404. In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells followed by GV3101 (128 ± 5.29 cfu per 106 cells and EHA105 (61 ± 5.03 cfu per 106 cells. However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

  18. Agrobacterium rhizogenes-Mediated Transformation – a Non-GMO Platform For Developing Compact Ornamentals

    DEFF Research Database (Denmark)

    Lütken, Henrik Vlk; Hegelund, Josefine Nymark; Lauridsen, Uffe Bjerre;

    of these compounds are potentially harmful to both the environment and human health. A new non-GMO molecular breeding strategy, as opposed to both the application of chemical growth retardants and conventional molecular breeding is Agrobacterium rhizogenes-mediated transformation. In this method, the soil borne...... for transformations, plants produced via this approach are not considered as GMOs in the European Union and Japan. We have developed an optimised Agrobacterium rhizogenes-mediated transformation platform useful for a wide range of ornamentals. Kalanchoë was the starting point and the effect of the rol-genes has now...

  19. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    Science.gov (United States)

    Michielse, C B; Arentshorst, M; Ram, A F J; van den Hondel, C A M J J

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

  20. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination.

    Science.gov (United States)

    Mishiba, Kei-ichiro; Chin, Dong Poh; Mii, Masahiro

    2005-07-01

    A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both beta-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l(-1) hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

  1. Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

    NARCIS (Netherlands)

    Ooms, G.; Burrell, M.M.; Karp, A.; Bevan, M.; Hille, J.

    1987-01-01

    Derivatives of potato (Solanum tuberosum cv.'s 'Maris Bard' and 'Desiree') transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixed-cultures

  2. Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium

    DEFF Research Database (Denmark)

    Trieu, A.T.; Burleigh, S.H.; Kardailsky, I.V.;

    2000-01-01

    Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium....... The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method....... Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T-2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M...

  3. Agrobacterium-mediated transformation: state of the art and future prospect

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation and its application. Many details of the key molecular events within the bacterial cells involved in T-DNA transfer have been elucidated, and it is notable that some plant factors which were elusive before are purified and characterized. Vast kinds of species, which were either recalcitrant to or not included in the host range of Agrobacterium, can now be transformed by this bacterium, and they include the very important cereal species, gymnosperms, yeast and many filamentous fungi. The simple in vivo transformation of tissue in intact plants and the "agrolistic" methods to transform recalcitrant plants are the two novel technical achievements. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragment can be transformed into the plant genome by Agrobacterium. Further studies will elucidate more plant-encoded factors involved in T-DNA transformation and there is a need to develop more powerful Agrobacterium-based transformation systems to meet different needs in basic research and crop improvement practice.

  4. Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum

    Directory of Open Access Journals (Sweden)

    Syamsidah Rahmawati

    2015-11-01

    Full Text Available This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica, Nipponbare (Japonica, and Rojolele (Javanica. Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05% was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtainedby Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations.Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefaciens

  5. Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.

    Science.gov (United States)

    Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki

    2013-01-01

    The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background.

  6. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    Science.gov (United States)

    Ozawa, Kenjirou

    2009-04-01

    Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

  7. Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

    Science.gov (United States)

    Ceasar, S Antony; Ignacimuthu, S

    2011-09-01

    A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

  8. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    Science.gov (United States)

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.

  9. Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration.

    Science.gov (United States)

    Bakshi, Souvika; Sadhukhan, Ayan; Mishra, Sagarika; Sahoo, Lingaraj

    2011-12-01

    An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

  10. Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens.

    NARCIS (Netherlands)

    Mikosch, T.S.P.; Lavrijssen, B.; Griensven, van L.J.L.D.

    2001-01-01

    Agrobacterium tumefaciens is known to transfer parts of its tumour-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus. A

  11. Visualizing virulence proteins and their translocation into the host during agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Sakalis, Philippe Alexandre

    2013-01-01

    The project focuses on visualizing Agrobacterium Mediated Transformation (AMT) of host cells by real time microscopy. With new visualization techniques the function of several proteins, which have recently been discovered in our lab to play a role during AMT, are studied.

  12. In Vitro Callogenesis and Agrobacterium-Mediated Transformation of Globe Artichoke

    NARCIS (Netherlands)

    Menin, B.; Moglia, A.; Comino, C.; Lanteri, S.; Herpen, van T.W.J.M.; Beekwilder, M.J.

    2012-01-01

    Micropropagation techniques have been widely applied in globe artichoke (C. cardunculus L. var. scolymus), however, efficient protocols for the establishment of in vitro callogenesis and organogenesis, a pre-requisite for Agrobacterium-mediated genetic transformation, have not been set up so far. We

  13. Agrobacterium-mediated transformation of the white-rot fungus Physisporinus vitreus.

    Science.gov (United States)

    Schubert, M; Stührk, C; Fuhr, M J; Schwarze, F W M R

    2013-11-01

    The biotechnologically important white-rot fungus Physisporinus vitreus was co-cultivated with Agrobacterium tumefaciens AGL-1 carrying plasmids with nourseothricin resistance as the selectable marker gene and red fluorescence protein as a visual marker. Mitotically stable transformed isolates were obtained showing red fluorescence protein activity.

  14. Agrobacterium-mediated transformation as a tool for functional genomics in fungi

    NARCIS (Netherlands)

    Michielse, C.B.; Hooykaas, P.J.J.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

    2005-01-01

    In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and t

  15. Agrobacterium-mediated transformation as a tool for functional genomics in fungi.

    NARCIS (Netherlands)

    Michielse, C.B.; Hooykaas, P.J.; Hondel, C.A. van den; Ram, A.F.

    2005-01-01

    In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and t

  16. 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.

  17. Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

    NARCIS (Netherlands)

    Schreuder, M.M.; Raemakers, C.J.J.M.; Jacobsen, E.; Visser, R.G.F.

    2001-01-01

    An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 10

  18. Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

    Indian Academy of Sciences (India)

    R Chakrabarty; N Viswakarma; S R Bhat; P B Kirti; B D Singh; V L Chopra

    2002-09-01

    A number of factors that are known to influence genetic transformation were evaluated to optimize Agrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the synthetic cryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.

  19. Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment.

    Science.gov (United States)

    Gao, Caixia; Long, Danfeng; Lenk, Ingo; Nielsen, Klaus Kristian

    2008-10-01

    Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.

  20. Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer.

    Science.gov (United States)

    Pickardt, T; Meixner, M; Schade, V; Schieder, O

    1991-02-01

    Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C1 pGV 3850 HPT, carrying a plasmid coding for hygromycin-phosphotransferase. On callus-induction medium containing 60 mg/l hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis.

  1. Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata).

    Science.gov (United States)

    The effect of Agrobacterium growth phase and density on transformation of citrus rootstock US-812 (Citrus reticulata x Poncirus trifoliata) epicotyl explants was determined. In the first experiment, Agrobacterium EHA105 containing pBINGUSint was grown in YEP medium to an OD600 of 1 and glycerol sto...

  2. Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

    Science.gov (United States)

    Bull, S E; Owiti, J A; Niklaus, M; Beeching, J R; Gruissem, W; Vanderschuren, H

    2009-01-01

    Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

  3. Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem's Delight orchid with higher efficiency.

    Science.gov (United States)

    Gnasekaran, Pavallekoodi; Antony, Jessica Jeyanthi James; Uddain, Jasim; Subramaniam, Sreeramanan

    2014-01-01

    The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600 nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 μM acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

  4. Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency

    Directory of Open Access Journals (Sweden)

    Pavallekoodi Gnasekaran

    2014-01-01

    Full Text Available The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

  5. Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes

    Institute of Scientific and Technical Information of China (English)

    吕德扬; 曹学远; 唐顺学; 田霞

    2000-01-01

    Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of trans-genie plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.

  6. Regeneration of foreign genes co-transformed plants of Medicago sativa L by Agrobacterium rhizogenes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium tumafeciens. Regeneration of transgenic plants was induced successfully from hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally transformed recipient. There was a negative correlation between age of hairy roots and embryogenesis frequency in alfalfa. Production of co-transformed plants with greater yield and super quality was important for development of new alfalfa varieties.

  7. A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens

    OpenAIRE

    Srivastava, Toolika; Das, Sandip; Sopory, Sudhir Kumar; Srivastava, P. S.

    2009-01-01

    Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration influenced transformation efficiency. Under optimal co-cultivation condit...

  8. Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration.

    Science.gov (United States)

    Subramanyam, Kondeti; Subramanyam, Koona; Sailaja, K V; Srinivasulu, M; Lakshmidevi, K

    2011-03-01

    A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.

  9. An efficient regeneration protocol for Agrobacterium-mediated transformation of melon (Cucumis melo L.).

    Science.gov (United States)

    Zhang, H J; Gao, P; Wang, X Z; Luan, F S

    2014-01-08

    An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation, using cotyledon node zone-stem connection region of melon, has been developed. The new Agrobacterium-mediated transformation methodology, independent of organ culture, used the entire germinated seed as explants. The transformation system was maximized to maintain the integrity of melon itself, thus avoiding the limitations of traditional tissue culture methods. The transformation was carried out under a non-sterile environment. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed by PCR and Southern blot analyses. The transformation frequency based on the PCR was 13%. Transgenic melon plants were usually detected by PCR in less than 1 month after Agrobacterium inoculation, and seeds could be harvested in 3 months. The growth characteristics and morphology of the transgenic plants were identical to the untransformed wild-type plants. This method would be beneficial for facilitating the characteristics of gene functions and for boosting the manipulation of melon transformation for commercial purposes.

  10. Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

    Science.gov (United States)

    Tang, Wei; Lin, Jinxing; Newton, Ronald J

    2007-05-01

    Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

  11. Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

    Directory of Open Access Journals (Sweden)

    Hiroaki Mano

    Full Text Available The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium. We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP. Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholinoethanesulfonic acid (MES buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max and pea (Pisum sativum. The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

  12. 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.

  13. 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.

  14. AGROBACTERIUM MEDIATED TRANSFORMATION OF PIGEONPEA (CAJANUS CAJAN L MILLLSP VAR LRG-41 FROM AXILLARY BUD

    Directory of Open Access Journals (Sweden)

    T. Raghavendra

    2014-02-01

    Full Text Available A reliable method of plant regeneration has been achieved from Axillary buds. Shoots appeared from explants when cultured on Murashige and skoog (MS medium supplemented with BAP (Benzyl amino purine, Napthalene acetic acid (NAA and Kinetin at various combinations. Elongated shoots were rooted with 70.6% rooting frequency in MS medium with indole buteric acid (IBA at 1.0mg/l. The rooted plantlets were established well in soilrite mixture medium with 91% success and days taken for acclimatization were 12.8 days. This protocol was used in Agrobacterium mediated transformation. The transformation was carried out using the Agrobacterium strain LBA4404 containing the binary vector pCAMBIA2301 harboring npt II as selectable marker and GUS as reporter gene.

  15. AGROBACTERIUM MEDIATED TRANSFORMATION OF PIGEONPEA (CAJANUS CAJAN L MILLLSP VAR LRG-41 FROM AXILLARY BUD

    Directory of Open Access Journals (Sweden)

    T. Raghavendra

    2014-03-01

    Full Text Available A reliable method of plant regeneration has been achieved from Axillary buds. Shoots appeared from explants when cultured on Murashige and skoog (MS medium supplemented with BAP (Benzyl amino purine, Napthalene acetic acid (NAA and Kinetin at various combinations. Elongated shoots were rooted with 70.6% rooting frequency in MS medium with indole buteric acid (IBA at 1.0mg/l. The rooted plantlets were established well in soilrite mixture medium with 91% success and days taken for acclimatization were 12.8 days. This protocol was used in Agrobacterium mediated transformation. The transformation was carried out using the Agrobacterium strain LBA4404 containing the binary vector pCAMBIA2301 harboring npt II as selectable marker and GUS as reporter gene.

  16. Successful Agrobacterium-mediated transformation of Populus tomentosa with apple SPDS gene

    Institute of Scientific and Technical Information of China (English)

    LIU Ting-ting; PANG Xiao-ming; LONG Cui; ZHANG Zhi-yi

    2008-01-01

    The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase (SPDS) genes derived from an apple by an Agrobacterium-mediatod method. Four transgenic clones were confirmed by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.

  17. Agrobacterium-mediated transformation of Cichorium intybus L. with interferon-a2b gene

    Directory of Open Access Journals (Sweden)

    Kvasko O. Yu.

    2009-04-01

    Full Text Available An efficient method for the plant regeneration and Agrobacterium mediated transformation with interferon-a2b gene has been developed for chicory C. intybus L. cv. Pala rossa. The regeneration with efficiency about 100 % was induced on the MS medium supplemented with 0.5–2.5 mg/l kinetin and 0.5 mg/l NAA. The transformed plantlets were recovered at a frequency 26,9 % on basal medium with 25 mg/l kanamycin. According to PCR-analysis the nptII and ifn-a2b genes were integrated into the genome of transformed plants.

  18. Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Kishchenko, E M; Komarnitskii, I K; Kuchuk, N V

    2011-01-01

    Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).

  19. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    Science.gov (United States)

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  20. A rapid and stable Agrobacterium-mediated transformation method of a medicinal plant Chelone glabra L.

    Science.gov (United States)

    Gao, Zhenrui; Li, Ying; Chen, Jinhua; Chen, Zhixing; Cui, Min-Long

    2015-03-01

    Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene (NPT II) as a selectable marker and a reporter gene β-glucuronidase (GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the β-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2-8 % after 2-3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra.

  1. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    Science.gov (United States)

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments. PMID:26338266

  2. A novel system for Agrobacterium-mediated transformation of wheat( Triticum aestivum L.) cells

    Institute of Scientific and Technical Information of China (English)

    XUYAO; BAOJIANLI; JINGFENJIA

    1993-01-01

    A new approach for transforming the cultured cells of wheat (Triticum aestivum L.cv.Ganmai 8)was developed vsing Agrobacterium tumefaciens. The features of the optimum procedure were:(a)both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA)cells for approximately 16h:(b)the gyratory magnetic field condition was used during cocultivation;(c)the cocultivating period and selecting condition were modified;(d)the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium.Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT Ⅱ and NOS genes.located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo.in transformed wheat cell colonies by adopting the techniques of dot blot ndPAGE or high voltage paper electrophoresis,Integration of the foreign genes into wheat genome was confirmed by Southerm blot hybridization.Moreover.a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.

  3. Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments

    Directory of Open Access Journals (Sweden)

    Almeida Weliton Antonio Bastos de

    2003-01-01

    Full Text Available Genetic transformation allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. The objective of this research was to establish a protocol for genetic transformation of Valencia and Natal sweet oranges (Citrus sinensis L. Osbeck and Rangpur lime (Citrus limonia L. Osbeck. Epicotyl segments of germinated in vitro plantlets (three weeks in darkness and two weeks in a 16-h photoperiod were used as explants. These were co-cultivated with Agrobacterium tumefaciens strain EHA-105 and different experiments were done to evaluate the transformation efficiency: explants were co-cultivated with Agrobacterium for one, three or five days; explants were incubated with Agrobacterium suspension for 5, 10, 20 or 40 minutes; co-cultivation medium was supplemented with acetosyringone at 0, 100 or 200 mmol L-1; Explants ends had a longitudinal terminal incision (2-3 mm; co-cultivation temperatures of 19, 23 or 27degreesC were imposed. The experimental design was completely randomized in all experiments with five replications, each consisted of a Petri dish (100 x 15 mm with 30 explants and resulted in a total of 150 explants per treatment. Longitudinal terminal incision in the explant ends did not improve shoot regeneration. However, transgenic plants of all three cultivars were confirmed from explants that had been subjected to inoculation time of 20 minutes, co-culture of three days at 23-27degreesC, in the absence of acetosyringone.

  4. Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat.

    Science.gov (United States)

    Ding, Liping; Li, Shengchun; Gao, Jianming; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

    2009-01-01

    Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.

  5. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    Science.gov (United States)

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

  6. Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

    Institute of Scientific and Technical Information of China (English)

    CAO Ying; Niimi Yoshiyuki; HU Shang-lian

    2006-01-01

    A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics (including ampicillin,carbenicillin, cefotaxime, and cefoperazone), meropenem showed the highest activity in suppressing all tested A.tumefaciens strains (minimum inhibitory concentration [MIC] < 0.5 mg L-1, which is equal to minimum bactericidal concentration [MBC]). Meropenem, at all tested concentrations, except for 10 mg L-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector pIG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L-1 meropenem and 25 mg L-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

  7. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

    Science.gov (United States)

    Kwon, Tackmin

    2016-01-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  8. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    Science.gov (United States)

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  9. Genetic Transformation of Metroxylon sagu (Rottb. Cultures via Agrobacterium-Mediated and Particle Bombardment

    Directory of Open Access Journals (Sweden)

    Evra Raunie Ibrahim

    2014-01-01

    Full Text Available Sago palm (Metroxylon sagu is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L. Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

  10. Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

    Directory of Open Access Journals (Sweden)

    José M. Alvarez

    2013-01-01

    Full Text Available An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII as a selectable marker gene and β-glucuronidase (uidA as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass was achieved when a vigorously growing embryonal mass (embryogenic line L01 was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

  11. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    Science.gov (United States)

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

  12. Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

    Science.gov (United States)

    Alvarez, José M; Ordás, Ricardo J

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

  13. Agrobacterium Mediated Transformation of Fld and GUS Genes into Canola for Salinity Stress

    Directory of Open Access Journals (Sweden)

    Niapour, Nazila

    2013-04-01

    Full Text Available Salinity is one of the major abiotic stress which limits wide spread canola cultivation. One way to overcome this problem could be transfection, to produce tolerable species. Cotyledonary and hypocotyls explants obtained from 4 and 7 days old seedling of Elite and RJS003 varieties were utilized in this study. Genetic transformation was implemented through Agrobacterium tumefaciens LBA4404 containing PBI121 plasmid and Agrobacterium tumefaciens C58, LBA4404, AGL0 and EHA 101 strains which contain P6u- ubi- fvt1 construct. The T-DNA region of P6u- Ubi- Fvt1 plasmid included HPT (Hygromycin phosphotransferase plant selectable marker and Fld (flavodoxin gene. PBI121 plasmid had NptII (Neomycin phosphotransferase plant Selectable marker and β-glucuronidase (GUS reporter genes. Transfected explants were analyzed by PCR and histochemical assay for Fld and Gus genes, respectively. Our data indicated that the cotyledonary explants of both cultivars were incompetent to be infected with Fld gens. However, the transformation in Elite hypocotyls explants with Agrobacterium tumefaciens C58 and LBA 4404 strains were confirmed through PCR product and histochemical evaluation for Fld and GUS genes, respectively. Therefore, the result of this manuscript may to certain degree fulfill the endeavor appointed to this oilseed.

  14. Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.

    Science.gov (United States)

    Guo, M; Zhang, Y L; Meng, Z J; Jiang, J

    2012-03-16

    Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

  15. Agrobacterium-mediated transformation of Cichorium intybus L. with interferon-a2b gene

    OpenAIRE

    Kvasko O. Yu.; Gerasymenko I. M.; Shachovsky A. M.; Matvieieva N. A.; Kuchuk N. V.

    2009-01-01

    An efficient method for the plant regeneration and Agrobacterium mediated transformation with interferon-a2b gene has been developed for chicory C. intybus L. cv. Pala rossa. The regeneration with efficiency about 100 % was induced on the MS medium supplemented with 0.5–2.5 mg/l kinetin and 0.5 mg/l NAA. The transformed plantlets were recovered at a frequency 26,9 % on basal medium with 25 mg/l kanamycin. According to PCR-analysis the nptII and ifn-a2b genes were integrated into the genome of...

  16. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    Science.gov (United States)

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  17. An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

    Directory of Open Access Journals (Sweden)

    Fatemeh Haddadi

    2015-02-01

    Full Text Available An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100% of shoot formation and the highest mean number of shoots (24 produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86% in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  18. Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces.

    Directory of Open Access Journals (Sweden)

    Claire J Knight

    Full Text Available BACKGROUND: Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus. METHODOLOGY/PRINCIPAL FINDINGS: We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum on a range of wounded plant tissues. Fungal transformants were obtained from co-cultivation on a range of plant tissue types, demonstrating that plant tissue provides sufficient vir gene inducers to allow A. tumefaciens to transform fungi in planta. CONCLUSIONS/SIGNIFICANCE: This work raises interesting questions about whether A. tumefaciens may be able to transform organisms other than plants in nature, or indeed should be considered during GM risk assessments, with further investigations required to determine whether this phenomenon has already occurred in nature.

  19. Setaria viridis floral-dip: A simple and rapid Agrobacterium-mediated transformation method

    Directory of Open Access Journals (Sweden)

    Polyana Kelly Martins

    2015-06-01

    Full Text Available Setaria viridis was recently described as a new monocotyledonous model species for C4 photosynthesis research and genetic transformation. It has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements that make it suitable for use as a model plant. We report an alternative method of S. viridis transformation using floral dip to circumvent the necessity of tissue culture phase for transgenic plant regeneration. S. viridis spikes at boot stage were selected to be immersed in Agrobacterium suspension. T1 seeds could be identified in 1.5–2 months after floral dipping. We demonstrated through molecular analysis and RFP expression that seeds and resulting plants from dipped inflorescences were transformed. Our results suggest the feasibility of S. viridis floral dip transformation as a time-saving and cost-effective compared with traditional methods. To our knowledge, this is the first report using floral dip in S. viridis as an Agrobacterium-mediated transformation method.

  20. An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system.

    Science.gov (United States)

    Haddadi, Fatemeh; Aziz, Maheran Abd; Abdullah, Siti Nor Akmar; Tan, Soon Guan; Kamaladini, Hossein

    2015-02-23

    An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  1. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato

    Indian Academy of Sciences (India)

    Manoj K Sharma; Amolkumar U Solanke; Dewal Jani; Yogendra Singh; Arun K Sharma

    2009-09-01

    We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and -glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

  2. IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation.

    Science.gov (United States)

    Bhattacharjee, Saikat; Lee, Lan-Ying; Oltmanns, Heiko; Cao, Hongbin; Veena; Cuperus, Joshua; Gelvin, Stanton B

    2008-10-01

    Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin alpha proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin alpha members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed. PMID:18836040

  3. Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens.

    Science.gov (United States)

    Saini, R; Sonia; Jaiwal, P K; Jaiwal, S

    2003-06-01

    Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.

  4. Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

    Directory of Open Access Journals (Sweden)

    Xue Han

    2013-01-01

    Full Text Available Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L 6-benzylaminopurine and (0.08 mg/L naphthaleneacetic acid, we have achieved the highest frequency (90% for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0 and an infection time (20–30 min. According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30% than older leaves (10%.

  5. DNA METHYLATION ANALYSIS DURING THE OPTIMIZATION OF Agrobacterium-MEDIATED TRANSFORMATION OF SOYBEAN.

    Science.gov (United States)

    Jiang, J; Wing, V; Xiet, T; Shi, X; Wang, Y P; Sokolov, V

    2016-01-01

    Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (Decrease in DNA methylation 1 and DNA methyltransferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.

  6. Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata.

    Science.gov (United States)

    Ma, Kai; Hu, Chun Gen; Xu, Bing; Yao, Jia Ling

    2013-09-01

    Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2 % of explants developed highly friable callus on medium containing 3.0 mg l(-1) 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2 mg l(-1) α-naphthalene acetic acid and 6.0 mg l(-1) 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105-harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter-was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.

  7. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    Science.gov (United States)

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  8. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    Science.gov (United States)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  9. Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.

  10. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    Science.gov (United States)

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-30

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

  11. An improved plant regeneration and Agrobacterium - mediated transformation of red pepper (Capsicum annuum L.).

    Science.gov (United States)

    Kumar, R Vinoth; Sharma, V K; Chattopadhyay, B; Chakraborty, S

    2012-10-01

    Capsicum annuum (red pepper) is an important spice cum vegetable crop in tropical and subtropical countries. Here, we report an effective and reproducible auxin free regeneration method for six different red pepper cultivars (ACA-10, Kashi Anmol, LCA-235, PBC-535, Pusa Jwala and Supper) using hypocotyl explants and an efficient Agrobacterium-mediated transformation protocol. The explants (hypocotyls, cotyledonary leaves and leaf discs) collected from axenic seedlings of six red pepper cultivars were cultured on either hormone free MS medium or MS medium supplemented with BAP alone or in combination with IAA. Inclusion of IAA in the regeneration medium resulted in callus formation at the cut ends of explants, formation of rosette leaves and ill defined shoot buds. Regeneration of shoot buds could be achieved from hypocotyls grown in MS medium supplemented with different concentrations of BAP unlike other explants which failed to respond. Incorporation of GA3 in shoot elongation medium at 0.5 mg/l concentration enhanced the elongation in two cultivars, LCA-235 and Supper, while other cultivars showed no significant response. Chilli cultivar, Pusa Jwala was transformed with βC1 ORF of satellite DNA β molecule associated with Chilli leaf curl Joydebpur virus through Agrobacterium tumefaciens. Transgene integration in putative transformants was confirmed by PCR and Southern hybridization analysis.

  12. HIGH FREQUENCY GENETIC TRANSFORMATION OF CICHORIUM INTYBUS L. USING nptII GENE AS A SELECTIVE MARKER.

    Science.gov (United States)

    Matvieieva, N; Shakhovsky, A; Kvasko, O; Kuchuk, N

    2015-01-01

    Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or "hairy" roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of "hairy" roots formation varied from 5.9 to 42.3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes- and A. tumefaciens-mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A. rhizogenes-mediated transformation the highest regeneration frequency up to 42.3% was demonstrated using p CB161 vector with ifn-α2b target gene and nptII selective gene. PMID:26419064

  13. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp.

    Science.gov (United States)

    Cha, Thye-San; Chen, Chin-Fong; Yee, Willy; Aziz, Ahmad; Loh, Saw-Hong

    2011-03-01

    The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.

  14. Agrobacterium-mediated transformation of chickpea with -amylase inhibitor gene for insect resistance

    Indian Academy of Sciences (India)

    S Ignacimuthu; S Prakash

    2006-09-01

    Chickpea is the world’s third most important pulse crop and India produces 75% of the world’s supply. Chickpea seeds are attacked by Callosobruchus maculatus and C. chinensis which cause extensive damage. The -amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into chickpea cultivar K850 through Agrobacterium-mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb -amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of -amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevil C. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1.

  15. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri.

    Science.gov (United States)

    Rathod, Jayant Pralhad; Prakash, Gunjan; Pandit, Reena; Lali, Arvind M

    2013-11-01

    Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.

  16. Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation.

    Science.gov (United States)

    Saika, Hiroaki; Toki, Seiichi

    2010-12-01

    We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning--a common problem with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied our efficient transformation system to Kasalath--a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting.

  17. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata: an important tool for functional study of genes and crop improvement

    Directory of Open Access Journals (Sweden)

    Evans eNyaboga

    2014-09-01

    Full Text Available Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp. with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by PCR, Southern blot analysis and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by RT-PCR analysis. Transformation efficiency varied from 9.4% to 18.2% depending on the cultivars, selectable marker genes and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop.

  18. Agrobacterium-Mediated Transformation of Glycine Max and Regene rati on of Transgenic Plants%根癌农杆菌介导β-1,4-半乳糖苷转移酶基因转化大豆及其转基因植株再生

    Institute of Scientific and Technical Information of China (English)

    张毅; 李弘剑; 张俊辉; 郭勇

    2001-01-01

    A reproducible transformation system was developed for soybean (Glycin e max) using as explants sections from the excised hypocotyls of seeds. A constr uct containing cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII) and human β-1,4-galactosyltransferase (hGT) was introduced into soybean culti var using Agrobacterium tumefaciens- mediated transfermation procedures. Regenerat i on was via organogenesis and transformed plants were selected on medium containi ng 50 mg/L kanamycin and 100 mg/L of aminopenicillanic acia. Transgenic soybeans were raised in the glasshouse. The hGT genes was integrated into the chomosomal genome of primary transgenic soybean plants, The transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase activity and Sout hern blotting analyses.%以大豆下胚轴为外植体,通过根癌农杆菌介导转化法,建立起良好的转化系统,将人的β-1,4-半乳糖苷转移酶基因(hGT)导入大豆。经转化的外植体在添加100mg/L氨苄青霉素和50mg/L卡那霉素的选择培养基中可诱导出愈伤组织和芽再生,在1/2MS培养基上诱导生根,并培养成再生苗,获得了完整的抗性再生植株。进行Southern blot分子杂交鉴定,证实了外源基因hGT已稳定地整合到植物基因组中。

  19. An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei.

    Science.gov (United States)

    Kummasook, Aksarakorn; Cooper, Chester R; Vanittanakom, Nongnuch

    2010-12-01

    We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 10(4) conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.

  20. Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice.

    Science.gov (United States)

    Saika, Hiroaki; Nonaka, Satoko; Osakabe, Keishi; Toki, Seiichi

    2012-11-01

    Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence.

  1. Agrobacterium-mediated Transformation of Rice (Oryza sativa L.) with Atrazine Chlorohydrolase Gene (atzA)

    Institute of Scientific and Technical Information of China (English)

    WANG Song-wen; SHI Li-li; SUN Zong-xiu; CAI Bao-li; FU Ya-ping; WANG Yang; SI Hua-min; LIU Xia; ZHANG Xin

    2005-01-01

    Atrazine chlorohydrolase gene (atzA) was cloned from Arthrobacter sp. AD1. A plant expression plasmid was constructed under the control of CaMV35s promoter and was used in rice transformation. The target gene was successfully introduced into mature embryos of a japonica rice cultivar Jindao 107 by Agrobacterium- mediated transformation and hundreds of transgenic plants were obtained. The exogenous atzA gene in the transgenic plants that expressed atrazine resistance was confirmed by Southern blot hybridization. The resistance experiments by spraying transgenic rice plants with 0.133% atrazine shown that most of the transgenic rice plants exhibited the resistance to herbicide atrazine. The segregation of exogenous atzA gene in T1 progeny corresponded to the Mendelian ratio.

  2. Regeneration and gene transformation systems of Robinia pseudoacacia 'Idaho' mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    Li Min; Cai Zao; Sun De-you; Yin Wei-lun; Chen Shou-yi; Wang Hua-fang

    2006-01-01

    Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.

  3. A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis

    Directory of Open Access Journals (Sweden)

    Polyana Kelly Martins

    2015-06-01

    Full Text Available The production and use of sugarcane in Brazil is very important for bioenergy production and is recognized as one of the most efficient in the world. In our laboratory, Setaria viridis is being tested as a model plant for sugarcane. S. viridis has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements that make it suitable for use as a model system. We report a highly efficient protocol for Agrobacterium-mediated genetic transformation of S. viridis. The optimization of several steps in tissue culture allowed the rapid regeneration of plants and increased the rate of transformation up to 29%. This protocol could become a powerful tool for functional genomics in sugarcane.

  4. Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production

    Directory of Open Access Journals (Sweden)

    Elfahmi

    2014-01-01

    Full Text Available Background: Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose. Aim: To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants. Setting and Designs: The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient β-glucuronidase (GUS. Materials and Methods: Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency. Results: The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of

  5. Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.

    Science.gov (United States)

    Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki

    2008-07-01

    Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

  6. 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.

  7. Agrobacterium mediated transformation of brassica juncea (l.) czern with chitinase gene conferring resistance against fungal infections

    International Nuclear Information System (INIS)

    Brassica juncea (Czern and Coss., L.) is an important oilseed crop. Since it is attacked by several bacterial and fungal diseases, therefore, we developed an easy and simple protocol for the regeneration and transformation of B. juncea variety RAYA ANMOL to give rise to transgenic plants conferring resistance against various fungal diseases. The transformation was carried out using Agrobacterium with Chitinase gene. This gene was isolated from Streptomyces griseus HUT6037. We used two types of explants for transformation i.e. hypocotyls and cotyledons. Only hypocotyls explants showed good results regarding callus initiation. Different hormonal concentrations were applied i.e. BAP 2, 4 and 6 mgL-1 and NAA 0.1, 0.2 and 0.3 mgL-1. However, high transformation efficiency was observed by supplementing the medium with combination of 2 mgL-1 BAP and 0.2 mgL-1 for initiation of callus. Similarly 10 mgL-1 kanamycin and 200 mgL-1 cefotaxime also proved successful for the selection of transformed callus. In order to confirm the presence of transgenic callus Polymerase chain reaction was performed using specific primers for Chitinase gene. (author)

  8. Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles.

    Directory of Open Access Journals (Sweden)

    Yi-Hong Wang

    Full Text Available BACKGROUND: Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. METHODOLOGY/PRINCIPAL FINDINGS: Previously we identified two genes (LeTGA1 and SOLly GLB1 induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected. CONCLUSION: This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.

  9. Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.

    Science.gov (United States)

    Gasparis, Sebastian; Bregier, Cezary; Orczyk, Waclaw; Nadolska-Orczyk, Anna

    2008-11-01

    This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes.

  10. Knock down Os1bglu1 β-glucosidase in rice by Agrobacterium-mediated transformation

    Directory of Open Access Journals (Sweden)

    Mariena Ketudat-Cairns

    2011-02-01

    Full Text Available This research attempted to study the function of Os1bglu1 by RNAi technique. The suppression of Os1bglu1genewas done using the 3’UTR region. The target gene fragment was cloned into the pHELLSGATE8 vector. The high percentagesof effective callus induction of 93% were obtained when the seeds were cultured on N6D medium for 4-6 weeks at 28°C. Thesuitable transformation conditions were to incubate the calli with Agrobacterium (OD 600 = 0.02 and blot dry to remove excessbacteria cells, then transferred to co-cultivation medium (pH 5.2 with 200 M acetosyringone and incubate for three days at25°C. The 20% transformation efficiency was obtained from the transformed calli with control plasmid, while transformationefficiency of only 15% was obtained from pHELLSGATE8 Os1bglu1 constructs. The transformed calli with control constructshowed higher growth rate than the transformed calli with pHELLSGATE8 Os1bglu1construct. The expression of Os1bglu1mRNA was not found in the transformed calli and siRNAs were found in the transformed calli. However no siRNAs weredetected in the control transformed calli. The regeneration efficiencies of 6% were obtained from only the calli transformedwith the control construct. The calli transformed with the knock down Os1bglu1 constructs were not able to regenerate. This may indicated that Os1bglu1 is involved in regeneration of rice from callus tissue.

  11. Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

    Directory of Open Access Journals (Sweden)

    Singh Surinder P

    2011-05-01

    Full Text Available Abstract Background Safflower (Carthamus tinctorius L. is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content and WT (high linoleic acid content genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

  12. Mutant acetolactate synthase (ALS) gene as a selectable marker for Agrobacterium-mediated transformation of soybean

    Institute of Scientific and Technical Information of China (English)

    Chen Shiyun; Zhang Yong

    2006-01-01

    Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean transformation systems with different selectable marker genes have been reported, e.g. antibiotic (kanamycin or hygromycin) resistant genes and herbicide ( glufosinate, glyphosate) resistant selectable marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase (ALS) gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide selection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant.PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.

  13. [Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

    Science.gov (United States)

    Zhou, Yue; Zhao, Xupeng; Wu, Xiuhua; Zhang, Yanling; Zhang, Lin; Luo, Keming; Tang, Shaohu

    2014-06-01

    Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

  14. Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

    Science.gov (United States)

    Zhang, Fuli; Chen, Can; Ge, Honglian; Liu, Jinmei; Luo, Yunling; Liu, Kun; Chen, Long; Xu, Kedong; Zhang, Yi; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

  15. RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation

    OpenAIRE

    Crane, Yan Ma; Gelvin, Stanton B

    2007-01-01

    We investigated the effect of RNAi-mediated gene silencing of 109 Arabidopsis thaliana chromatin-related genes (termed “chromatin genes” hereafter) on Agrobacterium-mediated root transformation. Each of the RNAi lines contains a single- or low-copy-number insertion of a hairpin construction that silences the endogenous copy of the target gene. We used three standard transient and stable transformation assays to screen 340 independent RNAi lines, representing 109 target genes, for the rat (res...

  16. Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens, not achievable with untransformed protoplasts.

    Science.gov (United States)

    Steffen, A; Eriksson, T; Schieder, O

    1986-04-01

    Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens "shooter" strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of "leaf disc and stem segment cloning" and co-cultivation experiments varied from 5×10(-3) to 5×10(-5). After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular "shooter" mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.

  17. Identification and localization of transformed cells in agrobacterium tumefaciens-induced plant tumors

    Science.gov (United States)

    Rezmer; Schlichting; Wachter; Ullrich

    1999-10-01

    Agrobacterium tumefaciens-induced tumors of dicotyledonous plants consist of well-defined vascular bundle-like structures originating from transformed cells. The current view that 25% of the tumor cells are transformed has been re-investigated by using beta-glucuronidase (gus)-gene-containing wild-type bacteria (A281 p35S gus-int). Regularly growing stem and leaf tumors showed irregular GUS-staining patterns in the different plant species, Ricinus communis L., Cucurbita maxima L., Vicia faba L. and Kalanchoe daigremontiana Hamet et Perrier. Variable staining and inconsistency between staining and tumor growth suggested an inhibition of gus expression. By polymerase chain reaction (PCR) and reverse transcriptase-PCR analyses it became evident that gus is also integrated into the DNA of unstainable tumor parts but not expressed. These results and area calculations of tissues unable to contain the bacterial transferred-DNA with gus provide strong evidence that in A. tumefaciens-induced tumors most cells, or even all, are transformed, i.e. ca. 100%. PMID:10550620

  18. Study on Transformation of Oriental Hybrid Lily by Agrobacterium-mediated

    Institute of Scientific and Technical Information of China (English)

    FAN Jinping; ZHANG Chen; ZHANG Yingying; ZHOU Xiaojie; CHE Daidi

    2009-01-01

    An efficient procedure was described for the transformation of the monocotyledonous oriental hybrid lily, Lilium cv. Siberia, by Agrobacterium-mediatsd genetic transformation via leaves regeneration. The leaves of leaflets which derived from bulbs were sliced into 1.0 cm long and were co-cultivated with A. tumefaciens strain LBA4404/pB2AE12, which harbored a vector carrying the neomycin phosphotransferase, DREB2A genes in the T-DNA region. The suitable genetic transformation condition was determined as follows: the bacterial concentration reached 0.5-0.6 (OD600), 15 min infection time, 20 mg·L-1 acetosyingone, and 10.6 mmol·L-1 NH4NO3 medium was used for co-cultivation 3 days, delayed 7 days for selecting by 30 mg·L-1 kanamycin containing regeneration medium. Efficient shoot regeneration was observed on MS medium supplemented with 1.0 mg·L-1 naphthaleneacetic acid, 0.5 mg·L-1 benzyladenine and 0.1 mg·L-1 Kinetin after about 6 weeks culture. The presence of DREB1A gene in the genomic DNA of regenerated plants was detected by means of PCR analysis.

  19. High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens.

    Science.gov (United States)

    Nizam, Shadab; Verma, Sandhya; Singh, Kunal; Aggarwal, Rashmi; Srivastava, Krishna Dutt; Verma, Praveen K

    2012-03-01

    Bipolaris sorokiniana, the causal agent of spot blotch of wheat, significantly reduces grain yield worldwide. In order to study pathogenic mechanisms of the fungus, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. To study different stages of hyphal fusion and pathogenic mechanisms of the fungus, two fluorescence markers viz. the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constitutively expressed. Southern hybridizations confirmed the presence of T-DNA in all hygromycin B or geneticin resistant transformants, and also showed random and single copy integration. Fluorescence microscopy suggested the high level expression of both DsRed and EGFP fluorescent proteins in spores and mycelia. The results signify that DsRed and EGFP can be used as efficient reporter gene for monitoring B. sorokiniana hyphal fusion as well as colonization in the host tissues. This work will be useful to develop methodologies for understanding the mechanisms of Bipolaris-wheat interaction and functional genomics of B. sorokiniana for various applications including insertional mutagenesis, targeted disruption of specific genes, ectopic complementation of loss-of-function strains and over-expression.

  20. [Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens].

    Science.gov (United States)

    WEI, Kai-Fa

    2009-11-01

    In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively. PMID:19933098

  1. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  2. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

    Science.gov (United States)

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T; Vogel, John P; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

  3. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum

    Science.gov (United States)

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; Vogel, John P.; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation. PMID:27252729

  4. Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

    Science.gov (United States)

    Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

    2015-01-01

    Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features. PMID:26681030

  5. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    Directory of Open Access Journals (Sweden)

    Ray eCollier

    2016-05-01

    Full Text Available The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

  6. Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

    Science.gov (United States)

    Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

    2015-12-14

    Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

  7. Agrobacterium-mediated transformation in chickpea (Cicer arietinum L.) with an insecticidal protein gene: optimisation of different factors.

    Science.gov (United States)

    Indurker, Shivani; Misra, Hari S; Eapen, Susan

    2010-07-01

    Agrobacterium-mediated transformation in chickpea was developed using strain LBA4404 carrying nptII, uidA and cryIAc genes and transformants selected on Murashige and Skoog's basal medium supplemented with benzyladenine, kinetin and kanamycin. Integration of transgenes was demonstrated using polymerase chain reaction and Southern blot hybridization of T0 plants. The expression of CryIAc delta endotoxin and GUS enzyme was shown by enzyme linked immunosorbent assay and histochemical assay respectively. The transgenic plants (T0) showed more tolerance to infection by Helicoverpa armigera compared to control plants. Various factors such as explant source, cultivar type, different preculture treatment period of explants, co-cultivation period, acetosyringone supplementation, Agrobacterium harboring different plasmids, vacuum infiltration and sonication treatment were tested to study the influence on transformation frequency. The results indicated that use of epicotyl as explant, cultivar ICCC37, Agrobacterium harboring plasmid pHS102 as vector, preculture of explant for 48 h, co-cultivation period of 2 days at 25°C and vacuum infiltration for 15 min produced the best transformation results. Sonication treatment of explants with Agrobacteria for 80 s was found to increase the frequency of transformation.

  8. A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation.

    Science.gov (United States)

    Cardoza, Rosa Elena; Vizcaino, Juan Antonio; Hermosa, Maria Rosa; Monte, Enrique; Gutiérrez, Santiago

    2006-08-01

    Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

  9. 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.

  10. Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.

    Science.gov (United States)

    Rodrigues, Maria Beatriz Calderan; Fávaro, Léia Cecília de Lima; Pallu, Ana Paula de Souza; Ferreira, Anderson; Sebastianes, Fernanda de Souza; Rodrigues, Maria Juliana Calderan; Spósito, Marcel Bellato; de Araújo, Welington Luiz; Pizzirani-Kleiner, Aline Aparecida

    2013-01-01

    Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

  11. Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

    Institute of Scientific and Technical Information of China (English)

    LIU Juan-xu; YU Yi-xun; LEI Jian-jun; CHEN Guo-ju; CAO Bi-hao

    2009-01-01

    This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper (Capsicum annuum L.). Chili pepper inbred lines (A, D, E, and I) were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR [MB (MS mineral+vitamine B5)+BA (6-Benzyladenine) 5.0 mg L-1 +IAA (indoleacetic acid) 1.0 mg L-1 +GA3 (gibberellic acid) 1.0 mg L-1 + sucrose 3% +agar 6.5 g L-1] for 2 d. The explants were infected by Agrobacterium tumefaciens when their OD600 (optical density at 600 nm) reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB + BA 5.0 mg L-1 + IAA 1.0 mg L-1 + GA3 1.0 mg L-1 + sucrose 3% + agar 6.5 g L-1 + AS (acetosyringone)200 μmol L-1], these cotyledons with petiole were cultured on selective differentiation medium in the media MT [MB medium supplemented with BA [5.0 mg L-1 + IAA 1.0 mg L-1 + GA3 1.0 mg L-1 + AgNO3 5.0 mg L-1 + CW (coconut water) 5% +Km (kanamycin) 65 mg L-1 + Cb (carbenicillin) 500 mg L-1 + 3% sucrose + agar 6.5 g L-1]. The Kmr (kanamycin resistant) bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen

  12. Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS) Gene

    OpenAIRE

    Erly Marwani; Agustina Tangapo; Fenny Martha Dwivany

    2015-01-01

    This study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS) and hygromycinphosphotransferase (hpt) genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS express...

  13. Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

    Science.gov (United States)

    Uçarlı, C; Tufan, F; Gürel, F

    2015-02-06

    Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

  14. Transformation of japonica rice with RHL gene and salt tolerance of the transgenic rice plant

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Overexpression of the yeast HAL2 gene increases salt tolerance of yeast and plant. Rice HAL2-like (RHL) gene was introduced into a japonica rice cultivar HJ19 with Agrobacterium tumefaciens-mediated transformation. Transgenic plants in R0 generation were selected on the principle of GUS-positive, RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R1 generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R1 generation showed that the RHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.

  15. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

    Science.gov (United States)

    Nyaboga, Evans N; Njiru, Joshua M; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.

  16. Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

    Institute of Scientific and Technical Information of China (English)

    LI Dong-dong; SHI Wei; DENG Xiu-xin

    2003-01-01

    Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cultivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg L-1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

  17. Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers.

    Science.gov (United States)

    Nanasato, Yoshihiko; Konagaya, Ken-ichi; Okuzaki, Ayako; Tsuda, Mai; Tabei, Yutaka

    2011-08-01

    An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige-Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T(0) generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T(1) seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T(1) generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.

  18. Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.

    Science.gov (United States)

    Bilichak, Andriy; Yao, Youli; Kovalchuk, Igor

    2014-06-01

    Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.

  19. Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules

    DEFF Research Database (Denmark)

    Holme, Inger; Brinch-Pedersen, Henrik; Lange, Mette;

    2012-01-01

    Agrobacterium-mediated transformation of in vitro cultured barley ovules is an attractive alternative to well-established barley transformation methods of immature embryos. The ovule culture system can be used for transformation with and without selection and has successfully been used to transfo...

  20. Agrobacterium-Mediated Stable Transformation of Medicinal Plant Andrographis paniculata Callus Expressing β-glucuronidase (GUS Gene

    Directory of Open Access Journals (Sweden)

    Erly Marwani

    2015-11-01

    Full Text Available This study was carried out to establish a stable genetic transformation in callus culture of Andrographispaniculata mediated by Agrobacterium tumefaciens. The leaf disks of A. paniculata were infected with A. tumefaciensLBA4404 carrying a binary vector pCAMBIA1304 that contain β-glucuronidase (GUS and hygromycinphosphotransferase (hpt genes. The infection was conducted by dipping method for one hour, followed byco-cultivation in the dark for three days. To examine transient GUS expression, the co-cultivated leaf disks wereassayed for β-glucuronidase activity and to obtain stable transformed callus, the co-cultivated leaf disks wereselected on the callus induction medium which contain 20 mg/l hygromycin for selection. The transformedcallus was periodically subcultured every three weeks into the fresh selection medium over the 15 weeksperiod. To test a stable transformation, the callus was subjected to PCR analysis for GUS gene detection. Theresults indicated that the co-cultivated leaf disks expressed GUS activity and proliferated to produce callus onthe selective medium. Analysis of PCR on the transformed callus indicated the presence 976 bp fragment thatconfi rmed the presence of β-glucuronidase gene. These fi ndings imply that the β-glucuronidase was stablyintegrated into A. paniculata callus culture.Keywords: Andrographis paniculata, Agrobacterium tumefaciens, andrographollide, transformed callus,β-glucuronidase gene.

  1. Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

    Science.gov (United States)

    Swain, S S; Sahu, L; Pal, A; Barik, D P; Pradhan, C; Chand, P K

    2012-02-01

    Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD(660) ≅ 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation. PMID:22806869

  2. FEASIBILITY OF HYGROMYCIN AS A SELECTION AGENT IN AGROBACTERIUM-MEDIATED TRANSFORMATION OF OILSEED RAPE (BRASSICA NAPUS L.

    Directory of Open Access Journals (Sweden)

    Tímea Kuťka Hlozáková

    2014-02-01

    Full Text Available In this work the feasibility of the antibiotic hygromycin as a selection agent in Agrobacterium-mediated transformation of oilseed rape (Brassica napus L. was evaluated. For this, two economically important commercial varieties Haydn and Hunter and tobacco as a model plant were subjected to Agrobacterium-mediated transformation. The 5-6 days-old oilseed rape hypocotyls and 4-6 weeks-old tobacco leaf segments were transformed with the binary vector pCambia1304. The T-DNA contained the reporter gfp:gus and the selectable marker htp genes. Regeneration of transformed cells was conducted under selection of 10 mg.l-1 (oilseed rape and 30 mg.l-1 (tobacco hygromycin. Putative transgenic plantlets were analysed by the mean of the histochemical GUS and PCR analyses. Transformation efficiency ranged from 1.0% (cv. Haydn to 40.4% (tobacco. No transgenic shoots were detected for the cv. Hunter. It points out the oilseed rape cultivar specificity plays significant role in choice of suitable selection agent.

  3. Transformation of indica rice (Oryza sativa L. cv. RD6 mediated by Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Manit Kosittrakul

    2004-01-01

    Full Text Available High percentage of callus induction at 97% was obtained when seeds of rice (Oryza sativa L. cv. RD6 were cultured on modified N6 medium supplemented with 3% (w/v sucrose, 22.5 μM 2,4-D and 0.8% agar under light condition. The suitable regeneration medium was N6 medium supplemented with 3% (w/v sucrose, 2.5 μM IAA, 18 μM BA and 0.8% agar. A test had been performed to determine the effect of antibiotics on the regeneration of rice cv. RD6. It was found that kanamycin concentration up to 150 mg l-1 and hygromycin concentration at 10 mg l-1 were effective for selection of transformants. Cefotaxime and carbenicillin concentration up to 250 mg l-1 had the highest phytotoxicity to plant regeneration. Agrobacterium-mediated gene transfer protocols for rice cv. RD6 were performed using A. tumefaciens strain LBA4404, which harbored the plasmid pBI121 containing genes for β- glucuronidase (GUS and kanamycin resistance (nptII, and strain EHA105, which harbored plasmid pCAMBIA1301 containing genes for β-glucuronidase (GUS and hygromycin resistance (hptII. GUS activities were found in rice calli after co-cultivation. A number of morphologically normal fertile transgenic rice plants were obtained. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transgenic plants in T0 and T1 generation. Mendelian segregation was observed in T1 progeny.

  4. Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Patil, Gunvant; Deokar, Amit; Jain, P K; Thengane, R J; Srinivasan, R

    2009-11-01

    To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l(-1) mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l(-1) mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.

  5. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L. Dunal.

    Directory of Open Access Journals (Sweden)

    Ganeshan Sivanandhan

    Full Text Available In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites. In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90% in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR, and Southern blott analysis. These transformed plants (T0 and T1 were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%. Withanolides (withanolide A, withanolide B, withanone and withaferin A contents of transformed plants (T0 and T1 were marginally higher than control plants.

  6. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2015-01-01

    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.

  7. Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

    Institute of Scientific and Technical Information of China (English)

    Hai-Hong Shang; Chuan-Liang Liu; Chao-Jun Zhang; Feng-Lian Li; Wei-Dong Hong; Fu-Guang Li

    2009-01-01

    Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a flbrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains, in contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.

  8. Construction of a plant medial expression vector containing snowdrop lectin (GNA) gene for Agrobacterium tumefaciens mediated transformation%用于农杆菌转化的雪花莲外源凝集素(GNA)基因植物中间表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    刘薇斌; 梁峰; 常团结

    2000-01-01

    雪花莲外源凝集素(GNA)是一类能特异地和甘露糖残基结合的蛋白质成分,对人畜毒性极低,但对同翅目昆虫有强烈的抗性.为利用雪花莲外源凝集素对同翅目害虫的抗性,将雪花莲外源凝集素基因置于Ubi启动子和Tnos终止子的控制之下,插入pCAMBIA3300的SmaI位点,构建了可用于农杆菌转化的植物中间表达载体-pCUGAN-Bar.将pCUGAN-Bar电击转入农杆菌,得到了带有pCUGAN-Bar的农杆菌菌株,可用于小麦和水稻的转化工作.

  9. Subcellular Localization of Agrobacterium tumefaciens T-DNA Binding Proteins VirD2 and VirE2 in Rice%根瘤农杆菌T-DNA结合蛋白VirD2和VirE2在水稻中的亚细胞定位研究

    Institute of Scientific and Technical Information of China (English)

    颜廷祥; 罗美中

    2012-01-01

    根瘤农杆菌侵染植物过程中,至少有5种毒性蛋白(Vir)进入宿主细胞发挥作用,而其中VirD2与VirE2的作用最为关键,研究二者在水稻中的亚细胞定位,对农杆菌介导的水稻遗传转化机制的阐明具有重要意义.利用水稻叶鞘原生质体瞬时表达系统,发现3种冠瘿碱型的VirD2均只定位于细胞核中,与在拟南芥中相同;而3种冠瘿碱型的VirE2均主要定位于细胞核中,但在细胞质中仍有较多分布,与在拟南芥中的定位不同.因此,推测不同冠瘿碱型的农杆菌对水稻侵染能力的差异与VirD2和VirE2亚细胞定位的关系不大;同时表明根瘤农杆菌介导的拟南芥及水稻遗传转化机制存在相似性,但也有不同之处.%In the process of Agrobacterium tumefaciens -mediated infection of plants,at least 5 virulence ( Vir) proteins play roles by entering the host cells,among which VirD2 and VirE2 are the key factors. Consequently,studying the sub-cellular localization of VirD2 and VirE2 in rice is crucial to understand the mechanism of Agrobacterium tumefaciens -mediated rice genetic transformation. Using the transient expression system of rice sheath protoplasts,we found that the VirD2 proteins of all 3 kinds of opine Agrobacterium are completely located in the nucleus,which is the same as in Arabidopsis; and the VirE2 proteins of all 3 kinds of opine Agrobacterium are mainly located in nucleus,but are also distributed in the cytoplasm,which is different from those in Arabidopsis. Therefore,we propose the different efficiency among the 3 kinds of opine Agrobacterium strains for rice genetic transformation is not mainly determined by sub-cellular localization of VirD2 and VirE2. In addition,our results demonstrate that the mechanism of Agrobacterium tumefaciens-mediated genetic transformation in rice and Arabidopsis share some similarities,but keep some differences.

  10. The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori

    NARCIS (Netherlands)

    Michielse, C.B.; Ram, A.F.J.; Hondel, C.A.M.J.J. van den

    2004-01-01

    The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decr

  11. AGROBACTERIUM-MEDIATED TRANSFORMATION OF СOMPOSITAE PLANTS. I. CONSTRUCTION OF TRANSGENIC PLANTS AND «HAIRY» ROOTS WITH NEW PROPERTIES

    OpenAIRE

    Matvieieva, N.

    2015-01-01

    The review explores some of the recent advances and the author's own researchs concerning biotechnological approaches for Agrobacterium tumefaciens and A. rhizogenes -mediated transformation of Compositae family plants. This paper reviews the results of genetic transformation of Compositae plants, including edible ( Cichorium intybus, Lactuca sativa ), oil ( Helianthus annuus ), decorative ( Gerbera hyb rida ), medical ( Bidens pilosa, Artemisia annua, Artemisia vulgaris, Calendula officinali...

  12. Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. x P. maximowiczii A. Henry.

    Science.gov (United States)

    Yevtushenko, Dmytro P; Misra, Santosh

    2010-03-01

    Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. x P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the beta-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4-10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1-2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra x P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

  13. Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

    Science.gov (United States)

    Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

    2008-04-01

    The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

  14. 2-Acetyl-1-pyrroline augmentation in scented indica rice (Oryza sativa L.) varieties through Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) gene transformation.

    Science.gov (United States)

    Kaikavoosi, Kayghobad; Kad, Trupti D; Zanan, Rahul L; Nadaf, Altafhusain B

    2015-12-01

    2-Acetyl-1-pyrroline (2AP) has been identified as a principal aroma compound in scented rice varieties. Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS) gene is reported to regulate the proline synthesis in plants and acts as the precursor of 2AP. Two scented indica rice varieties, namely Ambemohar 157 and Indrayani, were subjected to Agrobacterium tumefaciens-mediated genetic transformation containing P5CS gene. Overexpression of P5CS led to a significant increase in proline, P5CS enzyme activity and 2AP levels in transgenic calli, vegetative plant parts, and seeds over control in both the varieties. 2AP level increased more than twofold in transgenic seeds in both varieties. This is the first report of enhancement in 2AP content through overexpression of using P5CS gene, indicating the role of proline as a precursor amino acid in the biosynthesis of 2AP in scented rice.

  15. P90-T Screen for Arabidopsis thaliana Mutants Resistant to Agrobacterium tumefaciens—Mediated Transformation

    OpenAIRE

    Pierre-Charles, L.; Mir, K.; Muth, T.

    2007-01-01

    Agrobacterium tumefaciens is a typical soil bacterium that causes crown gall disease in a variety of plant species. A. tumefaciens is capable of recognizing wound sites on a plant by detecting chemicals produced during the wound response of the plant. Laceration of the plant tissue causes the production of phenols and sugar molecules, which in turn trigger not only the chemotaxis of the bacteria towards the injury, but the processing of the tumor-inducing plasmid (Ti plasmid) as well as the e...

  16. Transformation of Arabidopsis thaliana via Agrobacterium tumefacience with an endochitinase gene from Trichoderma, and enhanced resistance to Sclerotinia sclerotiorum

    Institute of Scientific and Technical Information of China (English)

    DAI Fu-ming; XU Tong

    2004-01-01

    @@ Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (ColO) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase ( ThEn-42 ) was amplified by Arabidopsis leaf-PCR or genomic DNA PCR. Unfertile, dwarf and normal phenotypes appeared in the T1 generation. In addition, an enhanced resistance to S. sclerotiorum was observed. The mortality percentage (7.7% to 33.3%) in transgenic plants was significantly lower than in non-transgenic plants (86. 7%) 10 days after inoculation with the pathogen.

  17. Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for 3-glucuronidase (GUS) and neomycin phosphotransferase (NPTI1). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

  18. Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.) using the expansin 10 (CsEXP10) gene.

    Science.gov (United States)

    Sun, Y D; Luo, W R; Sun, S Y; Ni, L

    2015-12-08

    The cucumber expansin 10 (CsEXP10) gene was previously cloned from young cucumber fruits but its role has not been defined. To determine the role of this gene in plant growth and development, a CsEXP10 gene transformation system was established. The open reading frame of the gene was inserted behind the CaMV35S promoter of vector pCAMBIA1301, and the construct was introduced into tomato plants by Agrobacterium-mediated transformation. In total, 19 kanamycin-positive lines were produced and nine independent transgenic lines were identified by β-glucuronidase and polymerase chain reaction (PCR) analysis. Quantitative real-time PCR analysis showed that levels of the CsEXP10 transcript were higher in transgenic lines than in a non-transgenic line.

  19. AGROBACTERIUM-MEDIATED TRANSFORMATION OF COMPOSITAE PLANTS. I. CONSTRUCTION OF TRANSGENIC PLANTS AND «HAIRY» ROOTS WITH NEW PROPERTIES

    Directory of Open Access Journals (Sweden)

    N. A.Matvieieva

    2013-02-01

    Full Text Available The review explores some of the recent advances and the author's own researchs concerning biotechnological approaches for Agrobacterium tumefaciens- and A. rhizogenes-mediated transformation of Compositae family plants. This paper reviews the results of genetic transformation of Compositae plants, including edible (Cichorium intybus, Lactuca sativa, oil (Helianthus annuus, decorative (Gerbera hybrida, medical (Bidens pilosa, Artemisia annua, Artemisia vulgaris, Calendula officinalis, Withania somnifera etc. plant species. Some Compositae genetic engineering areas are considered including creation of plants, resistant to pests, diseases and herbicides, to the effect of abiotic stress factors as well as plants with altered phenotype. The article also presents the data on the development of biotechnology for Compositae plants Cynara cardunculus, Arnica montana, Cichorium intybus, Artemisia annua "hairy" roots construction.

  20. Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

    Science.gov (United States)

    Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita

    2011-05-01

    We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.

  1. Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.).

    Science.gov (United States)

    Swain, Swasti S; Rout, Kedar K; Chand, Pradeep K

    2012-10-01

    Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T(L)-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T(R)-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, (1)H-NMR, and (13)C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species. PMID:22843061

  2. Transformation of Chinese Cabbage (Brassica rapa L. ssp.pekinensis) by Agrobacterium Micro-Injection into Flower Bud

    Institute of Scientific and Technical Information of China (English)

    YAN Ji-yong; HE Yu-ke; CAO Jia-shu

    2003-01-01

    We obtained two lines of Chinese head cabbage (Brassica rapa L. ssp. pekinensis) selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants (T0) with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One (DHZ-13-1) exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1) has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

  3. Evaluation of agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance.

    Science.gov (United States)

    Burns, C; Leach, K M; Elliott, T J; Challen, M P; Foster, G D; Bailey, A

    2006-02-01

    There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trp2 promoters. The Coprinus cinereus beta-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with AGL-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems. PMID:16444014

  4. Evaluation of agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance.

    Science.gov (United States)

    Burns, C; Leach, K M; Elliott, T J; Challen, M P; Foster, G D; Bailey, A

    2006-02-01

    There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trp2 promoters. The Coprinus cinereus beta-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with AGL-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.

  5. An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

    Directory of Open Access Journals (Sweden)

    Pileggi Marcos

    2001-01-01

    Full Text Available An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration. A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996, while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

  6. Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

    OpenAIRE

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-01-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest...

  7. 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.

  8. Agrobacterium-Mediated Multiple Gene Transformation in Rice Using a Single Vector

    Institute of Scientific and Technical Information of China (English)

    Ming-Xia CAO; Jian-Qiu HUANG; Zhi-Ming WEI; Quan-Hong YAO; Chang-Zhao WAN; Jia-An LU

    2005-01-01

    The homodimeric hemoglobin gene (VHb), the trans-zeatin synthetase gene (tzs), the modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), a selectable marker gene (hpt), and a reporter gene (gus), as linked expression cassettes, were stacked into the T-DNA region of a binary vector and introduced simultaneously into immature embryos of the rice (Oryza sativa L.) varieties Xiushui-11, Qiufeng,Youfeng, and Hanfeng by Agrobacterium tumefaciens. A total of 1 153 transgenic lines was obtained through selection for hygromycin B resistance. Approximately 90.2% of the transgenic lines harbored all the transgenes. Integration of multiple transgenes occurred at one to three genetic loci. Expression analysis revealed that the transgenes were coexpressed and inherited in a simple Mendelian fashion in transgenic plants and the frequency of coexpression was approximately 85%. On the basis of the cointegration and coexpression of the transgenes, most transgenic families were considered to be useful in a breeding program.

  9. The Agrobacterium rhizogenes GALLS gene encodes two secreted proteins required for genetic transformation of plants.

    Science.gov (United States)

    Hodges, Larry D; Lee, Lan-Ying; McNett, Henry; Gelvin, Stanton B; Ream, Walt

    2009-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5' end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus. PMID:18952790

  10. Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes.

    Science.gov (United States)

    Pappas, Katherine M; Winans, Stephen C

    2003-11-01

    Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants. PMID:14602634

  11. Evidence for stable transformation of wheat by floraldip in Agrobacterium tumefaciens

    Science.gov (United States)

    Hexaploid wheat is one of the world’s most important staple crops but genetic transformation is still challenging. We have developed a floral transformation protocol that does not utilize tissue culture. Three T-DNA wheat transformants have been produced in the germplasm line, Crocus, using this p...

  12. A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant.

    Science.gov (United States)

    Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

    2014-09-01

    In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.

  13. Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.

    Science.gov (United States)

    Ogawa, Yoichi; Dansako, Tomoko; Yano, Kentaro; Sakurai, Nozomu; Suzuki, Hideyuki; Aoki, Koh; Noji, Masaaki; Saito, Kazuki; Shibata, Daisuke

    2008-02-01

    We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

  14. 农杆菌介导的转谷氨酰胺合成酶基因小麦的抗除草剂特性研究%AGROBA CTERIUM TUMEFACIENS-MEDIATED TRANSGENIC WHEAT PLANTS WITH GLUTAMINE SYNTHETASES CONFER TOLERANCE TO HERBICIDE

    Institute of Scientific and Technical Information of China (English)

    黄其满; 刘伟华; 孙辉; 邓新; 苏金

    2005-01-01

    Cloning of cytosolic (GS1) cDNA and chloroplast glutamine synthetase (GS2) cDNA from Pisum satium was carried out previously in our laboratory. To identify the functions of the GS genes, we first constructed a plant expression vector, p2GS, harboring two different isoenzymes, GS1 and GS2 cDNAs, under the control of two constitutive promoters of rice, Actin1 (Act1) and maize Ubiquitin (Ubi) genes. Then, using an Agrobacterium tumefaciens-mediated transformation method, we introduced GS1 and GS2 genes into wheat (Triticum aestivum) plants, producing 2GS-transgenic wheat plants using immature embryos as the explants.Presence of the transgenes GS1 and GS2 in wheat plants was confirmed by PCR and Southern blot hybridization analyses. Forty-one independent transgenic wheat plants with tolerance to an herbicide (Phosphinothricin,PPT) were generated. Results from Basta tests showed that the 2GS-transgenic wheat plants were endowed with herbicide-tolerant properties. Almost all of the transgenic plants were normal in morphology, and seed production was similar to that of the control wheat plants. Our study suggest that PPT resistance is conferred by effective expression of glutamine synthetases in transformed wheat plants, and glutamine synthetase genes can serve as a selective marker gene of wheat transformation system in our study.%谷氨酰胺合成酶(Glutamine synthetase,GS,E.C.6.3.1.2)是植物氨同化过程中的关键酶,对植物的氮素吸收和代谢起着至关重要的作用.谷氨酰胺合成酶还是除草剂草胺膦(Phosphinothricin(PPT)或Basta)的靶标酶.前期工作已从我国特有的豌豆(Pisum satium)品种中克隆了细胞质型谷氨酰胺合成酶(GS1)cDNA和叶绿体型谷氨酰胺合成酶(GS2)cDNA.为了验证谷氨酰胺合成酶的功能,构建了同时含有GSl cDNA和GS2 cDNA的植物表达载体p2GS.以该表达载体通过农杆菌介导法,转化小麦(Triticum aestivum)的未成熟胚愈伤组织,经PPT筛选及分化再生培

  15. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

    Science.gov (United States)

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-07-27

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. PMID:26044711

  16. Agrobacterium-mediated transformation of Australian rice varieties and promoter analysis of major pollen allergen gene, Ory s 1.

    Science.gov (United States)

    Azria, Diah; Bhalla, Prem L

    2011-09-01

    A simple protocol for Agrobacterium-mediated transformation of Australian rice using mature embryos is described. Transgenic plants of two commercial genotypes of Australian rice, Amaroo and Millin, were produced. Transgenic plants were obtained by applying selection pressure to callus and to the regenerated shoots. Exclusion of the selective agent (hygromycin) during plant regeneration was found to be critical for recovery of transgenic plants from these commercial varieties. Transgenic plants were produced after 3 months. The developed system was also used to study spatial and temporal expression of a rice pollen-specific gene, Ory s 1. Expression of pOry s 1::uidA in transgenic rice demonstrated GUS expression in mature pollen, hence indicating potential use of this promoter to direct pollen-specific gene expression. Further Ory s 1 5' deletion study indicated that the pollen-specificity element may reside within -405 bp to the start of the transcription, while the region upstream of -405 contained a cis-acting regulatory element(s) responsible for quantitative expression of this gene.

  17. Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43).

    Science.gov (United States)

    Karthikeyan, Alagarsamy; Pandian, Shunmugiah Karutha; Ramesh, Manikandan

    2011-09-01

    A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)).

  18. Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

    Science.gov (United States)

    Sankari, Mohan; Hemachandran, Hridya; Anantharaman, Amirtha; Babu, Subramanian; Madrid, Renata Rivera; C, George Priya Doss; Fulzele, Devanand P; Siva, Ramamoorthy

    2016-07-01

    Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination. PMID:26922728

  19. Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease.

    Science.gov (United States)

    Elayabalan, Sivalingam; Kalaiponmani, Kalaimughilan; Subramaniam, Sreeramanan; Selvarajan, Ramasamy; Panchanathan, Radha; Muthuvelayoutham, Ramlatha; Kumar, Krish K; Balasubramanian, Ponnuswami

    2013-04-01

    One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana.

  20. Antioxidant potential of Agrobacterium-transformed and non-transformed Physalis ixocarpa plants grown in vitro and ex vitro 

    Directory of Open Access Journals (Sweden)

    Katarzyna Bergier

    2012-12-01

    Full Text Available Introduction:Oxidative stress is involved in pathogenesis of a number of chronic diseases hence is an increasing interest in plant-derived natural antioxidants with respect to their potential health benefits. Plants from the genus Physalis are particularly rich in secondary metabolites and show significant antioxidant potential. Recent development in transgenic research has opened new possibilities for enhanced production of secondary metabolites with plant cell and organ cultures. The hairy root-regenerated Physalis ixocarpa plants grown in vitro and ex vitro were compared to the non-transformed plants with respect to their antioxidant potential.Material/Methods:The total antioxidant capacity (TAC, the contents of total phenols and ascorbate were evaluated in fruits, flowers, leaves and roots of P. ixocarpa using the ferric reducing antioxidant power assay (FRAP, the Folin-Ciocalteu method and the 2,2’-dipyridyl method, respectively.Results/Discussion:The antioxidant profiles, in terms of TAC, ascorbate and phenols were organ-specific and depended on the culture conditions. Neither the total phenol content nor the ascorbate level appeared to determine the TAC of the studied plant extracts. The aqueous extracts exhibited lower antioxidant activities than the acetone ones indicating that lipophilic antioxidants made a major contribution to TAC of the plant tissues. Agrobacterium rhizogenes-mediated transformation changed the antioxidant status with respect to TAC, phenols and ascorbate and this effect was observed in the plants grown in vitro and ex vitro. 

  1. The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

    Science.gov (United States)

    Niu, Xiaolei; Zhou, Meiliang; Henkel, Christiaan V; van Heusden, G Paul H; Hooykaas, Paul J J

    2015-12-01

    During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process. PMID:26461850

  2. Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

    Science.gov (United States)

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-08-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

  3. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Science.gov (United States)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  4. Auxin autonomy in cultured tobacco teratoma tissues transformed by an auxin-mutant strain of Agrobacterium tumefaciens.

    Science.gov (United States)

    Campell, B R; Su, L Y; Pengelly, W L

    1992-08-01

    We have studied the mechanism of auxin autonomy in tobacco (Nicotiana tabacum L.) crowngall tissues transformed by the auxin-mutant (tms (-)) A66 strain of Agrobacterium tumefaciens. Normally, tms (-) tobacco tumor tissues require the formation of shoots to exhibit auxin-independent growth in culture. We have isolated from tms (-) tobacco cells several stable variants that are fully hormone-independent and grow rapidly as friable, unorganized tissues, thus mimicking the growth and morphology of tms (+) tobacco cells that produce high levels of auxin. However, none of the variants contained the high levels of auxin found in tms (+) tumor cells. The variants could be divided into two classes with respect to their response to applied auxin. The first class was highly sensitive to applied auxin: low concentrations (1 μM) of α-naphthaleneacetic acid (NAA) severely inhibited growth and markedly stimulated the accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The second class of variants showed a low sensitivity to applied auxin: growth was promoted by concentrations of NAA up to 10 μM, and growth inhibition and high ACC levels were observed only at high NAA concentrations (100 μM). Unorganized variants with low auxin sensitivity were also isolated from a variant line with high auxin sensitivity. The isolation of tumor cells that exhibited the growth phenotype of tms (+) cells while retaining the low auxin content and low auxin sensitivity of tms (-) cells indicates that full hormone autonomy, characteristic of wild-type crown-gall tumors, can be achieved by a mechanism that is independent of changes in the auxin physiology of the cells. PMID:24178208

  5. Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation.

    Science.gov (United States)

    Anand, Ajith; Rojas, Clemencia M; Tang, Yuhong; Mysore, Kirankumar S

    2012-07-01

    • Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. • We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. • We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. • We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome. PMID:22486382

  6. Developing transgenic maize (Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

    Institute of Scientific and Technical Information of China (English)

    SUN He; LANG Zhi-hong; LU Wei; ZHANG Jie; HE Kang-lai; ZHU Li; LIN Min; HUANG Da-fang

    2015-01-01

    Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacil us thuringiensis (Bt) cry gene and epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1Ah gene with the 2mG2-epsps gene and combined the wide-used manA gene as a selective marker to construct one coordinated expression vector cal ed p2EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40%of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing;meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

  7. The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

    NARCIS (Netherlands)

    Zheng Sijun, S.J.; Henken, B.; Ahn, Y.K.; Krens, F.A.; Kik, C.

    2004-01-01

    This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sou

  8. Brachypodium sylvaticum, a model for perennial grasses: transformation and inbred line development.

    Directory of Open Access Journals (Sweden)

    Michael A Steinwand

    Full Text Available Perennial species offer significant advantages as crops including reduced soil erosion, lower energy inputs after the first year, deeper root systems that access more soil moisture, and decreased fertilizer inputs due to the remobilization of nutrients at the end of the growing season. These advantages are particularly relevant for emerging biomass crops and it is projected that perennial grasses will be among the most important dedicated biomass crops. The advantages offered by perennial crops could also prove favorable for incorporation into annual grain crops like wheat, rice, sorghum and barley, especially under the dryer and more variable climate conditions projected for many grain-producing regions. Thus, it would be useful to have a perennial model system to test biotechnological approaches to crop improvement and for fundamental research. The perennial grass Brachypodiumsylvaticum is a candidate for such a model because it is diploid, has a small genome, is self-fertile, has a modest stature, and short generation time. Its close relationship to the annual model Brachypodiumdistachyon will facilitate comparative studies and allow researchers to leverage the resources developed for B. distachyon. Here we report on the development of two keystone resources that are essential for a model plant: high-efficiency transformation and inbred lines. Using Agrobacterium tumefaciens-mediated transformation we achieved an average transformation efficiency of 67%. We also surveyed the genetic diversity of 19 accessions from the National Plant Germplasm System using SSR markers and created 15 inbred lines.

  9. Brachypodium sylvaticum, a model for perennial grasses: transformation and inbred line development.

    Science.gov (United States)

    Steinwand, Michael A; Young, Hugh A; Bragg, Jennifer N; Tobias, Christian M; Vogel, John P

    2013-01-01

    Perennial species offer significant advantages as crops including reduced soil erosion, lower energy inputs after the first year, deeper root systems that access more soil moisture, and decreased fertilizer inputs due to the remobilization of nutrients at the end of the growing season. These advantages are particularly relevant for emerging biomass crops and it is projected that perennial grasses will be among the most important dedicated biomass crops. The advantages offered by perennial crops could also prove favorable for incorporation into annual grain crops like wheat, rice, sorghum and barley, especially under the dryer and more variable climate conditions projected for many grain-producing regions. Thus, it would be useful to have a perennial model system to test biotechnological approaches to crop improvement and for fundamental research. The perennial grass Brachypodiumsylvaticum is a candidate for such a model because it is diploid, has a small genome, is self-fertile, has a modest stature, and short generation time. Its close relationship to the annual model Brachypodiumdistachyon will facilitate comparative studies and allow researchers to leverage the resources developed for B. distachyon. Here we report on the development of two keystone resources that are essential for a model plant: high-efficiency transformation and inbred lines. Using Agrobacterium tumefaciens-mediated transformation we achieved an average transformation efficiency of 67%. We also surveyed the genetic diversity of 19 accessions from the National Plant Germplasm System using SSR markers and created 15 inbred lines. PMID:24073248

  10. Agro-transformation and evaluation of resistance to Phytophthora infestansin Solanum tuberosumL. variety Désirée

    Directory of Open Access Journals (Sweden)

    Jeanette Orbegozo

    2014-03-01

    Full Text Available The Oomycete Phytophthora infestans (Mont. de Bary, the causal agent of the disease known as late blight, is primarily responsible for the decreased in production performance and potato crops worldwide. The integration of the complete Rgenes sequences in the potato genome using Agro-transformation appears an alternative to be considered in the fight against this pathogen. The Rpi-blb2 gene (Rgene from the wild species Solanum bulbocastanumDunal shows a broad resistance to isolates ofP. infestans,making it an important candidate for plant breeding studies. This paper reports the integration of the Rpi-blb2gene into potato var. Désirée genome by Agrobacterium tumefaciens- mediated transformation system, the molecular characterization of 29 events transformed and whole plant infection with isolate POX67 of P. infestansfrom Peru. Désirée events [Rpi-blb2] 4 and Désirée [Rpi-blb2] 30, showed a substantial resistance to P. infestansinfection confirming complete transfer of the Rpi-blb2gene from a wild species to a cultivated species by genetic transformation.

  11. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    OpenAIRE

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-pro...

  12. AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION OF SORGHUM USING TISSUE CULTURE-BASED AND POLLEN-MEDIATED APPROACHES

    Directory of Open Access Journals (Sweden)

    Elkonin L.A.

    2012-08-01

    Full Text Available Genetic transformation is a powerful tool for genetic improvement of arable crops. Genetic engineering approaches are especially important for modification of starch and protein contents, vitamin and micronutrient concentration, improvement of nutritive value of protein fractions, and increase tolerance to environmental stresses. Application of transgenic technologies for genetic improvement of sorghum, a highly productive heat tolerant and drought resistant crop, is extremely important since climate aridization in many regions all over the globe hampers sustainable production of traditional cereals, such as wheat, maize and barley. However, sorghum, in spite of great number of investigations, is one of the most recalcitrant crop species to genetic modification. The most frequently reported problems are a low frequency of transformation and silencing of transgenes. Using the A. tumefaciens strain AGL0/p35SGIB with the bar and gus-intron genes under the nos and CaMV35S promoters, respectively, we studied different methods of Agrobacterium-mediated genetic transformation of the grain sorghum: in vitro culture-based techniques, by inoculation of immature embryos or embryo-derived calli, and pollen-mediated approach, by inoculation of flowering panicles. Four lines of grain sorghum – Milo-10, [9E] Milo-10 (CMS-line, KVV-114, and KVV-45 – were used. In both approaches, for activation of vir-genes agrobacterial cell suspension was grown in the AB or modified AB media with acetosyringone at room temperature. In vitro culture approach was effective for obtaining transgenic plants in the lines Milo-10 and KVV-45, which were able to produce embryogenic callus from immature embryos after their co-cultivation with agrobacterial cell suspension. Callus cultures tolerant to glufosinate ammonium (GA and capable to plant regeneration were obtained. The frequency of immature embryos producing PCR-positive transgenic plants varied in different experiments

  13. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

    Science.gov (United States)

    Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra

    2014-01-01

    Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

  14. Establishment of Suspension Cell Culture from Agrobacterium-transformed Hairy Root Cells of Psammosilene tunicoides, an Endangered and Rare Medicinal Plant of China

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

    Full Text Available Psammosilene tunicoides is an important medicinal plant endemic in China. Its annual yield is severely limited due to slow growth, poor seed germination and excessive collection. To satisfy the growing market demands, it’s necessary to seek alternatives to field cultivation and wild resources of this endangered plant. Using Agrobacterium -transformed hairy roots as initial explants, here, we reported the development of a suspension cell culture system for P. tunicoides. Results showed the Agrobacterium -transformed hairy roots-derived suspension cells are fast in growth and strong in capacity for accumulation of bioactive metabolites. We established that 1/2MS was a suitable medium for culturing the hairy root-derived suspension cells and the optimal combination of phytohormones is 1.5 mg/L 2, 4-D+0.5 mg/L 6-BA+0.25 mg/L NAA+0.1 mg/L KT. Under this condition, the maximal biomass was achieved at the 20th day of culture with an average growth rate of 0.72 g/L/d; and the intracellular saponine content reached 0.92%, comparable to that of mother hairy roots. Compared with the normal P. tunicoides suspension cells, the hairy roots-derived suspension cells exhibited features of fast growth, short culture period and high concentration of saponines, suggesting that the large scale culture of hairy root-derived cells could be a feasible alternative to the wild resources of P. tunicoides.

  15. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

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    Waheed Arshad

    Full Text Available Tomato (Solanum lycopersicum L. is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.

  16. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

    Science.gov (United States)

    Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra

    2014-01-01

    Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.

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

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    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

  18. Advances in Agrobacterium-mediated plant transformation with enphasys on soybean Avanços na transformação genética de plantas mediada por Agrobacterium com ênfase em soja

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    Paulo Celso de Mello-Farias

    2008-02-01

    Full Text Available Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.A soja é uma das maiores fontes de proteína vegetal da humanidade. É também muito importante na alimentação animal e como matéria-prima industrial. Grandes avanços foram recentemente alcançados em sua transformação genética. O objetivo desta revisão é fornecer uma discussão acerca de importantes fatores que influenciam a transformação de soja mediada por Agrobaterium, incluindo tecidos alvo, condições do tecido vegetal, métodos de injúria, sistemas de regeneração, marcadores de seleção e genes repórter.

  19. Agrobacterium: nature's genetic engineer.

    Science.gov (United States)

    Nester, Eugene W

    2014-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun's old observations and also explain why Agrobacterium is nature's genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

  20. Functional analysis of agrobacterium virulence genes

    NARCIS (Netherlands)

    Niu, Xiaolei

    2013-01-01

    Agrobacterium tumefaciens is a gram-negative soil bacterium that induces plant tumors by transferring a segment of DNA, called T-DNA, into plant cells. Under laboratory conditions, Agrobacterium can also transform many different non-plant organisms such as the yeast Saccharomyces cerevisiae. During

  1. Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

    OpenAIRE

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

    2015-01-01

    DOGR1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l-1 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration med...

  2. Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper).

    Science.gov (United States)

    Sainger, Manish; Chaudhary, Darshna; Dahiya, Savita; Jaiwal, Ranjana; Jaiwal, Pawan K

    2015-10-01

    An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their

  3. Introduction of cryIB-cryIAa Hybrid Gene Into Rice (Oryza sativa Genom cv. Rojolele using Agrobacterium-Mediated Transformation

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    SYAMSIDAH RAHMAWATI

    2006-03-01

    Full Text Available Rojolele is one of Indonesian local variety from Javanica group that susceptible to yellow stem borer (Scirpophaga incertulas. Previous study showed that Rojolele can be cultured and regenerated in vitro. Two cry genes, cryIB-cryIAa were fused and introduced into rice cv. Rojolele in an attempt to improve resistance and to obtain durable resistance rice against the yellow stem borer. Two-week old embryogenic calli of Rojolele rice were inoculated with Agrobacterium tumefaciens harbored with binary vector pCAMBIA 1301, 1303, or 1304 carrying cryIB-cryIAa hybrid gene, hygromycin resistant gene (hpt, and -glucuronidase (gus gene interrupted with an intron. The transformed calli were selected gradually on medium containing hygromycin (50, 100 mg/l and regenerated on medium containing 0.5 mg/l IAA and 0.3 mg/l BAP. GUS activity in infected calli was detected by histochemical assay 3 days after inoculation. The highest (100% transformation efficiency were obtained from calli transformed with pCAMBIA 1303 and 1304. Thirty four out of 77 transformed shoots were tested positive for the cryIB-cryIAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T1 progeny revealed that two out of six lines tested showed a Mendelian segregation pattern. These two lines were also potentially resistant to yellow stem borer based on bioassay in planta.

  4. The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

    Science.gov (United States)

    Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2012-02-01

    The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

  5. Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

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    Ernandes Manfroi

    2015-01-01

    Full Text Available AbstractLow transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037. Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciensovergrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.

  6. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

    Science.gov (United States)

    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. PMID:25786575

  7. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    Science.gov (United States)

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

  8. Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector

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    Nikolić Radomirka

    2007-01-01

    Full Text Available Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa­ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1 over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42% plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by β-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

  9. Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

    Song, Guo-Qing; Sink, K C

    2006-03-01

    Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro. PMID:16369768

  10. Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation Influência de antibióticos sobre o tecido embriogênico e a supressão de Agrobacterium tumefaciens na transformação genética de soja

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    Beatriz Wiebke

    2006-01-01

    Full Text Available The influence of different antibiotic treatments in soybean genetic transformation was evaluated. First, an assay was performed to verify how different antibiotic treatments affect soybean embryogenic tissues. The effect of carbenicillin at 500 mg L-1 was genotype-dependent. This antibiotic did not affect embryo survival of cv. IAS5, but a three-fold increase of embryo proliferation was observed for cv. Bragg, when compared to the control. On the other hand, cefotaxime at 350 and 500 mg L-1 caused death of embryogenic tissues of both cultivars. Finally, the association of cefotaxime (250 mg L-1 + vancomycin (250 mg L-1 did not affect negatively the somatic embryos of tested cultivars until 63 days of treatment. Thereafter, a second experiment was carried out to determine the efficacy of different antibiotic treatments in suppressing LBA4404 Agrobacterium tumefaciens strain in genetic transformation. On tissue culture conditions, carbenicillin at 500 and 1000 mg L-1 was not active against Agrobacterium. On the other hand, treatments with cefotaxime at 350 and 500 mg L-1, and cefotaxime + vancomycin efficiently suppressed Agrobacterium during 49 days. Data of both experiments suggested cefotaxime + vancomycin for 49-63 days as the most appropriate treatment. This is the first work reporting the effect of antibiotics on soybean tissues. By identifying an antibiotic combination that suppressed A. tumefaciens with minimal phytotoxic effects, we are able to recommend it for improvement of soybean Agrobacterium-mediated transformation procedure.Foi avaliada a influência de diferentes tratamentos com antibióticos durante a transformação genética de soja. Inicialmente, desenvolveu-se um estudo para identificar como diferentes tratamentos com antibióticos afetam o tecido embriogênico da soja. O efeito da carbenicilina a 500 mg L-1 foi genótipo-dependente. Esse antibiótico não afetou a sobrevivência dos embriões da cv. IAS5, enquanto a

  11. Establishment of an Efficient Regeneration System Amenable to Agrobacterium Mediated Transformation of Two Elite Indica Rice Varieties of North East India

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    Mohitosh Dey

    2015-12-01

    Full Text Available An efficient plant regeneration system from embryogenic callus of two elite indica rice (Oryza sativa spp. indica varieties of Northeast India, Ketokijoha and Monoharsali is established. The effect of auxin, 2,4-dicholorophenoxy acetic acid (2,4-D on callus induction was optimized. Friable, nodular and creamish-white embryogenic calli were induced from mature seeds on NB medium supplemented with 2.5 mg/l 2,4-D. Plants were regenerated from 40-50 days old embryogenic callus on NB medium containing 0.5 mg/l BAP (6-benzylaminopurine and 0.25 mg/l ABA (abscisic acid. Regenerated plants with multiple tillers were rooted on half strength MS medium and rooted plants were acclimatized with 94% survival rate. Higher frequency of callus induction as well as plant regeneration was recorded in Ketokijoha as compared to Monoharsali. The calli of both the varieties were found amenable to Agrobacterium-mediated transformation as evident from strong GUS (β-glucuronidase expression. The results may find wide application for genetic improvement for valuable traits these elite indica rice varieties of Northeast India.

  12. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

    Science.gov (United States)

    Ogita, Shinjiro; Usui, Miki; Shibutani, Nanae; Kato, Yasuo

    2009-07-01

    A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

  13. A Review of Some Assisted Strategies for Improving the Efficiency of Agrobacterium-Mediated Plant Transformation%提高植物农杆菌转化效率辅助策略研究进展

    Institute of Scientific and Technical Information of China (English)

    叶兴国; 王新敏; 王轲; 杜丽璞; 林志珊; 徐惠君

    2012-01-01

    农杆菌介导是目前转基因植物研究采用的主要方法,不同植物间利用农杆菌转化的转化效率差异很大,高效农杆菌转化体系对于转基因植物产业化和功能基因组学等研究具有重要意义.总体而言,影响农杆菌转化效率的因素主要包括基因型、外植体及其生理状态、农杆菌菌株、培养基成分、共培养条件等.对于一些难转化的植物来说,辅助因素对转化效率的提高起着至关重要的作用.本文综述了影响农杆菌转化效率的一些辅助因素及其作用,包括植物材料微创伤处理和干燥处理、培养基中抗氧化剂和表面活性剂使用、农杆菌中额外拷贝Vir和植物细胞中VIP(VirE2 interacting protein)过表达、载体上核基质附着序列引入等,以期为进一步改进难转化作物如小麦、大豆等农杆菌介导的遗传转化效率提供参考.%Agrobacterium-mediated transformation is one of the main methods for plant transformation, however, its efficiency varies among different plant species. It is important to develop an efficient Agrobacterium-mediated transformation system in various crop species for the development of new economically important traits and for the study of functional genomics. Many factors influence the efficiency of Agrobacterium-mediated transformation of plants, such as genotypes, explant sources and their physiological status, Agrobacterium strains, culture media, and conditions of co-cultivation. Some assisted techniques or factors have been thought to be useful in improving the efficiency of some plants which are highly recalcitrant to conventional Agrobacterium-mediated transformation. In this paper, some of the factors, in particular microwounding, desiccation of target plant tissues, addition of antioxidants and surfactants to the culture media, over-expressions of additional copies of Vir gene in Agrobacterium and VIP (VirE2 interacting protein) gene in plant, and construction of

  14. Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer ability

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    Satoko eNonaka

    2014-12-01

    Full Text Available Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.

  15. Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

    OpenAIRE

    Singh Surinder P; Hac Luch; Belide Srinivas; Green Allan G; Wood Craig C

    2011-01-01

    Abstract Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describ...

  16. Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compounds

    DEFF Research Database (Denmark)

    Møller Hansen, Martin; Lauridsen, Uffe Bjerre; Hegelund, Josefine Nymark;

    .liu-clarke@bioforsk.no Keywords: Natural transformation - rol-genes – roseroot – rosavin - salidroside Abstract Introduction Rhodiola rosea commonly known as roseroot has since ancient times been used against depression and for improving mental abilities mainly due to its two bioactive compounds salidroside and rosavin. Due...... into segments and the leaves were separated. Both the leaves and the stem segment were inoculated with A. rhizogenes. The inoculated explants were placed on co-cultivation media consisting of ½xMS containing acetosyringone for 3 days in darkness. The explants were washed in a timentin solution and moved...

  17. Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOGR1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens

    Science.gov (United States)

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

    2015-01-01

    DOGR1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l-1 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm. PMID:26442041

  18. 木薯FEC诱导及农杆菌介导转化的研究进展%Advances in Induction of Cassava FEC and Agrobacterium-mediated Transformation

    Institute of Scientific and Technical Information of China (English)

    程琴; 金刚; 李慧敏; 彭欣怡; 黎萍; 王丽萍

    2014-01-01

    在木薯生物技术中,转基因植株的生产很普遍,它主要通过器官发生和体细胞胚胎发生途径,木薯遗传转化的关键技术是FEC的诱导和形成,农杆菌介导的FEC转化在转基因木薯领域被广泛应用。概述木薯再生途径中体胚和FEC制备、诱导及增值,并对农杆菌介导的转化及其在木薯中的应用等进行综述。%Production of transgenic plants is gradually becoming routine in cassava biotechnology. It is primarily through organogenesis and somatic embryogenesis, the key technology of cassava genetic transformation is friable embryogenic calli(FEC)induction and formation, Agrobacterium-mediated transformation of FEC is the most widely used method to generate transgenic cassava plants.This article provides an overview of cassava regeneration way including embryo and FEC preparation, induction and appreciation, the Agrobacterium-mediated transformation and its application in cassava were summarized.

  19. Evaluación y selección de un protocolo vía Agrobacterium para la incorporación de resistencia al cogollero en la variedad de tomate Unapal-Arreboles Evaluation and selection of a protocol for Agrobacterium-mediated genetic transformation of tomato variety Unapal-Arreboles for resistance to budworm

    Directory of Open Access Journals (Sweden)

    Hernando Ramírez

    2009-04-01

    Full Text Available Se evaluó y seleccionó una metodología para la transformación genética de la variedad de tomate UNAPAL-Arreboles con el gen cry1Ab para la incorporación de resistencia al cogollero (Tuta absoluta, utilizando el sistema de Agrobacterium. Se regeneraron 59 plantas transgénicas a partir de 3.200 explantes (1.84%. La integración estable, expresión y herencia de los genes nptII y gus-intrón, se demostraron mediante análisis histoquímico y molecular en los clones To28, To33 y To47 y en la correspondiente generación T1. Sin embargo, los análisis molecular e inmunológico indicaron ausencia del gen cry1Ab sugiriendo que la secuencia de este gen se puede haber modificado.A plant transformation methodology was selected and evaluated to incorporate the cry1Ab gene by Agrobacterium-mediate genetic transformation into tomato variety UNAPAL-Arreboles for resistance to budworm (Tuta absoluta. A total of 59 transgenic plants were regenerated from 3.200 explants (1.84%. Histochemical gus assay and molecular analysis of three independent events To28, To33 and To47 and corresponding T1 derived generations, demonstrate the stable integration, expression and inheritance of the nptII and gus-intron genes. However, the molecular and immunological analysis of these same clones, indicate that the cry1Ab gene is not present in the transformed plants, suggesting that the sequence of this gene may be modified as result of possible recombinant events.

  20. Advances in transforming kudzu (Pueraria phaseoloides) and carrot (Daucus carota var. Danvers 126) roots with different Agrobacterium rhizogenes strains for increasing MA fungi growth

    OpenAIRE

    Marisol Medina Sierra; Francisco Hernando Orozco P.; María Elena Márquez F.

    2011-01-01

    En el presente trabajo se transformaron raíces de kudzú (Pueraria phaseoloides) y de zanahoria (Daucus carota) en diferentes medios de cultivo, mediante el empleo de cinco cepas diferentes de Agrobacterium rhizogenes; de comportamiento diferente tanto en la transformación de zanahoria por las cepas de A. rhizogenes A.r.15834, A.r.8196 y A.r.2659; como en la transformación de kudzú por las cepas A.r.15834 y A.r.1724. Por otro lado, se logró la multiplicación en medio White modificado (WM) de l...

  1. 木质素合成酶基因反义 COMT对亚麻的转化及检测%Transformation and Detection of Linum usitatissimum with COMT Gene

    Institute of Scientific and Technical Information of China (English)

    黄文功; 康庆华; 姜卫东; 姚玉波; 詹亚光

    2016-01-01

    The COMT gene mainly participates in the biosynthesis of S lignin.The content of lignin will reduce when the activity of COMT gene is suppressed in the transgenic plants.In this study,the BSP and COMT were inserted into the vector pCAM2301 to construct a recombinant expression gene.The re-combinant gene was transformed into fiber flax by Agrobacterium tumefacien mediated callus transforma-tion.The transformed calluses were subjected to kanamycin screening and resistance callus was induced in induction and differentiation culture.The transformed seedlings were obtained and verified by PCR. The COMT gene was integrated into genome of the flax.We gained the positive genetic flax would lay a solid foundation for the research of flax fiber and variety breeding.%COMT基因主要参与S木质素的生物合成,转基因植物在COMT活性被抑制时,木质素含量减少,利用杨树皮储藏蛋白( BSP )启动子和该基因构建成反义植物表达载体pBSP-anti-COMT-2301,利用农杆菌介导法将其转入亚麻,经过卡那霉素筛选获得了抗性的愈伤组织,诱导分化后获得了亚麻苗。经特异引物的PCR扩增,证明目的基因已经整合到亚麻基因组中,本研究获得的阳性转基因亚麻植株为亚麻纤维研究及品种选育奠定了坚实基础。

  2. Evaluation on the effectiveness of 2-deoxyglucose-6-phosphate phosphatase (DOG(R)1) gene as a selectable marker for oil palm (Elaeis guineensis Jacq.) embryogenic calli transformation mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

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

    2015-01-01

    DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm. PMID:26442041

  3. Evaluation on the Effectiveness of 2-Deoxyglucose-6-phosphate phosphatase (DOGR1 Gene as a Selectable Marker for Oil Palm (Elaeis guineensis Jacq. Embryogenic Calli Transformation Mediated by Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Abang Masli eDayang Izawati

    2015-09-01

    Full Text Available DOGR1, which encodes for 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOGR1 gene, was transformed into oil palm embryogenic calli mediated by Agrobacterium tumefaciens strain LBA4404. Transformed embryogenic calli were exposed to 400 mg l–1 2-deoxyglucose (2-DOG as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOGR1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOGR1 gene and 2-DOG for regenerating transgenic oil palm.

  4. 马铃薯遗传转化体系的优化及玉米淀粉分支酶基因SBEⅡb的导入%Optimization of genetic transformation system of potato and introduction of maize starch branching enzyme gene SBEⅡb into potatoFAN

    Institute of Scientific and Technical Information of China (English)

    范亚丽; 阮颖; 刘春林

    2012-01-01

    The highly effective potato transformation systems were established and optimized using tube potato plant-lets as transformation materials in this study. The maize starch branching enzyme gene(SBE Ⅱ b)was transformed into potato using Agrobacterium tumefaciens-mediated transformation method. 762 stem sections were infec ted by Agrobaterium-mediated tumefacien method. 4 transgenic plants were obtained by PCR measurement, implying that SEE Ⅱ b gene had been integrated into potato genome. It was also found that GUS gene could normal ly be expressed in stem sections and potato microtubers of the trasgenetic potato plants.%以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系.通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株.经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达.

  5. Research Progress on the Influencing Factors of Agrobacterium-mediated Genetic Transformation of Cannabis sativa L.%农杆菌介导的汉麻遗传转化影响因素的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵越; 魏国江; 潘冬梅; 韩承伟; 韩喜财; 徐磊

    2015-01-01

    Genetic transformation technology is the basis to develop new hemp varieties and study functional ge‐nomics .The efficiency of A grobacterium‐mediated Cannabis sativa L .genetic transformation was effected by genotype ,explant size ,agrobacterium strains ,pretreatment time ,infection time ,co‐culture time ,antioxidants ,a‐cetyl clove ketone and so on .These influencing factors and the research progress were analyzed and summa‐rized ,so as to provide the scientific basis for promoting Cannabis sativa L .genetic transformation research .%遗传转化技术是培育汉麻新品种和进行功能基因组学研究的基础,采用农杆菌介导法对汉麻进行遗传转化,汉麻的基因型、外植体大小、农杆菌菌株、预处理时间、侵染时间、共培养时间、抗氧化剂、乙酰丁香酮等都会对遗传转化效率产生影响。分析并概括了这些影响因素及其研究进展情况,为促进汉麻农杆菌介导的遗传转化研究提供科学依据。

  6. Agrobacterium: Nature’s Genetic Engineer

    Directory of Open Access Journals (Sweden)

    Eugene William Nester

    2015-01-01

    Full Text Available Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago.Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle or TIP, the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single stranded DNA (T-DNA with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is coated and protected from nucleases by a bacterial secreted protein,VirE2. A nuclear localization signal in the endonuclease guides the T-strand into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The genes associated with T-strand formation and transfer (vir map to the Ti plasmid and are only expressed when the bacteria are at a plant’s wound site. Plant signals are recognized by a two-component system which activates vir genes. However, chromosomal genes with pleiotrophic functions also play important roles in plant transformation. The T-DNA encodes enzymes of auxin, cytokinin and opine synthesis, the latter a food source for Agrobacterium. The data now explain Braun’s observations made 75 years ago and also explain why Agrobacterium is Nature’s Genetic Engineer. Since any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells, Agrobacterium has become the major vector in plant genetic engineering.

  7. The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

    OpenAIRE

    Sharifi, Sara; Sattari, Taher Nejad; Zebarjadi, Alireza; Majd, Ahmad; Ghasempour, Hamidreza

    2013-01-01

    We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed ...

  8. Establishment of Cucurbita moschata Genetic Transformation System by Agrobacterium tumefaciens Transfection%农杆菌介导南瓜遗传转化体系的建立

    Institute of Scientific and Technical Information of China (English)

    付洪冰; 崔崇士; 赵曦; 刘琦

    2010-01-01

    以南瓜金辉一号(Cucurbita moschata 'Jinhui 1')为实验材料,利用根癌农杆菌(Agrobacterium tumefaciens)介导转化南瓜子叶节,研究了预培养时间、侵染时间、乙酰丁香酮(AS)浓度和共培养时间,抗生素羧苄青霉素(Carb)、头孢霉素(Cef)以及筛选剂卡那霉素(Kan)等因素对离体不定芽的影响,建立了南瓜最适遗传转化体系.结果表明:外植体预培养0天,侵染时间30分钟,AS浓度为100 mg·L-1,共培养5天可获得最高遗传转化效率;最适除菌剂为Cef,其最适浓度为500mg·L-1;最适Kan筛选浓度为100 mg·L-1:在MS培养基上培养抗性芽生根,经PCR和Southern blot检测,证明为转基因植株.

  9. Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

    Science.gov (United States)

    Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Albeck, Shira; Unger, Tamar; Peleg, Yoav; Jacobovitch, Jossef; Michael, Yigal; Daube, Shirley; Sharon, Michal; Robinson, Carol V; Svergun, Dmitri I; Fass, Deborah; Tzfira, Tzvi; Elbaum, Michael

    2007-02-01

    Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand. PMID:17060320

  10. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

    DEFF Research Database (Denmark)

    Romans-Fuertes, Patricia; Sondergaard, Teis Esben; Sandmann, Manuela Ilse Helga;

    2016-01-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate kno...... and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread....

  11. 农杆菌介导的sbe2a基因RNAi载体对玉米遗传转化的研究%Genetic Transformation with RNAi Vector of sbe2a Gene on Maize via Agrobacterium

    Institute of Scientific and Technical Information of China (English)

    关淑艳; 赵丽娜; 刘慧婧; 刘广娜; 王丕武

    2011-01-01

    将淀粉分支酶基因sbe2a的RNAi表达载体转入玉米自交系H99和丹598胚性愈伤组织.探讨了不同菌浓度、不同侵染时间和不同的共培养时间对玉米转化率的影响,确定了最佳转化条件:菌液浓度OD值为0.5~0.7,侵染时间为25 min,共体培养时间为3d.对转化的愈伤组织分化诱导出苗后进行PCR检测,获得了2株阳性植株,初步证明外源基因已经整合到玉米的基因组中.%Starch branch enzyme sbe2a gene of RNAi expression vector was transformed into embryogenic callus in maize inbred lines of H99 and Dan598 via Agrobacterium transformation system. The different concentration of bacteria, the different time of infection and the different time of co-culture on the infection rate of conversion of corn were determined. The results showed that the bacteria concentration (OD600 0. 5 ~ 0.7), infection time (25min)and co-culture time (3d) were optimal. Differentiated transformed callus were detected by PCR, indicating that 2 positive transgenic plants were obtained.

  12. Establishment and Optimization of Puna Chicory Genetic Transformation System with Agrobacterium-mediated Method%农杆菌介导普那菊苣遗传转化体系的建立

    Institute of Scientific and Technical Information of China (English)

    张丽君; 程林梅; 杜建中; 李贵全; 孙毅

    2011-01-01

    以普那菊苣(Cichorium intybus L.cv.Puna)叶片为试验材料,接种于含不同激素浓度配比的MS培养基上进行愈伤组织、芽分化以及根再生的诱导,分析了不同激素浓度及其配比对愈伤组织诱导和芽分化以及根再生效果的影响.以已经建立的再生体系为基础,以农杆菌菌株LBA4404(含质粒pBin438- TaNHX2)侵染转化普那菊苣,探索普那菊苣高效遗传转化体系.结果表明:对外植体适宜的预培养时间为2~3 d,与农杆菌的共培养时间也应控制在2~3 d;侵染时间控制在8 min左右;卡那霉素(Km)阳性筛选的适宜选择浓度为60mg·L-1.乙酰丁香酮(AS)200 μmol·L-1是促进农杆菌转化的最佳浓度,200 W超声波处理、20次负压处理也可提高农杆菌转化率效果.26 mg·L- 1 Km是野生型普那菊苣苗能够存活的上限,头孢唑林钠和头孢噻肟钠在500~1000 nmg·L-1浓度范围内、羧苄青霉素300 mg·L-1和氨苄青霉素在40~60 mg·L-1浓度范围内均能较好的诱导出愈伤组织和芽.将来自小麦(Triticum aestivum)的Na+/H+逆向转运蛋白(vacuolar Na+/H+ exchanger or antiporter,简称NHX,NHE或NHA)导入普那菊苣;经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株.%Chicory (Cichorium intybus L. Cv. Puna) leaf segments from aseptic seedlings were used as experimental materials. The explants were inoculated onto the MS medium with various phytohormone combinations to induce callus formation, and bud and root regeneration. Effects of phytohormone concentrations and combinations on the induction of callus, buds and roots were analyzed. Agrobacterium tumefa-ciens LBA4404 (harboring plasmid pBin438-TaNHX2) was used to infect Puna Chicory explants based on the regeneration system that had been established for the high efficiency transformation of the cultivar. Result showed that both suitable pre-culture time and co

  13. Do leaf surface characteristics affect Agrobacterium infection in tea [Camellia sinensis (L.) O Kuntze]?

    Indian Academy of Sciences (India)

    Nitish Kumar; Subedar Pandey; Amita Bhattacharya; Paramvir Singh Ahuja

    2004-09-01

    The host range specificity of Agrobacterium with five tea cultivars and an unrelated species (Artemisia parviflora) having extreme surface characteristics was evaluated in the present study. The degree of Agrobacterium infection in the five cultivars of tea was affected by leaf wetness, micro-morphology and surface chemistry. Wettable leaf surfaces of TV1, Upasi-9 and Kangra jat showed higher rate (75%) of Agrobacterium infection compared to Upasi-10 and ST-449, whereas non-wettable leaves of A. parviflora showed minimum (25%) infection. This indicated that the leaves with glabrous surface having lower (larger surface area covered by water droplet), higher phenol and wax content were more suitable for Agrobacterium infection. Caffeine fraction of tea promoted Agrobacterium infection even in leaves poor in wax (Upasi-10), whereas caffeine-free wax inhibited both Agrobacterium growth and infection. Thus, study suggests the importance of leaf surface features in influencing the Agrobacterium infection in tea leaf explants. Our study also provides a basis for the screening of a clone/cultivar of a particular species most suitable for Agrobacterium infection the first step in Agrobacterium-mediated genetic transformation.

  14. Construction of expression vector and transformation of FpDREB2A gene into Robinia pseudoacacia 'Idaho' mediated with Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    Zeng Hui-ming; Wang Hua-fang

    2006-01-01

    The transcription factor gene FpDREB2A of Fraxinus pennsylvanica Marsh var. subintegerrima (Vahl.) Fern was conmefaciens GV3101. Callus was screened with G418. Morphogenesis of shoots and roots of Idaho locust transformed genes was carried out on antibiotic media. The transformed plants were verified by PCR and Southern blotting tests that the FpDREB2A gene had been inserted into the genome DNA of Idaho locust.

  15. Improvements in the transformation of Arabidopsis thaliana C24 leaf-discs by Agrobacterium tumefaciens

    DEFF Research Database (Denmark)

    van der Graaff, Eric; Hooykaas, P J

    1996-01-01

    We report here an efficient Arabidopsis leafdisc transformation protocol yielding an average transformation frequency of 1.6 transgenic shoots per leaf explant 4 weeks after the bacterial infection period. Subsequent cultivation in vitro is such that a high percentage (85-90%) of the primary tran...... harboring an activator T-DNA construct in a gene tagging approach to isolate genes involved in morphogenesis and auxin signal transduction....

  16. Advances in transforming kudzu (Pueraria phaseoloides and carrot (Daucus carota var. Danvers 126 roots with different Agrobacterium rhizogenes strains for increasing MA fungi growth

    Directory of Open Access Journals (Sweden)

    Marisol Medina Sierra

    2011-12-01

    Full Text Available Kudzú (P. phaseoloides and carrot (D. carota roots were transformed in this survey into different kinds of culture medium by using five different A. rhizogenes strains. These presented different behaviour both in carrot transformation by A. rhizogenes 15834, A.r.8196 and A.r.2659 strains as well as kudzu transformation by A.r.15834 and A.r.1724 strains. Transformed carrot root growth was increased in WM culture medium, whilst transformed kudzu root growth did not increase in either the same medium or in modified MS medium. Transformed carrot roots were used for G. intrarradices increase and sporulation; however, wild AMF strains, isolated from a mining area (the lower Cauca area of Antioquia, did not grow either in roots from this specie or those from kudzu, in spite of this plant having great affinity for wild AMF strains. The results represent an advance in the procedure for DNA isolation and keeping AMF collections, required for other research.

  17. Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation.

    Science.gov (United States)

    Sun, He; Lang, Zhihong; Zhu, Li; Huang, Dafang

    2012-10-01

    The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.

  18. 农杆菌介导植酸酶基因转化大豆的研究%Transformation of Phytase Gene into Soybean via Agrobacterium-mediated Method

    Institute of Scientific and Technical Information of China (English)

    刘琦; 夏善勇; 张军; 段忠卫; 刘鑫磊; 李希臣

    2011-01-01

    以大豆胚尖为受体,利用农杆菌介导法将来源于泡盛曲霉的植酸酶基因phy转入黑农37和吉林小粒豆中.此法取材方便,操作简单,产生嵌合体的可能件低.在筛选培养基中经草胺膦(1.0 mg·L-1)筛选,获得抗性植侏.对获得的草胺磷抗性植株采用除草剂(10%Basta)筛查表型鉴定与目的基因、筛选标记基因PCR分子检测相结合的方法,检测结果直观、可信度高.最终获得5株阳性植株,转化率为0.5%.%The Phy gene cloned from Aspergillus awamori was transformed into soybean varieties Heinong37 and Jilinxiaoli soybean through Agrobacterium-mediated method with soybean embryonic tips as acceptors. The method has advantage of easy operation and lower chimera rate. The regenerated plants were screened by glufosinate-ammonium( 1.0 mg · L-1 )of medium.The regenerated plantlets resistant to glufosinate-ammonium were detected by combining the PCR analysis of the target gene as well as selective marker gene and the herbicide screening. The result was intuitive and credible. Finally, 5 positive plants were obtained, and the transformation efficiency reached 0. 5%.

  19. Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Brinch-Pedersen, Henrik; Lange, Mette;

    2008-01-01

    vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars...... with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically...

  20. Agrobacterium rhizogenes mediated transformation of Rhodiola sp. - an approach to enhance the level of bioactive compounds

    DEFF Research Database (Denmark)

    Himmelboe, Martin; Lauridsen, Uffe Bjerre; Hegelund, Josefine Nymark;

    2015-01-01

    Rhodiola rosea, commonly known as roseroot, has since ancient times been used against depression and for improving mental abilities because of its bioactive compounds. Due to excessive exploitation, the natural populations have been declining. Natural transformation with root-loci (rol)-genes from...... grown Rhodiola sp. for future sustainable production of bioactive compounds. For the experiment, in vivo grown stems and leaves of R. rosea and two accessions of R. pachyclados were inoculated with A. rhizogenes strain ATCC43057 in sterile conditions. Subsequent growth of putatively transformed roots...

  1. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    OpenAIRE

    ÇEVİK, Bayram; Richard F. Lee; NIBLETT, Charles L.

    2012-01-01

    Citrus paradisi Macfad. ‘Duncan’ was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from a CTV genome and used to generate the wild-type and 2 mutant RdRp constructs for plant transformation. One mutant had the key amino acids GDD changed to AAA (RdRp-mGDD), and the second mutant had a deletio...

  2. Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

    Science.gov (United States)

    Wang, Meng; Wang, Diandong; Zhang, Xi; Wang, Xu; Liu, Wencui; Hou, Xiaomeng; Huang, Xiaoyin; Xie, Bingyan; Cheng, Xinyue

    2016-05-01

    RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode

  3. Inactivation of a transgene due to transposition of insertion sequence (IS136) of Agrobacterium tumefaciens

    Indian Academy of Sciences (India)

    Preeti Rawat; Sanjeev Kumar; Deepak Pental; Pradeep Kumar Burma

    2009-06-01

    Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, although the fidelity of the binary vector was confirmed following transformation into Agrobacterium. Such transpositions are rare but can occur and it is thus important to check the fidelity of the binary vector at different times of Agrobacterium growth in order to avoid failure in achieving transgene expression.

  4. Behavior of IncQ Plasmids in Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Schilperoort, Rob

    1981-01-01

    Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

  5. A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

    Directory of Open Access Journals (Sweden)

    Mizuri Marutani-Hert

    Full Text Available BACKGROUND: Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. METHODOLOGY/PRINCIPAL FINDINGS: Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. SIGNIFICANCE: The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

  6. USING OF Agrobacterium-MEDIATED TRANSFORMATION FOR THE BIOTECHNOLOGICAL IMPROVEMENT OF COMPOSITAE PLANTS. ІІ. SYNTHESIS OF BIOACTIVE COMPOUNDS IN TRANSGENIC PLANTS AND «HAIRY» ROOTS

    Directory of Open Access Journals (Sweden)

    N. A. Matvieieva

    2015-04-01

    Full Text Available The review focused on the data concerning current state in the field of Compositae “hairy” roots and transgenic plants construction using A.tumefaciens- and A. rhizogenes-mediated transformation to obtain biologically active compounds, including recombinant proteins. The article presents data on the results of genetic transformation of Cichorium intybus, Lactuca sativa, Artemisia annua, Artemisia vulgaris, Calendula officinalis, Withania somnifera and other Compositae plants as well as studies on the artemisinin, flavonoids, polyphenols, fructans and other compounds accumulation in transgenic plants and roots. The data show that the use of biotechnological approaches for construction of "hairy" roots and transgenic plants with new features are of great interest. The possibility of increase in the accumulation of naturally synthesized bioactive compounds and recombinant proteins production via A. tumefaciens and A. rhizogenes-mediated transformation have been shown. In vitro cultivation of transgenic plants characterized by high level of bioactive compounds accumulation and synthesis of recombinant proteins makes it possible to obtain guaranteed pure raw material. Using of biotechnological approaches preserved natural populations of plants is particularly important for rare and endangered plant species.

  7. Agrobacterium-mediated Transformation of an Economically Important Potato Cultivar Using Internodal Stem Explants%一种加工型马铃薯品种的农杆菌转化体系研究

    Institute of Scientific and Technical Information of China (English)

    汤莉; 汤晖; 王素英; 杨晓丽; LEE Haeng-Soon; KWAK Sang-Soo

    2007-01-01

    大西洋马铃薯是经济价值很高的炸片型加工品种,逆境胁迫下,易产生褐变、空心等问题,影响加工品质.为获取抗逆境胁迫的优质转基因新品种,采用根癌农杆菌介导法,以大西洋马铃薯的茎段为外植体,建立了快速,简便,高效的遗传转化体系.从共培养到转化植株获得只需7~8周,转化频率达80%.结果表明茎段是较好的转化受体,硫代硫酸银可以有效促进不定芽分化并提高再生频率.PCR、Southern杂交分析证明外源基因已经成功整合到马铃薯再生植株的基因组中.该转化体系为大量开发转基因马铃薯植株,进而筛选优质的马铃薯炸片加工型新品种奠定基础.%Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7~8 weeks), high efficiency and easy

  8. Global analysis of differentially expressed genes and proteins in the wheat callus infected by Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Xiaohong Zhou

    Full Text Available Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs and differentially expressed proteins (DEPs were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq and two-dimensional electrophoresis (2-DE in conjunction with mass spectrometry (MS. A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.

  9. Microbiological transformations. Part 48 : Enantioselective biohydrolysis of 2-, 3- and 4-pyridyloxirane at high substrate concentration using the Agrobacterium radiobacter AD1 epoxide hydrolase and its Tyr215Phe mutant

    NARCIS (Netherlands)

    Genzel, Y; Archelas, A; Lutje Spelberg, Jeffrey H.; Janssen, DB; Furstoss, R

    2001-01-01

    The epoxide hydrolase (EH) from Agrobacterium radiobacter AD1 wild type (ArWT) and its Tyr215Phe mutant were compared for the biocatalyzed hydrolytic kinetic resolution (BHKR) of 2-, 3- and 4-pyridyloxirane. The radioselectivity of the oxirane ring opening as well as the substrate concentration limi

  10. 农杆菌介导的高效玉米遗传转化体系的建立%Establishment of high efficiency genetic transformation system of maize mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    魏开发

    2009-01-01

    In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25℃ for co-culture temperature, 0.7 OD×5 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively.%为了建立玉米高频再生及高效遗传转化体系,对影响玉米胚性愈伤组织诱导的11个因素及影响胚性愈伤分化的9个因素用正交实验方法进行研究.结果显示,基因型对胚性愈伤诱导有极显著影响.6-BA.培养基、AgNO3、2,4-D、ABA对胚性愈伤诱导的影响达到显著水平.多重比较分析显示ABA 2 mg/L每间隔1代添加对胚性愈伤诱导率有显著影响.在影响分化的因素中,基因型和6-BA浓度表现出极强

  11. 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.

  12. Agrobacterium-Mediated Transformation of Resveratrol Synthase Gene (PcPKS5) into Huping Jujube (Zizyphus jujuba)%农杆菌介导虎杖芪合酶基因遗传转化壶瓶枣的研究

    Institute of Scientific and Technical Information of China (English)

    罗在柒; 郭辉力; 杨亚东; 杨明峰; 马兰青; 王有年

    2015-01-01

    synthase ( STS ) gene, PcPKS5,contains all functionally divergent plant specific type III PKSs and is involved in resveratrol synthesis. The resveratrol synthase genes are expressed in many transgenic crops such as rapeseed and wheat,successfully generating transgenic plants that have enhanced anti-fungal functions. To allow resveratrol accumulation in fruit organs and improve resistance of jujube to fungal pathogens,the PcPKS5 was transformed into Huping jujube under the control of the CaMV 35S promoter,and the obtained transgenic plants were tested if they increased resveratrol accumulation. This study aimed to assess the effects of heterologous overexpression of the resveratrol synthase gene ( PcPKS5 ) in Huping jujube plant resistance and nutritional quality. [Method]Stems with leaves and shoot tips of Huping jujube were infected with agrobacterium carrying PcPKS5 and GUS, and three positive plants were identified. [Result]The PcPKS5 gene previously cloned from Polygonum cuspidatum in our laboratory was amplified in the TOP10 bacterial strain and ligated to the pMD 18-T vector. Two primers were designed based on the gene bank sequence EU647245 and synthesized by Sangon Biotech Shanghai Co. Ltd. to clone the STS gene for plant expression plasmid construction. Agrobacterium strain EHA105 harboring the pCAMBIA3301-121 plasmid with the PcPKS5 genes controlled by the cauliflower mosaic virus ( CaMV) 35S promoter and termination sequences was used as the vector system for transformation. The infection lasted 15 min,a high percentage of GUS positive leaves was observed. The optimized conditions for transformation were 15 min infection and 2 days co-culture in the dark. The control bacterial concentration was OD600 0. 6 as well,and AS was added at 60 mg·L -1 . Experimental result showed that a total of 197 plants regenerated from nearly 20 000 buds were obtained during the glufosinate-ammonium resistance screening. However,only three actual resistant transgenic plants were

  13. Horizontal gene transfer from Agrobacterium to plants

    Directory of Open Access Journals (Sweden)

    Tatiana V. Matveeva

    2014-08-01

    Full Text Available Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A.rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named cellular T-DNA (cT-DNA. It represents an imperfect inverted repeat and contains homologues of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14 and an opine synthesis gene (Ngmis. A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologues of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

  14. Differentiation of Phytopathogenic Agrobacterium spp.

    Directory of Open Access Journals (Sweden)

    Nemanja Kuzmanović

    2011-01-01

    Full Text Available Due to the difficulties in differentiation of phytopathogenic Agrobacterium spp. and lack of a standardized protocol, we carried out selection and evaluation of suitable methods based on the bacterial physiological, genetic and pathogenic properties. Strains of Agrobacterium tumefaciens, A. rhizogenes and A. vitis were differentiated using standard bacteriological and molecular methods. The biochemical and physiological tests confirmed authenticity of the strains. Two duplex PCR methods were conducted with four different primer pairs. In all strains, presence of plasmid virD2 and virC pathogenicity genes was detected. Chromosomal pehA gene was determined in A. vitis strain. Pathogenicity was confirmed on carrot slices and young plants of tomato and sunflower. Strains of A. tumefaciens and A. vitis were pathogenic on all test plants, while strain of A. rhizogenes induced characteristic symptoms only on carrot slices. The tests used in this study provided reliable discrimination between the three species and confirmed their identity as tumorigenic (TiAgrobacterium tumefaciens and A. vitis, and rhizogenic (Ri A. rhizogenes.

  15. Transgenic shoots and plants as a source of natural phytochemical products

    OpenAIRE

    Katarzyna Floryanowicz-Czekalska; Halina Wysokińska

    2014-01-01

    Genetic engineering has allowed the production of plants and in vitro cultures with an altered content of secondary metabolites. In the present work it is hoped to give some detailed background information on obtaining bioactive compounds based on the use of genetically transformed shoots and the whole plants. Agrobacterium tumefaciens-mediated shoots have recently been a matter of great interest as a source of chemicals synthesized in the aerial parts of plants. The possibilities for the fut...

  16. Scientific Opinion on application (EFSA-GMO-BE-2011-101) for the placing on the market of herbicide-tolerant genetically modified oilseed rape MON 88302 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

    OpenAIRE

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

    2014-01-01

    Oilseed rape MON 88302 was developed by Agrobacterium tumefaciens-mediated transformation to express the CP4 EPSPS protein, which confers tolerance to glyphosate. The molecular characterisation of oilseed rape MON 88302 did not raise safety issues. Agronomic and phenotypic characteristics of oilseed rape MON 88302 tested under field conditions revealed no biologically relevant differences between oilseed rape MON 88302 and its conventional counterpart, except for days-to-first flowering. No d...

  17. Scientific Opinion on application (EFSA-GMO-BE-2011-101) for the placing on the market of herbicide-tolerant genetically modified oilseed rape MON 88302 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

    OpenAIRE

    EFSA Panel on Genetically Modified Organisms (GMO)

    2014-01-01

    Oilseed rape MON 88302 was developed by Agrobacterium tumefaciens-mediated transformation to express the CP4 EPSPS protein, which confers tolerance to glyphosate. The molecular characterisation of oilseed rape MON 88302 did not raise safety issues. Agronomic and phenotypic characteristics of oilseed rape MON 88302 tested under field conditions revealed no biologically relevant differences between oilseed rape MON 88302 and its conventional counterpart, except for days-to-first flowering. No d...

  18. Scientific Opinion on the application (EFSA-GMO-BE-2012-110) for the placing on the market of tissue-selective herbicide-tolerant genetically modified maize MON 87427 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

    OpenAIRE

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

    2015-01-01

    Maize MON 87427 was developed by Agrobacterium tumefaciens-mediated transformation to express the CP4 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) protein, in all tissues except for the male reproductive tissues, conferring tissue-selective tolerance to glyphosate. The molecular characterisation of maize MON 87427 did not give rise to safety issues. Agronomic and phenotypic characteristics as well as compositional data of maize MON 87427 did not raise food/feed and environmental safet...

  19. Plant responses to Agrobacterium tumefaciens and crown gall development

    Directory of Open Access Journals (Sweden)

    Jochen eGohlke

    2014-04-01

    Full Text Available Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumours. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide (‘omic’ approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumour formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant.

  20. Optimization of Conditions for Agrobacterium tumefciens-Mediated Transformation and Regeneration of Maize Inbred Line A188 Callus%根癌农杆菌介导的玉米自交系A188愈伤组织再生条件的优化

    Institute of Scientific and Technical Information of China (English)

    仇平平; 辛相启; 刘文浩; 苏文敏; 竺晓平

    2012-01-01

    The factors affecting the Agrobacterium tumefciens - mediated transformation and regeneration of maize (Zea mays L. ) embryogenic callus were optimized, and the genetic transformation system was established for the maize inbred line A188. The results indicated that 1.0-1.5 mm immature embryos were easier to induce embryogenic callus; the addition of acetosyringone in infection solution and common medium could increase the transformation efficiency significantly; appropriate bacterial concentration ( OD600 = 0.4) , incubation time (3 days) and co -culture temperature (24℃) were benefit to increasing formation efficiency.%以A188玉米自交系的愈伤组织为材料,研究了影响农杆菌介导玉米转化和再生的各种因素,建立了该自交系的遗传转化体系.结果表明,1.0~1.5 mm的幼胚最易诱导出胚性愈伤组织;在侵染液和共培养基中加入乙酰丁香酮能显著提高转化效率;合适的菌液浓度(OD600=0.4)、共培养时间(3 d)、共培养温度(24℃)有利于提高转化效率.

  1. Implementación de un protocolo para la producción de raíces pilosas (hairy roots de uña de gato (Uncaria tomentosa mediante transformación con Agrobacterium rhizogenes Implementation of a protocol for the production of hairy roots of cat’s claw (Uncaria tomentosa by Agrobacterium rhizogenes mediated transformation

    Directory of Open Access Journals (Sweden)

    Giovanni Garro Monge

    2012-11-01

    Full Text Available Los beneficios para la salud registrados a partir del uso de metabolitos secundarios de la planta llamada uña de gato (Uncaria tomentosa han generado una fuerte demanda comercial, así como la extracción intensiva de esta especie en los países en los cuales se distribuye, con el consecuente deterioro de este recurso genético en su hábitat natural. Es por eso que resulta necesario implementar protocolos de cultivo de células y tejidos de esta especie, con el fin de lograr la síntesis de los compuestos en forma controlada. La corteza de las raíces es uno de los tejidos en los que se concentra la producción de estos compuestos, razón por la cual la producción de raíces de cabellera (hairy roots resulta ser una técnica alternativa para la producción a escala de los metabolitos de interés. En este proyecto se implementó un protocolo de agroinfección de microestacas de U. tomentosa utilizando cepas silvestres de Agrobacterium rizhogenes (AR1500 y A4RS, así como el mantenimiento en medio líquido de las raíces pilosas obtenidas. En colaboración con el Laboratorio de Biología Molecular del programa PIPRA (UC Davis, se determinó la eficacia del protocolo de agroinfección, así como el uso de otras herramientas moleculares para la detección de expresión génica, las cuales mostraron resultados satisfactorios en los ensayos de agroinfección, bajo las metodologías establecidas en el proyecto.The beneficial health proper ties registered of secondary metabolites produced by the plant cat’s claw (Uncaria tomentosa had generated a strong market demand and intensive extraction of this species in the countries where distributed, with the deterioration of this genetic resource in its natural habitat. Because of this, is necessary to implement protocols for cell and tissue culture of this species in order to achieve the synthesis of compounds in a controlled manner.The root bark is one of the tissues where the production of these

  2. Transgenic shoots and plants as a source of natural phytochemical products

    Directory of Open Access Journals (Sweden)

    Katarzyna Floryanowicz-Czekalska

    2014-02-01

    Full Text Available Genetic engineering has allowed the production of plants and in vitro cultures with an altered content of secondary metabolites. In the present work it is hoped to give some detailed background information on obtaining bioactive compounds based on the use of genetically transformed shoots and the whole plants. Agrobacterium tumefaciens-mediated shoots have recently been a matter of great interest as a source of chemicals synthesized in the aerial parts of plants. The possibilities for the future exploitation of Agrobacterium tumefaciens transformation techniques are enormous. However, we need more knowledge of genes and enzymes controlling secondary metabolic synthesis.

  3. 根癌农杆菌介导的向日葵遗传转化体系的建立%Establishment of Sunflower (Helianthus annuus) Transformation by Agrobacterium-mediated

    Institute of Scientific and Technical Information of China (English)

    郝彦玲; 朱本忠; 朱鸿亮; 栾春光; 罗云波; 段晓昱

    2005-01-01

    采用携带gus和hpt基因双元表达载体pCAMBIA1301的根癌农杆菌(Agrobacterium tumefaciens)EHA105对向日葵(Helianthus annuus)品种新葵杂6号的茎尖分生组织进行遗传转化,以共培养后7 d外植体的gus基因纯合表达频率为指标测定转化率.结果表明,潮霉素适宜的筛选浓度为10mg/L,根癌农杆菌侵染浓度OD600=0.8,果胶酶(0.05%)与纤维素酶(0.1%)共同消化外植体30 min,共培养温度24℃,共培养培养基中添加乙酰丁香酮(ACS)100μmol/L,向日葵茎尖gus基因纯合表达率高达32.8%.对抗性苗进行PCR和Southern blot检测,初步证明T-DNA上的hpt基因已整合向日葵的基因组中.

  4. Agrobacterium-mediated transformation of maize shoot apical meristem by introducing fused gene Chi-linker-Glu and bar%农杆菌介导Chi-linker-Glu融合基因和bar基因转化玉米茎尖的研究

    Institute of Scientific and Technical Information of China (English)

    方永丰; 李永生; 彭云玲; 王芳; 王威; 穆延召; 王汉宁

    2012-01-01

    Most maize disease are caused by fungi pathogens, such as northern leaf blight, southern leaf blight, ear and stalk rots, and so on. In normal years, this disease makes maize yield lost by 10% and this percentage may be increased by 30%-40% in its popular years. However, it is very difficult for conventional breeding to develop an elite maize material with the resistance to all the fungi disease. Therefore, we can use transgenic method to transfer some genes related to the resistance of these disease into maize in order to obtain resistance materials. Chitinase and β-1,3-glucanase are two important enzymes which play a key role in the hydrolysis reaction of fungal cell wall, so it is useful in the control of fungi disease, meanwhile, the herbicide resistant gene bar are usually used as a selectable marker in maize transformation. The purpose of this research is to introduce the fused gene of chitinase and β-1,3-glucanase and bar as well into elite maize inbred Zheng58 for improving its resistance to fungi disease. The wounded shoot apical meristem (SAM) of germinated seedlings were used as the material for Agrobacterium mediated transformation. We report here: 1) An optimized transformation system for Agrobacterium mediated transformation of shoot apical meristem, using the optimal concentration of bacterial culture (the value of OD600 was 0. 6) for infection, addition of 150 μmol/L acetosyringone (AS) in the bacterial suspension, the whole infection process was carried out in a vacuum desiccators with a negative pressure of 50 kPa for 12 minutes. 2) Identified by herbicide screening procedure and PCR detection, 13 transformed plants were obtained among the 32 herbicide-resistant plants, and the overall transformation was 2. 6%. The preliminary evidences showed that the foreign genes had been introduced into the maize genome. This method circumvented the long period of tissue culture step and limitation of different seasons, in addition, many elite inbred lines

  5. Enhanced Virulence Gene Activity of Agrobacterium in Muskmelon (Cucumis melo L. cv. ‘Birdie’

    Directory of Open Access Journals (Sweden)

    Abul K.M. MOHIUDDIN

    2011-05-01

    Full Text Available Muskmelon (Cucumis melo L. cultivar ‘Birdie’, was evaluated for its response to the tumorigenic Agrobacterium tumefaciens and the oncogenic A. rhizogenes strains. Stem and petiole of three week-old in vitro-grown muskmelon plants were inoculated with five strains of A. tumefaciens and A. rhizogenes each and observed phenotypic expressions i.e. induction of crown galls and hairy roots. This phenotypic expression was efficaciously increased when virulence gene activity of different strains of two Agrobacterium species was enhanced. Intensive studies on enhancement of virulence gene activity of Agrobacterium found to be correlated to the appropriate light intensity (39.3 μmol m-2 s-1 with a specific concentration of monocyclic phenolic compound, acetosyringone (20 μM. The gene activity was also influenced by several other physical factors e.g. plant tissue type, Agrobacterium species and their strains, and plant tissue-Agrobacterium interaction. Among the different A. tumefaciens strains, LBA4404 showed the best virulence gene activity in both stem and petiole through the formation of higher rate of crown galls. On the other hand, strain 15834 of A. rhizogenes showed better gene activity in stem and 8196 in petiole through the formation of higher rate of hairy roots as well as higher average number of hairy roots. Among the two different types of explants, petiole was more susceptible to both Agrobacterium species. Thus it was concluded that future muskmelon transformation study can efficiently be carried out with LBA4404, 15834 and 8196 strains using petiole explants by adding 20 μM of acetosyringone in the medium.

  6. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor

    OpenAIRE

    Heidmann, I.; Lange; Lambalk, J.; Angenent, G.C.; Boutilier, K.

    2011-01-01

    Pepper (Capsicum L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using Agrobacterium tumefaciens (agrobacterium) is a powerful biotechnology tool that could be used in pepper to develop community-based functional genomics resources and to introduce important agronomic traits. However, pepper is considered to be highly recalcitrant for agrobacterium-mediated transformation, and c...

  7. An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba × P. berolinensis and Populus davidiana × P. bolleana.

    Science.gov (United States)

    Wang, Haihai; Wang, Cuiting; Liu, Hua; Tang, Renjie; Zhang, Hongxia

    2011-11-01

    Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT-PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding. PMID:21717184

  8. TRANSFORMATION

    Energy Technology Data Exchange (ETDEWEB)

    LACKS,S.A.

    2003-10-09

    Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants was revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).

  9. Transformation

    DEFF Research Database (Denmark)

    Peters, Terri

    2011-01-01

    Artiklen diskuterer ordet "transformation" med udgangspunkt i dels hvorledes ordet bruges i arkitektfaglig terminologi og dels med fokus på ordets potentielle indhold og egnethed i samme teminologi.......Artiklen diskuterer ordet "transformation" med udgangspunkt i dels hvorledes ordet bruges i arkitektfaglig terminologi og dels med fokus på ordets potentielle indhold og egnethed i samme teminologi....

  10. Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.

    Science.gov (United States)

    Ali, Shawkat; Bakkeren, Guus

    2015-01-01

    The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi. PMID:25239747

  11. TRANSFORMER

    Science.gov (United States)

    Baker, W.R.

    1959-08-25

    Transformers of a type adapted for use with extreme high power vacuum tubes where current requirements may be of the order of 2,000 to 200,000 amperes are described. The transformer casing has the form of a re-entrant section being extended through an opening in one end of the cylinder to form a coaxial terminal arrangement. A toroidal multi-turn primary winding is disposed within the casing in coaxial relationship therein. In a second embodiment, means are provided for forming the casing as a multi-turn secondary. The transformer is characterized by minimized resistance heating, minimized external magnetic flux, and an economical construction.

  12. Phenanthrene-degrading pathway of Agrobacterium sp. Phx1

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lei; YUAN Hongli; WANG Shuangqing; HUANG Huaizeng

    2005-01-01

    The metabolic pathway of phenanthrene-degrading strain Agrobacterium sp. Phx1 was investigated. Phx1 almost was able to transform 100 υg/mL of phenanthrene completely in 1 day in broth media of beef extract-peptone (BP), Luria-Bertani (LB) and mineral salts media (MS), and LB and BP could promote the growth and degradation efficiency of Phx1. The GC-MS was employed to analyze the metabolites of the 1st, 3rd, 7th days of phenanthrene degradation in MS. As a result, the 1-Hydroxy-2-naphthoic acid (1H2N) and 1-naphthol (NOL) were detected in the metabolites of the 1st day. Only NOL was observed on the 3rd day and it disappeared on the 7th day. The accumulated NOL did not pertain to the defined pathway of phenanthrene degradation by bacteria. The further HPLC study confirmed the finding in GC-MS analysis and found the production of catechol (CAT) from o-phthalic acid (OPA) in the phenanthrene metabolizing, which has never been reported in the defined degrading pathways. This production was also evidenced by the production of CAT using OPA as substrate. All of our results showed that the Agrobacterium sp. Phx1 had a novel phenanthrene-degrading pathway.

  13. Transformation of Rhodiola rosea with rol-genes from Agrobacterium rhizogenes to enhance the production of bioactive compounds and analyses of key genes in their pathways

    DEFF Research Database (Denmark)

    Stofberg, Jimmi; Antypa, Natalia-Meropi; Müller, Renate;

    .03 % (v/v) tween20 for 5 min. Leaf cuttings were then inoculated with A. rhizogenes strain ATCC4305 pRiA4 for more approximately 30 min. The inoculated leaf cuttings were then co-cultivated with the bacteria for 2 days in darkness, washed in MilliQ water with 10 g l-1 timentin and transferred to a 1x MS...... medium containing 100 mg l-1 timentin. After this explants were monitored for development of HRs. Approximately 60 % of the transformed explants developed HRs compared to 27 % for the untreated controls. Our preliminary data showed that the transformed HR formation peaked between 5 and 10 weeks after...

  14. Agrobacterium-mediated transformation of Musa spp.cv.Tianbao with cDNA encodina S6PDH(NADP-dependent sorbitol-6-phosphate dehydrogenase) from Prunus salicina var.cordata%根癌农杆菌介导的榇S6PDH基因转化香蕉研究

    Institute of Scientific and Technical Information of China (English)

    匡云波; 赖钟雄

    2012-01-01

    研究以香蕉栽培品种“天宝蕉”(Musa spp.cv.Tianbao)横切薄片(Thin cross-sections,TCSs)为材料,采用根癌农杆菌介导的方法,进行棒S6PDH基因转化香蕉的研究.结果表明,在横切薄片继代增殖培养基M4中添加5%~7%(V/V)的椰汁明显增强了香蕉芽苗的生长势;GUS基因瞬时表达检测表明,长势旺盛的香蕉芽苗(直径为7~8 mm)适宜作为香蕉遗传转化的受体材料,横切薄片厚度以2mm左右为佳;采用两步法进行抗性芽的筛选得到37个抗性芽苗,生根移栽后获得31株成活苗;目的基因S6PDH和报告基因GUS的PCR检测表明其中4株是转基因植株.该研究为将蔷薇科山梨醇代谢途径引入香蕉以提高其耐渗透胁迫的能力奠定了重要的基础.%Musa spp. cv. Tianbao was transformed with cDNA encoding S6PDH(NADP-dependent sorbitol-6-phosphate dehydrogenase) isolated from Prunus sa/icina var. cordata by an Agrobacterium-mediated thin cross-sections (TCSs) transformation system. The condition of the buds was effectively improved when the TCSs were transferred onto the medium M4 adding 5%-7%(V/V) coconut water. And the highest GUS transient expression occurred while 2 mm thin TCSs from the healthy and strong buds were used as the recepted material. Total 37 putative transformants were selected via the two-step method and 31 putative transformants survived after transplanting. Finally, four transgenic lines were conformed by PCR analysis of S6PDH gene and GUS gene. Sorbitol synthesis pathway which was unique to the Rosaceae plants had been introduced into Musa spp.cv.Tianbao, laying the groundwork to increase its tolerance to environmental stress.

  15. Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

    Science.gov (United States)

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

    2015-01-01

    Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant. PMID:25102992

  16. Agrobacterium may delay plant nonhomologous end-joining DNA repair via XRCC4 to favor T-DNA integration.

    Science.gov (United States)

    Vaghchhipawala, Zarir E; Vasudevan, Balaji; Lee, Seonghee; Morsy, Mustafa R; Mysore, Kirankumar S

    2012-10-01

    Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-ray cross complementation group4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate. PMID:23064322

  17. Response of Kalanchoe daigremontiana to wounding and infection with Agrobacterium tumefaciens

    OpenAIRE

    Tkalec, Mirta; Car, Diana; GOSPOČIĆ, JANKO; KRIŽAIĆ, IVA; DUŽ, KAROLINA; VIDAKOVIĆ-CIFREK, ŽELJKA

    2012-01-01

    Background and Purpose: Transformation of plant tissue with Agrobacterium tumefaciens includes wounding of plant and subsequent infection by bacteria. Polyphenol oxidase activity and oxidative stress parameters – the content of H2O2, as well as activity and isoenzymes of antioxidative enzymes catalase, pyrogallol and guaiacol peroxidase were investigated as markers of plant response to wounding and infection. Materials and Methods: Five tissue types – healthy tissue, wounded tissue, tissue in...

  18. 利用农杆菌介导法获得转柠檬酸合成酶基因粳稻及其耐低磷的研究%Genetic Transformation of Japonica Rice Mediated with Agrobacterium tumefaciens Harboring the Citrate Synthase Gene and Tolerance of the Transgenic Plants to Low Phosphorus in Soil

    Institute of Scientific and Technical Information of China (English)

    于志晶; 蔡勤安; 李淑芳; 刘丽; 林秀峰; 马瑞

    2012-01-01

    The citrate synthase gene was transformed into main cultivar (super rice 'Jijing 88') of Jilin Province, mediated with Agrobacterium tumefaciens. Total 162 obtained transgenic plants (TO) were validated by PCR and southern blot. Five transgenic plants (T3) with tolerance to low phosphorus in soil and good agronomic characteristics were obtained by PPT selection, PCR and Southern blot confirmation and tolerance test to low phosphorus. The citrate synthase activity and citrate acid contents in root tips and leaves of the 5 transgenic plants were higher than the control. The growth and yield of the transgenic plants were superior to the control.%利用根癌农杆菌介导法将柠檬酸合成酶CS基因导入吉林省主栽超级粳稻品种吉粳88中.经PCR检测,获得162株转基因阳性植株.转基因植株后代进一步经过PPT抗性筛选、分子检测和耐低磷筛选,获得5株(T3代)耐低磷性状明显且农艺性状较好的转基因植株.对转基因植株柠檬酸合成酶活性和柠檬酸含量的测定以及形态学和产量性状调查结果表明:转基因植株优于非转基因对照植株.

  19. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    Directory of Open Access Journals (Sweden)

    Benoît Lacroix

    Full Text Available VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.

  20. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Directory of Open Access Journals (Sweden)

    Sujatha eSubramoni

    2014-07-01

    Full Text Available As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its T-DNA (Transferred DNA from its Tumour-inducing (Ti plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA, cytokinin (CK and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including -amino butyric acid (GABA and salicylic acid (SA to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene (ET to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium-plant interactions.

  1. 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

  2. Studies on the Agrobacterium Mediaed Transformation of the Bivalent Gene Conferring Glyphosate Tolerance into Maize Lines%农杆菌介导的转双价基因耐草甘膦玉米研究

    Institute of Scientific and Technical Information of China (English)

    余桂容; 刘艳; 杜文平; 宋军; 张莲; 陆伟; 徐利远

    2013-01-01

    A bivalent plant expression vector pCMG2R79-EPSPS expressing the glyphosatetolerant G2-epsps gene and R79-epsps gene was constructed and transformed into the immature embryos of the elite maize lines 78599 and Zong31, for gaining more higher glyphosate-tolerance of transgenic maize plants. To this day, 32 regeneration plants was acquired and the specific PCR detection showed that 7 plants of them were transferred with the target gene. The positive rate was 21.9%. Further analysis was performed on the T1 and T2 generation and it was.obtained 6‰concentration glyphosate-resistant plants, that was 3 times concentration levels than the glyphosate commer-cial using did, by spraying herbicide glyphosate in field. It would provide a kind of better parent materials for breeding the new resistant herbicide glyphosate maize varieties.%  本研究构建了含有耐草甘膦基因G2-epsps和R79-epsps的双价植物表达载体pCMG2R79-EPSPS,以优良玉米自交系78599和综31的幼胚为受体材料,采用农杆菌介导法将耐草甘膦双价基因G2R79-epsps转入玉米幼胚细胞,以期获得耐草甘膦性更高的转基因玉米植株。目前已获得转基因再生玉米植株32株,经特异PCR检测表明其中7株植株转入了目标基因,阳性率达到21.9%。对得到的阳性植株的T1、T2代进行了跟踪检测,经田间喷施除草剂试验结果表明,获得了可耐草甘膦6‰的玉米材料,即生产上草甘膦田间使用浓度3倍水平的材料,为抗除草剂玉米新品种选育提供了较好的基础材料。

  3. Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The legume forage Alhagi pseudoalhagi was transformed by the Agrobacterium rhizogenes strain A4 using cotyledon and hypocotyl segments as infection materials. Regenerated plants were achieved from sterile calli derived from hairy roots, which occurred at or near the infection sites. The regenerated plants from hairy root were characterized by normal leaf morphology and stem growth but a shallow and more extensive root system than normal plants. Opine synthesis, PCR and Southern blot confirmed that TDNA had been integrated into the A. pseudoalhagi genome. Acetosyringone (AS) was found to be vital for successful transformation of A. pseudoalhagi.

  4. Agrobacterium-mediated transformation of LJAMP2 gene into ‘Red Sun' kiwifruit and its molecular identification%农杆菌介导LJAMP2基因导入‘红阳’猕猴桃及分子鉴定

    Institute of Scientific and Technical Information of China (English)

    周月; 赵许朋; 吴秀华; 张艳玲; 张林; 罗克明; 汤绍虎

    2014-01-01

    为了获得抗溃疡病的‘红阳’猕猴桃转基因植株,以红阳猕猴桃试管苗叶盘为转基因受体材料,通过根癌农杆菌介导将CaMV35S启动子调控下的LJAMP2基因导入红阳猕猴桃.450个叶盘与携带表达载体质粒pBI121的根癌农杆菌菌珠LBA4404共培养2d后,转入含25 mg/L Kan的筛选培养基培养40 d,15d转接1次,之后30d继代1次.结果表明,在MS+3.0 mg/L BA+1.0 mg/L NAA筛选培养基中,Kanr芽率达85%以上,在1/2 MS+0.8 mg/L IBA培养基中,Kanr芽生根率达100%.共获得Kanr再生植株40株,经GUS组织染色和PCR分析证明,其中23株为转基因植株.阳性率为57.50%,转化率达5.11%.抗溃疡病基因LJAMP2已成功导入红阳猕猴桃,为红阳猕猴桃的抗病基因工程育种奠定了基础.%Bacterial canker caused by Pseudomonas syringae pv.Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses.In order to obtain transgenic plants with resistance for ‘Red Sun' kiwifruit to canker disease,a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurusjaponicus) was introduced into ‘Red Sun' kiwifruit through Agrobacterium-mediated transformation.After two days of co-cultivation with A.tumefaciens strain LBA4404 harboring 35S:LJAMP2,the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA.The regeneration efficiency of kanamycin-resistant shoots reached to 85%.All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained.PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive,suggesting that the LJAMP2 gene was integrated into the genome of ‘Red Sun' kiwifruit.Taken together,we established an efficient genetic transformation method for ‘Red Sun' kiwifruit using A

  5. Cowpea [Vigna unguiculata (L.) Walp].

    Science.gov (United States)

    Behura, Ratikanta; Kumar, Sanjeev; Saha, Bedabrata; Panda, Manasa Kumar; Dey, Mohitosh; Sadhukhan, Ayan; Mishra, Sagarika; Alam, Shamsher; Sahoo, Debee Prasad; Sugla, Twinkle; Sahoo, Lingaraj

    2015-01-01

    Agrobacterium tumefaciens-mediated transformation is an efficient method for incorporating genes and recovering stable transgenic plants in cowpea because this method offers several advantages such as the defined integration of transgenes, potentially low copy number, and preferential integration into transcriptional active regions of the chromosome. Cotyledonary node explants of cowpea present an attractive target for T-DNA delivery followed by regeneration of shoots via axillary proliferation without involvement of a de novo regeneration pathway. In this chapter, we describe a detailed protocol for Agrobacterium-mediated transformation of the cowpea variety Pusa Komal. The seedling cotyledonary node explants are used for cocultivation with an Agrobacterium strain EHA105 harboring standard binary vector, pCAMBIA2301 or pNOV2819, and putative transformed plants are selected using aminoglycoside antibiotic or mannose as sole carbon source, respectively. The entire process includes explant infection to transgenic seed generation in greenhouse. PMID:25300846

  6. Cherry.

    Science.gov (United States)

    Song, Guo-Qing

    2015-01-01

    Agrobacterium tumefaciens-mediated transformation of sour chgahtvy (Prunus cerasus L.) "Montmorency" and sweet cherry rootstocks "Gisela 6" and "Gisela 7" (P. cerasus × P. canescens) is described. Briefly, leaf explants from in vitro shoots are cocultivated with A. tumefaciens either directly (for "Gisela 6" and "Gisela 7") or after pretreatment (for "Montmorency") on cocultivation medium; selection and regeneration of transformed shoots are carried out on selection medium containing 50 mg/L kanamycin (Km) and 250 mg/L timentin (or cefotaxime) for 3-5 months. In this protocol, the optimal media for shoot proliferation and shoot regeneration from leaf explants are genotype dependent. PMID:25416255

  7. Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    A Srivastava; S K Raj

    2008-06-01

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

  8. МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЙ АНАЛИЗ ТРАНСГЕННЫХ РАСТЕНИЙ КАРТОФЕЛЯ С ГЕНОМ GOX PENICILLIUM FUNICULOSUM

    OpenAIRE

    Савчин, Д. В.; Панюш, А. С.; Картель, Н. А.

    2011-01-01

    Glucose oxidase (GOX) enzyme can generate H2O2 by the oxidation of glucose in the presence of molecular oxygen. H2O2 has the potential to enhance biotic and abiotic stress tolerance of higher plants. The gene encoding glucose oxidase was isolated from the genomic DNA of Penicillium funiculosum. The GOX gene was cloned under the control of a CaMV 35S promoter into the plant transformation vector pBI121. Potato was transformed via an Agrobacterium tumefaciens-mediated procedure by s...

  9. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor

    NARCIS (Netherlands)

    Heidmann, I.; Lange, de B.; Lambalk, J.; Angenent, G.C.; Boutilier, K.

    2011-01-01

    Pepper (Capsicum L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using Agrobacterium tumefaciens (agrobacterium) is a powerful biotechnology tool that could be used in pepper to develo

  10. Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration[W

    Science.gov (United States)

    Vaghchhipawala, Zarir E.; Vasudevan, Balaji; Lee, Seonghee; Morsy, Mustafa R.; Mysore, Kirankumar S.

    2012-01-01

    Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)–mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-RAY CROSS COMPLEMENTATION GROUP4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate. PMID:23064322

  11. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

    Directory of Open Access Journals (Sweden)

    Mohammad M. Rana

    2016-07-01

    Full Text Available Tea (Camellia sinensis L. is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose. Additionally, the reporter genes β-glucuronidase (gusA and cyan fluorescent protein (cfp were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

  12. Protoplast Culture and Plantlet Regeneration from Cell Line of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi Desv%骆驼刺发根农杆菌转化系的原生质体培养和植株再生

    Institute of Scientific and Technical Information of China (English)

    张改娜; 贾敬芬

    2009-01-01

    The protoplasts were isolated from calli which were induced from hairy root segments of Agrobacterium rhizogenes A4-transformed Alhagi pseudalhagi. After cultured in the DPD medium supplemented with 1.5 mg·L~(-1) 2,4-D, 0.2 mg·L~(-1) 6-BA, 0.3 mol·L~(-1) mannitol, 500 mg·L~(-1) casein hydrolysate (CH) and 2% (W/V) sucrose, the protoplasts underwent sustained divisions and formed calli. The protoplast density of 4×10~5 mL~(-1) and (450±3) mOsm·kg~(-1) osmotic pressure in culture medium were proved to be appropriate for obtaining higher division frequency of protoplasts. A lot of protoplasts could be obtained by the enzymatic hydrolysis of yellowish subcultured calli after cultured on MS medium supplemented with 1.5 mg·L~(-1) NAA, 1.0 mg·L~(-1) 6-BA, 500 mg·L~(-1) CH and 2% (W/V) sucrose for 7-10 d. Lower temperature (4 ℃) pretreatment of subcultured calli enhanced ratios of protoplast isolation and subsequent divisions. The division frequency of protoplasts was about 50%. After transferred on the MS medium added with 1-2 mg·L~(-1) 6-BA (or KT) and 0.2 mg·L~(-1) NAA, the protoplast-derived calli differentiated and formed the regenerated plantlets. Paper electrophoresis analysis indicated that the protoplast-derived calli and regenerated plantlets still contained special product-opine in transgenic root hairs.%从发根农杆菌A4转化的荒漠植物-骆驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7~10 d的淡黄色松软愈伤组织,可获得大量有活力的原生质体.原生质体在附加有1.5 mg·L~(-1) 2,4-D、0.2 mg·L~(-1) 6-BA、0.3 mol·L~(-1)甘露醇、2%(W/V)蔗糖和500 mg·L~(-1)水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂.培养基的最适渗透压为(450±3)mOsm·kg~(-1),原生质体的最适植板密度为4×10~5个·mL~(-1).制备原生质体的愈伤组织以低温(4℃)预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50%.

  13. Promoter deletions are essential for transformation of lettuce by the T-cyt gene: the phenotypes of transgenic plants

    NARCIS (Netherlands)

    Curtis, H.E.C.; Jordi, W.; Davelaar, E.; Power, J.B.; Laat, de A.M.M.; Davey, M.R.

    1999-01-01

    The Agrobacterium T- cyt gene was transferred into lettuce, Latuca sativa‘Saladin’ using a genotype-independent transformation procedure employing a supervirulent Agrobacterium tumefaciens strain carrying the binary vector pMOG23. Kanamycin-resistant shoots were initiated from inoculated explants on

  14. Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens Transient gene expression in sugarcane using Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Martha Liliana Bonilla Betancourt

    2008-10-01

    Full Text Available En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105 con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404 con pCambia 2301. Se usó el medio de infiltración (IM con acetosiringona y se evaluó el tiempo de cocultivo y la densidad óptica de la bacteria al momento de la inducción. Los genotipos mostraron respuesta diferencial con las combinaciones cepa-plásmido: obtuvieron mayor expresión del gen GUS cuando el genotipo CC85-92 se transformó con la cepa AGL-1-pCambia 1305.2. CC84-75 y CC87-505 mostraron mayor expresión cuando se transformaron con la cepa EHA105-pCambia 1305.2. Mayor eficiencia en la expresión se obtuvo cuando la bacteria se indujo en IM después de siete días de cocultivo y cuando la densidad óptica de la bacteria fue de 0.2(600nm al momento de la inducción. Se demostró superioridad de los explantes en la eficiencia de transformación.The aim of the present study was to develop a transformation method mediated by Agrobacterium in Colombian cultivars of sugarcane. Transformation was evaluated in each step through transient GUS expression. Embryogenic calli and meristematic explants of CC85-92, CC84-75 y CC87-505 cultivars, were transformed using Agrobacterium tumefaciens AGL-1, LBA 4404 and EHA 105 strains, harboring pCambia 1305.2 plasmid. Furthermore, strains LBA 4404 and EHA 105 harboring pCambia 2301 were also tested. Bacterian activator medium, named infiltration media (IM with acetosyringone was used. Co-cultivation time and bacteria optical density before induction were tested. Sugarcane cultivars evaluated showed differential response to different strain-plasmid combinations, obtaining

  15. Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

    Science.gov (United States)

    Sakalis, Philippe A; van Heusden, G Paul H; Hooykaas, Paul J J

    2014-02-01

    Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli. PMID:24376037

  16. The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

    Science.gov (United States)

    Sharifi, Sara; Sattari, Taher Nejad; Zebarjadi, Alireza; Majd, Ahmad; Ghasempour, Hamidreza

    2014-01-01

    We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10-14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g(-1) of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L(-1) naphthaleneacetic acid and 2 mg L(-1) 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots. PMID:24554840

  17. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.

    Science.gov (United States)

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-02-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 μm sec⁻¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  18. Genetic transformation of carnation (Dianthus caryophylus L.).

    Science.gov (United States)

    Nontaswatsri, Chalermsri; Fukai, Seiichi

    2010-01-01

    This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

  19. Ecological dynamics and complex interactions of Agrobacterium megaplasmids

    Directory of Open Access Journals (Sweden)

    Thomas Gene Platt

    2014-11-01

    Full Text Available As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring it’s At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.

  20. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  1. Improving Plant Transformation Using Zygote as the Recipient Cell

    Institute of Scientific and Technical Information of China (English)

    Y.H. Yang; H.Q. Tian

    2007-01-01

    @@ Since the first transgenic plant was obtained from tobacco in the 1980's, the transformation of higher plants has been a vigorous field of study using applications of molecular biology. To date, transgenic methods of introducing foreign genes into higher plants include techniques of electrofusion, eletroporation, microinjection and transformation mediated by PEG and Agrobacterium.

  2. The molecular structure of agrobacterium VirE2-single stranded DNA complexes involved in nuclear import.

    Science.gov (United States)

    Citovsky, V; Guralnick, B; Simon, M N; Wall, J S

    1997-09-01

    Nuclear import of DNA is a central event in genetic transformation of plant cells by Agrobacterium tumefaciens. Agrobacterium elicits tumors on plant hosts by transporting a single-stranded (ss) copy of the bacterial transferred DNA (T-DNA) from its Ti (tumor-inducing) plasmid into the plant cell nucleus. Presumably, the process of T-DNA nuclear import is mediated by two agrobacterium proteins, VirD2 and VirE2, which are thought to directly associate with the transported T-DNA. Both proteins have been shown to contain functional nuclear localizations signals (NLS). Recently, VirE2 alone has been shown to actively transport ssDNA into the plant cell nucleus. To understand the process of DNA nuclear import, it is important to know the structure of the transport intermediate. To this end, complexes of VirE2 and ssDNA were analyzed by scanning transmission electron microscopy (STEM). This analysis suggests that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure. The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import. Detailed mass analysis of VirE2-ssDNA filaments is presented and a structural model is proposed. PMID:9299322

  3. Agrobacterium VirD2 protein interacts with plant host cyclophilins.

    Science.gov (United States)

    Deng, W; Chen, L; Wood, D W; Metcalfe, T; Liang, X; Gordon, M P; Comai, L; Nester, E W

    1998-06-01

    Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer. PMID:9618535

  4. Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and lignan production in Linum tauricum ssp. tauricum

    Directory of Open Access Journals (Sweden)

    Iliana Ionkova

    2009-01-01

    Full Text Available Hairy root cultures were induced from leaf explants of Linum tauricum ssp. Tauricum by infection with Agrobacterium rhizogenes. Different bacterial strains of Agrobacterium rhizogenes - TR 105 and ATCC 15834 were evaluated for induction of transformed hairy roots in Linum tauricum ssp. Tauricum. These different strains varied in their virulence for induction of hairy roots in this species. Acetosyringon in cultivation medium was used to increase of frequency of hairy root induction. Growth kinetics of transgenic roots indicated a similar pattern of growth, with maximum growth occurring between 17 and 20 days. The transformed nature of tissue was confirmed by the production of opines. The lignin production of different clones was found to be growth-related. The cultures produced to 2.6% of the lignin 4′-demethyl-6-methoxypodophylotoxin (4′-DM-MPTOX and to 3.5% of the lignin 6-methoxypodophyllotoxin (6MPTOX on a dry weight basis, which was 10 to 12 times higher than in Linum tauricum ssp. Tauricum cell suspensions. Transformed cultures showed significant differences in lignin content. The highest amount of 4′-DM-MPTOX and MPTOX was found in transformed line induced by strain ATCC 15834. Rapidly growing root lines were selected to increase the efficiency of he production of lignans.

  5. Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated virE2 of Agrobacterium.

    Science.gov (United States)

    Krastanova, Stoyanka V; Balaji, Vasudevan; Holden, Michele R; Sekiya, Mary; Xue, Baodi; Momol, Esengul A; Burr, Thomas J

    2010-12-01

    A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines. PMID:20182792

  6. First Report of Tumorigenic Agrobacterium radiobacter on Raspberry in Serbia

    Directory of Open Access Journals (Sweden)

    Svetlana Milijašević

    2007-01-01

    Full Text Available During the spring of 2003, gall symptoms on the roots and crowns of young raspberry plants cv. Vilamette were observed near Valjevo. Phytopathogenic bacteria were isolated from diseased plant samples. Based on the pathogenic, morphological, differential biochemicaland physiological characteristics, the isolated strains were identified as tumorigenic Agrobacterium radiobacter (biovar 1 Agrobacterium. In order to confirm the identity of isolated strains by polymerase chain reaction (PCR primers complementary to tms2 genelocated on the Ti plasmid were used. In the first PCR protocol using a tms2F1 + tms2R2 primer pair, 617 bp products specific for tumorigenic Agrobacterium strains were amplified. The second PCR protocol, using a tms2F1 + tms2B primer pair, amplified the expected 458 bp products. On the basis of multiplex PCR with primers complementary to chromosomal gene coding for 23S rRNA, the isolated strains were classified as biovar 1 Agrobacterium (A. radiobacter. This is the first report of tumorigenic A. radiobacter on raspberry in Serbia.

  7. Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein.

    Science.gov (United States)

    Das, A

    1988-05-01

    The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmid are essential for transformation of plant cells. Overproduction of a virE-encoded gene product in Escherichia coli was achieved by construction of an operon fusion with the E. coli tryptophan (trp) operon. The virE2 gene product in E. coli partitioned into the insoluble membrane fraction. The protein was solubilized by treatment with 4 M urea at 0 degree C. DNA-protein binding experiments showed that a strong single-stranded (ss) DNA-binding activity was present in protein fractions containing the virE2 gene product. The binding was highly specific with little or no binding observed with either double-stranded DNA or ssRNA. No significant binding to Ti plasmid DNA sequences was observed. Protein blotting studies indicated that the ssDNA-binding activity was associated with the 68-kDa virE2 polypeptide. PMID:2452439

  8. 爬行卫矛再生体系建立及根癌农杆菌介导的遗传转化%Establishment of Direct Adventitious Shoot Regeneration System of Euonymus fortunei var. radicans and Its Genetic Transformation Mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    尚爱芹; 陈颖; 赵梁军; 田颖川

    2008-01-01

    以爬行卫矛(Euonymus fortunci var.radicans)无菌苗下胚轴为外植体,在附加不同浓度植物生长调节剂的MS基础培养基上诱导培养,通过器官直接发生途径获得再生植株.结果表明,0.5 mg/LBAP和0.01 mg/L NAA组合的诱导效果最佳,其不定芽再生率高达92%,外植体平均不定芽数目为4.2个.将含有雪花莲凝集素基因(Galanthus nivalis agglutinin,GNA)的植物表达载体pBCGm导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,通过农杆菌介导法遗传转化爬行卫矛的下胚轴,经PCR和PCR-Southern检测,GNA基因已整合到转化植株的基因组中.遗传转化过程中,外植体不经预培养,菌液浓度OD=0.6,共培养3 d,有利于获得最高遗传转化效率.

  9. Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system

    Directory of Open Access Journals (Sweden)

    Xu Ruqiang

    2008-01-01

    Full Text Available Abstract Background In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway® cloning technology (Invitrogen Co. has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem. Results An integrated protocol for cloning genes of interest from PCR to Agrobacterium transformants via the Gateway® System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections. Conclusion The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to Agrobacterium via the Gateway® System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications.

  10. T-DNA transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

    Institute of Scientific and Technical Information of China (English)

    Chi Yan; Ping Wenxiang; Li Shanshan; Zhu Jing; Ma Xi; Gao Fengshan; Zhou Dongpo

    2008-01-01

    Agrobacterium tumefaciens-mediated DNA transformation method was applied to transform Nodulisporium sylviforme fusant HDF-68, a taxol-producing fungus. We constructed a binary vector pBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of N.sylviforme with A.tumefaciens containing pBI121-43 led to 110~130 hygromycin-resistant transformants per million conidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N.sylviforme fusant HDF-68, and the development of such system lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.

  11. Spike-dip transformation of Setaria viridis.

    Science.gov (United States)

    Saha, Prasenjit; Blumwald, Eduardo

    2016-04-01

    Traditional method of Agrobacterium-mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium-mediated genetic transformation of S. viridis using spike dip. Pre-anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5-day-old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025% Silwet L-77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β-glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron-interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high-throughput transformation and potentially facilitates translational research in a monocot model plant. PMID:26932666

  12. Transgenic Crops by Direct Treatment of Exogenous DNA Without Agrobacterium tumefaciens Plasmid and Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    ZhangGuodong

    1995-01-01

    Gene transfter methods are developing quickly recently,but each method has its limitations.We introduce a new gene transfer technique in this paper,which is simple,effective,and easy to operate,but does not get enough attention from scientists.This technique is used to transform plants by injecting exogenous DNA to stigma,style,ovary,young fruit or meristem of the recipient,or soaking the recipient's seeds in exogenous DNA solution.Los of heritable variations were found in many characters of many crops,It may be used to creaste new germplasms or realize gene exchange between different species,gerera,or families,even between animals and plants,A brief discussion was given to the mechanism of exogenous DNA introduction,integration into and expression in the recipient.We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform plants genetically,but each method has its limitations that are delaying the application of the techniques to certaincommercially important crops.The first tecnhique exploits a natural genetic engineer,Agrobacterium tumefaciens,which contains a tumor-inducing(Ti) plasmid that transfers a DNA segment(the T-DNA) from the plasmid to the nuclear genome of infected plants(or in vitro to plant tissue).The method is restricted to dicotyledenous plants;monocotyledenous plants are usually not susceptible to agrobacterial infection.The second technique involves direct transfter of DNA to plant protoplast ,prepared by enzymatic digestion of cell walls,for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse,generating transient'holes'in the protoplast membrane.This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts,But so far it is impossible to achieve plant regeneration from protoplasts in many crops.Both techniques use dominant selectable markers(for example,kanamycin resistance) to

  13. Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Qi Jia

    2012-01-01

    Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

  14. 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...

  15. Complex Regulation of Arsenite Oxidation in Agrobacterium tumefaciens

    OpenAIRE

    Kashyap, Des R.; Botero, Lina M.; Franck, William L.; Daniel J Hassett; McDermott, Timothy R.

    2006-01-01

    Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor h...

  16. Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

    OpenAIRE

    Matthysse, A G

    1983-01-01

    During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were att...

  17. Taro (Colocasia esculenta (L.) Schott).

    Science.gov (United States)

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Zhu, Yun J

    2015-01-01

    Genetic engineering of taro is an effective method to improve taro quality and the resistance to various diseases of taro. Agrobacterium tumefaciens-mediated transformation of taro is more efficient than the particle bombardment transformation method based on current research. The development of a regeneration system starting from taro shoot tip explants could produce dasheen mosaic virus (DsMV)-free plantlets. Highly regenerative calluses could be developed from DsMV-free, in vitro plantlets on the Murashige and Skoog (MS) medium with 2 mg/L BA and 1 mg/L NAA (M5 medium). The Agrobacterium tumefaciens-mediated transformation method is reported in this chapter. The highly regenerative calluses were selected and cocultivated with the Agrobacterium strain EHA105 harboring the binary vector PBI121 with either a rice chitinase gene chi11 or a wheat oxalate oxidase gene gf2.8. After cocultivation for 3-4 days, these calluses were transferred to selection medium (M5 medium) containing 50 mg/L Geneticin G418 and grown for 3 months in the dark. Transgenic shoot lines could be induced and selected on the MS medium containing 4 mg/L BA (M15 medium) and 50 mg/L Geneticin G418 for 3 months further in the light. Molecular analyses are used to confirm the stable transformation and expression of the disease resistance gene chi11 or gf2.8. Pathologic bioassays could be used to demonstrate whether the transgenic plants had increased disease resistance to taro pathogens Sclerotium rolfsii or Phytophthora colocasiae.

  18. Taro (Colocasia esculenta (L.) Schott).

    Science.gov (United States)

    He, Xiaoling; Miyasaka, Susan C; Fitch, Maureen M M; Zhu, Yun J

    2015-01-01

    Genetic engineering of taro is an effective method to improve taro quality and the resistance to various diseases of taro. Agrobacterium tumefaciens-mediated transformation of taro is more efficient than the particle bombardment transformation method based on current research. The development of a regeneration system starting from taro shoot tip explants could produce dasheen mosaic virus (DsMV)-free plantlets. Highly regenerative calluses could be developed from DsMV-free, in vitro plantlets on the Murashige and Skoog (MS) medium with 2 mg/L BA and 1 mg/L NAA (M5 medium). The Agrobacterium tumefaciens-mediated transformation method is reported in this chapter. The highly regenerative calluses were selected and cocultivated with the Agrobacterium strain EHA105 harboring the binary vector PBI121 with either a rice chitinase gene chi11 or a wheat oxalate oxidase gene gf2.8. After cocultivation for 3-4 days, these calluses were transferred to selection medium (M5 medium) containing 50 mg/L Geneticin G418 and grown for 3 months in the dark. Transgenic shoot lines could be induced and selected on the MS medium containing 4 mg/L BA (M15 medium) and 50 mg/L Geneticin G418 for 3 months further in the light. Molecular analyses are used to confirm the stable transformation and expression of the disease resistance gene chi11 or gf2.8. Pathologic bioassays could be used to demonstrate whether the transgenic plants had increased disease resistance to taro pathogens Sclerotium rolfsii or Phytophthora colocasiae. PMID:25416252

  19. Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.).

    Science.gov (United States)

    Vermeulen, A; Vaucheret, H; Pautot, V; Chupeau, Y

    1992-06-01

    Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. PMID:24203132

  20. An efficient protocol for regeneration and transformation of Symphyotrichum novi-belgii

    DEFF Research Database (Denmark)

    Mørk, Eline Kirk; Henriksen, Karin; Brinch-Pedersen, Henrik;

    2012-01-01

    A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS...

  1. Effective elimination of chimeric tissue in transgenics for the stable genetic transformation of lesquerella fendleri

    Science.gov (United States)

    In order to improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 carrying binary vector pCAMBIA 1301.1...

  2. 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.

  3. The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA-binding site.

    Science.gov (United States)

    Maes, Michal; Amit, Einav; Danieli, Tsafi; Lebendiker, Mario; Loyter, Abraham; Friedler, Assaf

    2014-11-01

    Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants. PMID:25212215

  4. A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Traore Sy

    2011-12-01

    Full Text Available Abstract Background Cloning genes into plasmid vectors is one of the key steps for studying gene function. Recently, Invitrogen™ developed a convenient Gateway® cloning system based on the site-specific DNA recombination properties of bacteriophage lambda and the cytotoxic protein ccdB, which is lethal to most E. coli strains. The ccdB protein, however, is not toxic to Agrobacterium tumefaciens, an important player often used for studying gene function in planta. This limits the direct application of the Gateway® cloning system in plant transformation-mediated research. Results In this study, we constructed a novel Gateway®-compatible destination vector, pEG101-SacB/R, by replacing the ccdB gene with a SacB-SacR gene cassette as the negative selectable marker. Conclusion Our results demonstrate that the new pEG101-SacB/R destination vector can be used for Gateway® cloning in Agrobacterium tumefaciens. pEG101-SacB/R will be a valuable tool for high-throughput functional analysis of genes in planta.

  5. Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.

    Science.gov (United States)

    Bharat, Tanmay A M; Zbaida, David; Eisenstein, Miriam; Frankenstein, Ziv; Mehlman, Tevie; Weiner, Lev; Sorzano, Carlos Oscar S; Barak, Yoav; Albeck, Shira; Briggs, John A G; Wolf, Sharon G; Elbaum, Michael

    2013-07-01

    Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity. PMID:23769668

  6. Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato.

    Directory of Open Access Journals (Sweden)

    Pudota B Bhaskar

    Full Text Available Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.

  7. A Simplified Seed Transformation Method for Obtaining Transgenic Brassica napus Plants

    Institute of Scientific and Technical Information of China (English)

    SONG Li; ZHAO De-gang; WU Yong-jun; TIAN Xiao-e

    2009-01-01

    We report here a seed transformation of sonication-assisted,no-tissue culture to rapidly produce transgenic Brassica napus plants.This method comprises the steps of treating seeds by ultrasonic wave,inoculating Agrobacterium tumefaciens with a recombinant ChlFN-a gene and germinating directly of treatment seed on wet filter papers.The obtained transformants were verified by GUS histochemical assay and nested PCR amplification.It suggests that seed transformation has a potential use in genetic transformation of rape.

  8. Linear Chromosome-generating System of Agrobacterium tumefaciens C58

    Science.gov (United States)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood

    2012-01-01

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide. PMID:22582388

  9. How to get exogenous DNA to cross the cell membrane of plants. Comment on “Physical methods for genetic transformation in plants” by Rivera et al.

    Science.gov (United States)

    Cruz Hernández, Andrés; Campos Guillén, Juan

    2012-09-01

    Physical methods for genetic transformation in plants. The most commonly applied methods in plant transformation include Agrobacterium infection and protoplast or microprojectile bombardment. A plant transformation system is a prerequisite for the development of a plant improvement program. The global area utilized for biotech crops increases every year.

  10. PLANT TRANSFORMATION: ADVANCES AND PERSPECTIVES

    Directory of Open Access Journals (Sweden)

    Alves Adriana Cristina

    1999-01-01

    Full Text Available Genetic transformation is a powerful tool for plant breeding and genetical, physiological or biochemical research, consequently it is an extremely dynamic field. Transgenic plants are commonly used to complete or substitute mutants in basic research, helping the studies of complex biological situations such as pathogenesis process, genome organization, light reception and signal transduction. In this review, recent approaches for foreign gene introduction (e.g. Agrobiolistics, whole tissue electroporation, in planta Agrobacterium transformation, screening (reporter gene possibilities and performance and transformant selection (ipt selective marker are discussed. Transgene expression and mechanisms underlying (transgene inactivation are presented. Practical applications of genetically modified plants, field tests and commercial transgenic crops worldwide and in Brazil are listed, as well as the main traits and species modified. Potential uses of transgenic plants for animal compound production, biological remediation and synthetic polymer assembly are also shown.

  11. Mutants of Agrobacterium tumefaciens with elevated vir gene expression

    International Nuclear Information System (INIS)

    Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG

  12. Improved Agrobacterium-mediated transformation protocol for VgDGAT1a gene based on shoot tip culture of cotton%农杆菌介导的VgDGAT1a基因棉花茎尖转化体系优化研究

    Institute of Scientific and Technical Information of China (English)

    王安可; 何秋伶; 潘晶晶; 孙英超; 祝水金; 陈进红

    2013-01-01

    In past two decades , abundant researches have been published on transformation , regeneration , and genetic enhancement of cotton , especially Gossypium hirsutum . Generally , genes conferring agronomic advantages have been introduced into plants via A grobacterium‐mediation or particle bombardment , and then the transgenic plants are regenerated through somatic embryogenesis from callus . However , the embryogenic callus‐based regeneration is difficult and time‐consuming in cotton . Therefore , it is necessary to establish an effective system for the A grobacterium‐mediated genetic transformation of shoot tip in cotton . Triacylglycerols ( TAG) are a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bones . Diacylglycerol acyltransferase ( DGAT ; EC3 .2 .1 .20) catalyzes the last step of TAG biosynthesis and it is the only key enzyme evolved in . DGA T1 and DGA T2 , as two types of DGA T in eukaryotes , belong to different gene families . And previous studies have reported that the expression of DGA T1 increased seed oil content and mass . In order to get new cotton germplasm with high oil content , we used the shoot tips of Zhongmiansuo 49 as explants and introduced an improved vector carrying a selection marker H ptⅡ gene and a target V gDGA T1a gene into cotton via A grobacterium‐mediated transformation . This improved A grobacterium‐mediated transformation and regeneration system were established by optimizing different parameters such as pre‐culture period of seeds , concentration of A grobacterium in solution , immersing time , co‐cultivation period and components of MSB [ Murashige and Skoog ( MS) medium + vitamins of Gamborg s (B5) medium] . Cotton seeds ( Gossypium hirsutum L . cv . Zhongmiansuo 49 ) were decorticated manually and surface sterilized in 0 .1% HgCl for 8 min , followed by rinsing with sterile distilled water for five times . The cotyledons were removed from 3‐day‐old to

  13. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  14. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium -mediated system

    Institute of Scientific and Technical Information of China (English)

    翟文学; 李晓兵; 田文忠; 周永力; 潘学彪; 曹守云; 赵显峰; 赵彬; 章琦; 朱立煌

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  15. Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

    Institute of Scientific and Technical Information of China (English)

    HUANG Jia-quan; SUN Zhong-hai

    2005-01-01

    The Arabidopsis ICE1 (inducer of CBF expression 1) gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon (Citrus Limon (L.) Burm. F. cv. Eureka) genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICE1 gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

  16. Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

    Science.gov (United States)

    Hodges, Larry D; Cuperus, Josh; Ream, Walt

    2004-05-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2. PMID:15126468

  17. Study on Introduction of Avian Influenza Antigen Gene NA into Lettuce%禽流感抗原基因NA导入生菜的研究

    Institute of Scientific and Technical Information of China (English)

    范亚丽; 李进; 阮颖; 刘春林

    2012-01-01

    With lettuce cotyledons as transformation receptor, using Agrobacterium tumefaciens mediated genetic transformation method to introduce avian influenza antigen gene NA into lettuce. Six transgenic plants were obtained and were proved by PCR detection. The results of PT-PCR proved that the target genes have been integrated into lettuce genome and can normally express.%以生菜子叶为转化受体材料,利用农杆菌介导的方法将禽流感抗原基因NA导人生菜,通过用特异引物进行PCR检测,获得6株转基因植株.经PT-PCR检测,证明外源基因已整合到了生菜基因组中,且能够正常表达.

  18. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  19. Ammonium removal by Agrobacterium sp. LAD9 capable of heterotrophic nitrification-aerobic denitrification.

    Science.gov (United States)

    Chen, Qian; Ni, Jinren

    2012-05-01

    Characteristics of ammonium removal by a newly isolated heterotrophic nitrification-aerobic denitrification bacterium Agrobacterium sp. LAD9 were systematically investigated. Succinate and acetate were found to be the most favorable carbon sources for LAD9. Response surface methodology (RSM) analysis demonstrated that maximum removal of ammonium occurred under the conditions with an initial pH of 8.46, C/N ratio of 8.28, temperature of 27.9°C and shaking speed of 150rpm, where temperature and shaking speed produced the largest effect. Further nitrogen balance analysis revealed that 50.1% of nitrogen was removed as gas products and 40.8% was converted to the biomass. Moreover, the occurrence of aerobic denitrification was evidenced by the utilization of nitrite and nitrate as nitrogen sources, and the successful amplifications of membrane bound nitrate reductase and cytochrome cd(1) nitrite reductase genes from strain LAD9. Thus, the nitrogen removal in strain LAD9 was speculated to comply with the mechanism of heterotrophic nitrification coupled with aerobic denitrification (NH(4)(+)-NH(2)OH-NO(2)(-)-N(2)O-N(2)), in which also accompanied with the mutual transformation of nitrite and nitrate. The findings can help in applying appropriate controls over operational parameters in systems involving the use of this kind of strain.

  20. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn).

    Science.gov (United States)

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum 'Hokkai T10' cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3(,) H1, FtF3'H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat. PMID:27014239

  1. Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn

    Directory of Open Access Journals (Sweden)

    Aye Aye eThwe

    2016-03-01

    Full Text Available The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum ‘Hokkai T10’ cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as FtPAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3, H1, FtF3,H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 µg/mg DW, respectively, cyanidin 3-O-glucoside (800, 750, and 650 µg /g DW, respectively, and cyanidin 3-O-rutinoside (2410, 1530, and 1170 µg /g DW, respectively. A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat.

  2. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn).

    Science.gov (United States)

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum 'Hokkai T10' cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3(,) H1, FtF3'H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat.

  3. Improved genetic transformation of cork oak (Quercus suber L.).

    Science.gov (United States)

    Alvarez-Fernández, Rubén; Ordás, Ricardo-Javier

    2012-01-01

    An Agrobacterium-mediated transformation system for selected mature Quercus suber L. trees has been established. Leaf-derived somatic embryos in an early stage of development are inoculated with an AGL1 strain harboring a kanamycin-selectable plasmid carrying the gene of interest. The transformed embryos are induced to germinate and the plantlets transferred to soil. This protocol, from adult cork oak to transformed plantlet, can be completed in about one and a half years. Transformation efficiencies (i.e., percentage of inoculated explants that yield independent transgenic embryogenic lines) vary depending on the cork oak genotype, reaching up to 43%.

  4. Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

    Directory of Open Access Journals (Sweden)

    Das Pralay

    2011-05-01

    Full Text Available Abstract Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide, respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

  5. Current status of genetic transformation technology developed in cucumber (Cucumis sativus L.)

    Institute of Scientific and Technical Information of China (English)

    WANG Shun-li; Seong Sub Ku; YE Xing-guo; HE Cong-fen; Suk Yoon Kwon; Pil Son Choi

    2015-01-01

    Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cu-cumber, and its frequency has been up to 23%. For example, signiifcantly enhancement of the transformation efifciency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efifcient positive selection system in lots of experiments. This review is to summarize some key factors in-lfuencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efifciency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.

  6. Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

    DEFF Research Database (Denmark)

    Romans Fuertes, Patricia; Søndergaard, Teis Esben; Sandmann, Manuela Ilse Helga;

    2016-01-01

    Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens- mediated transformation (ATMT) approach to generate...... knock-out mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway...... biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium...

  7. Salt tolerance conferred by over-expression of OsNHX1 gene in Poplar 84K

    Institute of Scientific and Technical Information of China (English)

    WANG Shuyao; CHEN Qijun; WANG Wenlong; WANG Xuechen; LU Mengzhu

    2005-01-01

    OsNHX1 gene (Na+/H+ antiporter gene of Oryza sativa L.) was introduced into Poplar 84K with Agrobacterium tumefaciens-mediated transformation. PCR, Southern and Northern blot analysis showed that OsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 200 mmol/L NaCl, while the Na+ content in the leaves of the transgenic plants grown at 200 mmol/L NaCl was significantly higher than that in plants grown at 0 mmol/L NaCl. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.

  8. Overexpression of a Cytosolic Ascorbate Peroxidase Gene, OsAPX2, Increases Salt Tolerance in Transgenic Alfalfa

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qian; MA Cui; XUE Xin; XU Ming; LI Jing; WU Jin-xia

    2014-01-01

    Alfalfa (Medicago sativa L.) is an important forage crop in the world and it is of great signiifcance for the improvement of its salt tolerance. To improve salt tolerance in alfalfa, a rice ascorbate peroxidase gene (OsAPX2) was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation with marker gene bar. The different T-DNA insertions in T1 transgenic alfalfa were identiifed by Southern hybridization. Three independent T2 transgenic lines were selected for stress analysis and the results showed that all of them were salt tolerant compared with wild-type plants. The transgenic plants had low levels of H2O2, malondialdehyde and relative electrical conductivity under salt and drought stresses. Moreover, the contents of chlorophyll and proline, and APX activity were high in transgenic plants under salt and drought stresses. Taken together, the overexpression of OsAPX2 enhances salt tolerance in alfalfa through scavenging reactive oxygen species.

  9. Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani.

    Science.gov (United States)

    Jha, Sanjay; Tank, Harsukh G; Prasad, Bishun Deo; Chattoo, Bharat B

    2009-02-01

    Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice. PMID:18618285

  10. Transfer DREB into Lolium perenne L. To improve its drought tolerance

    Institute of Scientific and Technical Information of China (English)

    Ma Xinrong; Sun Zhenyuan; Jiang Changshun; Dong Zhaoyong; Zhang Yizheng

    2006-01-01

    A method of Agrobacterium tumefaciens mediated transformation for perennial ryegrass was developed using the calli of ryegrass derived from mature embryos. The calli were inoculated with a disarmed A. tumefaciens strain EHA105 harboring binary vector p2328. Vector p2328 contained transcription factor DREB1B and neomycin phosphotransferase (npt II) genes which were driven by promoters of rd29B and CaMV35S, respectively. The inoculated calli were selected on paromomycin- or kanamycin-containing media till the established plants being transferred to soil. Six transgenic plants with DREB1B had been obtained from perennial ryegrass strain Tove. PCR and Southern-blotting showed that npt II and DREB1B genes were integrated in perennial ryegrass genome. Stress treatment confirmed that transgenic plants with higher drought tolerance were obtained.

  11. Advances in Research on Genetically Engineered Plants for Metal Resistance

    Institute of Scientific and Technical Information of China (English)

    Ri-Qing Zhang; Chun-Fang Tang; Shi-Zhi Wen; Yun-Guo Liu; Ke-Lin Li

    2006-01-01

    The engineering application of natural hyperaccumulators in removing or inactivating metal pollutants from soil and surface water in field trials mostly presents the insurmountable shortcoming of low efficiency owing to their little biomass and slow growth. Based on further understanding of the molecular mechanism of metal uptake, translocation, and also the separation, identification, and cloning of some related functional genes, this article highlights and summarizes in detail the advances in research on transgenic techniques, such as Agrobacterium tumefaciens-mediated transformation and particle bombardment, in breeding of plants for metal resistance and accumulation, and points out that deepening the development of transgenic plants is one of the efficient approaches to improving phytoremediation efficiency of metal-contaminated environments. From the viewpoint of sustainable development, governments should strengthen support to the development of genetic engineering for metal resistance and accumulation in plants.

  12. Transformation of miniature potted rose (Rosa hybrida cv. Linda) with PSAG12-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene

    DEFF Research Database (Denmark)

    Zakizadeh, Hedayatollah; Lütken, Henrik Vlk; Sriskandarajah, Sridevy;

    2013-01-01

    Transgenic plants of Rosa hybrida ‘Linda’ were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the PSAG12-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P35S-INTGUS gene) were used for transformation of...

  13. The Transformations of Transformations.

    Science.gov (United States)

    Lin, Francis Y.

    2000-01-01

    Harris's original idea of transformations has been changed several times in Chomsky's work. This article explicates these transformations, arguing that though their motivations are highly understandable, these transformations are not necessary for understanding the workings of natural languages. (Author/VWL)

  14. Transformation of Morinda citrifolia via simple mature seed imbibition method.

    Science.gov (United States)

    Lee, J J; Ahmad, S; Roslan, H A

    2013-12-15

    Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.

  15. Functional diversity and mutational analysis of Agrobacterium 6B oncoproteins.

    Science.gov (United States)

    Helfer, A; Pien, S; Otten, L

    2002-07-01

    Many Agrobacterium T-DNA genes belong to a diverse family of T-DNA genes, the rolB family. These genes cause various growth abnormalities but their modes of action remain largely unknown. So far, none of the RolB-like proteins has been subjected to mutational analysis. The RolB-like oncoprotein 6B, which induces tumours on species such as Nicotiana glauca and Kalanchoe tubiflora, was chosen to investigate the role of the most conserved amino acid residues within the RolB family. We first determined which of the natural 6B variants had the strongest oncogenic activity; to this end, six 6b coding sequences (A- 6b, AB- 6b, C- 6b, CG- 6b, S- 6b and T- 6b) were placed under the control of the strong constitutive 2x35S promoter and compared for tumour induction on N. glauca, N. tabacum and K. daigremontiana. Oncogenicity increased in the order C- 6b/CG- 6b, A- 6b/AB- 6b, and S- 6b/T- 6b. The most conserved amino acid residues in the strongly oncogenic T-6B protein were mutated and shown to be required for oncogenicity and accumulation of the T-6B protein in planta but not in bacteria. Hybrids between T-6B and the weakly oncogenic A-6B protein revealed an additional oncogenic determinant required for the formation of large tumours. PMID:12172796

  16. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens.

    Science.gov (United States)

    Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M; Narberhaus, Franz

    2012-04-01

    Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium.

  17. Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum.

    Science.gov (United States)

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Jones, Jeffrey B; Obradović, Aleksa

    2015-09-01

    Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (<92%) between the whole genome sequences of strain KFB 330(T) and related Agrobacterium species. The major cellular fatty acids of the novel strains were 18:1 w7c (72.8-77.87%) and 16:0 (6.82-8.58%). Phenotypic features allowed their differentiation from closely related species. Polyphasic characterization showed that the three strains represent a novel species of the genus Agrobacterium, for which the name Agrobacterium arsenijevicii sp. nov. is proposed. The type strain of A. arsenijevicii is KFB 330(T) (= CFBP 8308(T) = LMG 28674(T)).

  18. Rapid deletion plasmid construction methods for protoplast and Agrobacterium based fungal transformation systems

    Science.gov (United States)

    Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Gene deletion is a critical tool for functional analysis. The targeted deletion of genes requires both a suitable method for the trans...

  19. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches

    NARCIS (Netherlands)

    Boer, P. de; Bronkhof, J.; Duki, K.; Kerkman, R.; Touw, H.; Berg , M. van den; Offringa, R.

    2013-01-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for dir

  20. Agrobacterium-mediated transformation of Mycosphaerella fijiensis, the devastating Black Sigatoka pathogen of bananas

    NARCIS (Netherlands)

    Díaz-Trujillo, C.; Adibon, H.; Kobayashi, K.; Zwiers, L.H.; Souza, M.T.; Kema, G.H.J.

    2010-01-01

    Mycosphaerella fijiensis, M. musicola en M. eumusae veroorzaken de Sigatoka-ziekte in banaan. Op dit moment is de toepassing van fungiciden de enige optie om deze ziekte te bestrijden. Het PRPB (Pesticide Reduction Program for Banana) investeert in de ontwikkeling van technieken voor de genotype- en

  1. Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein.

    Science.gov (United States)

    Schrammeijer, B; Risseeuw, E; Pansegrau, W; Regensburg-Tuïnk, T J; Crosby, W L; Hooykaas, P J

    2001-02-20

    The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process. PMID:11250154

  2. Model Transformations? Transformation Models!

    NARCIS (Netherlands)

    Bézivin, J.; Büttner, F.; Gogolla, M.; Jouault, F.; Kurtev, I.; Lindow, A.

    2006-01-01

    Much of the current work on model transformations seems essentially operational and executable in nature. Executable descriptions are necessary from the point of view of implementation. But from a conceptual point of view, transformations can also be viewed as descriptive models by stating only the

  3. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor.

    Science.gov (United States)

    Heidmann, Iris; de Lange, Brenda; Lambalk, Joep; Angenent, Gerco C; Boutilier, Kim

    2011-06-01

    Pepper (Capsicum L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using Agrobacterium tumefaciens (agrobacterium) is a powerful biotechnology tool that could be used in pepper to develop community-based functional genomics resources and to introduce important agronomic traits. However, pepper is considered to be highly recalcitrant for agrobacterium-mediated transformation, and current transformation protocols are either inefficient, cumbersome or highly genotype dependent. The main bottleneck in pepper transformation is the inability to generate cells that are competent for both regeneration and transformation. Here, we report that ectopic expression of the Brassica napus BABY BOOM AP2/ERF transcription factor overcomes this bottleneck and can be used to efficiently regenerate transgenic plants from otherwise recalcitrant sweet pepper (C. annuum) varieties. Transient activation of BABY BOOM in the progeny plants induced prolific cell regeneration and was used to produce a large number of somatic embryos that could be converted readily to seedlings. The data highlight the utility of combining biotechnology and classical plant tissue culture approaches to develop an efficient transformation and regeneration system for a highly recalcitrant vegetable crop. PMID:21305301

  4. Genetic transformation of Slovak cultivar of potato (Solanum tuberosum L.): efficiency and the behavior of the transgene

    NARCIS (Netherlands)

    Moravcikova, J.; Libantova, J.; Matusikova, I.; Libiakova, G.; Nap, J.P.H.; Mlynarova, L.

    2003-01-01

    Transgenic potato plants (Solanum tuberosum L.) of selected Slovak cultivars (cvs.) and one breeding line were obtained by Agrobacterium-mediated transformation. In vitro-grown plants of cvs. Albina, Eta, Malvina, Vila and line 116/86 were tested for their ability to regenerate transgenic plants. In

  5. GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens

    OpenAIRE

    Chevrot, Romain; Rosen, Ran; Haudecoeur, Elise; Cirou, Amélie; Shelp, Barry J.; Ron, Eliora; Faure, Denis

    2006-01-01

    The concentration of GABA increases rapidly in wounded plant tissues, but the implication of this GABA pulse for plant–bacteria interactions is not known. Here we reveal that GABA stimulated the inactivation of the N-(3-oxooctanoyl)homoserine lactone (OC8-HSL) quorum-sensing signal (or “quormone”) by the Agrobacterium lactonase AttM. GABA induced the expression of the attKLM operon, which was correlated to a decrease in OC8-HSL concentration in Agrobacterium tumefaciens cultures. The Agrobact...

  6. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export.

    Science.gov (United States)

    Lee, L Y; Gelvin, S B; Kado, C I

    1999-01-01

    When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF

  7. alpha-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes.

    Science.gov (United States)

    Burtin, D; Martin-Tanguy, J; Tepfer, D

    1991-02-01

    alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.

  8. α-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes

    Science.gov (United States)

    Burtin, D.; Martin-Tanguy, J.; Tepfer, D.

    1991-01-01

    α-dl-Difluoromethylarginine (DFMA) and α-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway. Images Figure 1 PMID:16668006

  9. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    Science.gov (United States)

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

  10. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    Science.gov (United States)

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. PMID:26593465

  11. 紫花苜蓿 MsWRKY33转录因子的分离及遗传转化研究%Isolation of MsWRKY33 transcription factor and its genetic transformation in Medi-cago sativa

    Institute of Scientific and Technical Information of China (English)

    冯光燕; 王学敏; 付媛媛; 方志红; 高洪文; 张新全

    2015-01-01

    MsWRKY33 gene may be involved in the regulation of environmental stress responses in alfalfa.The fragment encoding 10-239AA was inserted into pET-30α(+)to construct the expression vectors,and SDS-PAGE analysis revealed that the MsWRKY33 protein could be expressed in prokaryotic cells.The full length cDNA of MsWRKY33 was amplified from alfal-fa RNA and the plant expression vector pBI121-MsWRKY33 was constructed based on the pBI121 vector. Transgenic plants were obtained through somatic embryogenesis by Agrobacterium tumefaciens-mediated transformation.The npt Ⅱ gene and vector sequence were tested by PCR in the genome of transgenic plants and indicated that the target gene had been transferred.The qRT-PCR testing showed that the MsWRKY33 gene was over expressed in transgenic plants.This study will lay a foundation for further study of the function of the WRKY transcription factor in stress-tolerance regulation in alfalfa.

  12. Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues

    NARCIS (Netherlands)

    Tang, Lixia; van Hylckama Vlieg, Johan E.T.; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB

    2002-01-01

    Halohydrin dehalogenase from Agrobacterium radiobacter AD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of beta-mercaptoethanol or glycerol. Gel f

  13. Agrobacterium infection : translocation of virulence proteins and role of VirF in host cells

    NARCIS (Netherlands)

    Jurado Jácome, Esmeralda

    2011-01-01

    The VirB/D4 Type four secretion system (T4SS) is a bacterial multiprotein complex that spans the bacterial envelope, which mediates the translocation of T-DNA and effector virulence proteins into recipient cell. My research revealed that the Agrobacterium VirE3 and VirD2 proteins are effector protei

  14. Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens:an Assessment of Factors Influencing the Efficiency of Gene Transfer

    Institute of Scientific and Technical Information of China (English)

    Gao Liping; Bao Manzhu

    2004-01-01

    To develop a transformation protocol of Rosa hybrida 'Samantha' via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.5-0.8 for 20 min and co-culture in darkness under 23 °C on medium with 1/2 MS salts and 300 μmol·L-1 AS for 3 d.

  15. Formation of Se (0 Nanoparticles by Duganella sp. andAgrobacterium sp. isolated from Se-laden soil of North-East Punjab, India

    Directory of Open Access Journals (Sweden)

    Bajaj Mini

    2012-07-01

    Full Text Available Abstract Background Selenium (Se is an essential trace element, but is toxic at high concentrations. Depending upon the geological background, the land use or on anthropogenic pollution, different amounts of Se may be present in soil. Its toxicity is related to the oxyanions selenate and selenite as they are water soluble and bioavailable. Microorganisms play an important role in Se transformations in soil and its cycling in the environment by transforming water-soluble oxyanions into water insoluble, non-toxic elemental Se (0. For this study, soil samples were collected from selenium-contaminated agricultural soils of Punjab/India to enrich and isolate microbes that interacted with the Se cycle. Results A mixed microbial culture enriched from the arable soil of Punjab could reduce 230 mg/l of water soluble selenite to spherical Se (0 nanoparticles during aerobic growth as confirmed by SEM-EDX. Four pure cultures (C 1, C 4, C 6, C 7 of Gram negative, oxidase and catalase positive, aerobic bacteria were isolated from this mixed microbial consortium and identified by 16 S rDNA gene sequence alignment as two strains of Duganella sp. (C 1, C 4 and two strains of Agrobacterium sp.(C 6, C 7. SEM/TEM-EDX analyses of the culture broth of the four strains revealed excretion of uniformly round sharply contoured Se (0 nanoparticles by all cultures. Their size ranged from 140–200 nm in cultures of strains C 1 and C 4, and from 185–190 nm in cultures of strains C 6 and C 7. Both Duganella sp. revealed better selenite reduction efficiencies than the two Agrobacterium sp. Conclusions This is the first study reporting the capability of newly isolated, aerobically growing Duganella sp. and Agrobacterium sp. from soils of Punjab/India to form spherical, regularly formed Se (0 nanoparticles from water soluble selenite. Among others, the four strains may significantly contribute to the biogeochemical cycling of Se in soil. Bioconversion of toxic

  16. Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells.

    Science.gov (United States)

    Aguilar, Julieta; Zupan, John; Cameron, Todd A; Zambryski, Patricia C

    2010-02-23

    The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells. PMID:20133577

  17. Obtained transgenic wheat expressing pac1 mediated by Agrobacterium is resistant against Barley yellow dwarf virus-GPV

    Institute of Scientific and Technical Information of China (English)

    YAN Fei; ZHENG Yinying; ZHANG Wenwei; XIAO Hong; LI Shifang; CHENG Zhuomin

    2006-01-01

    In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleotide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expression product of cloned pac1 in E. coli showed activity to degrade the double-strand RNA. Harboring the binary vector pBI121, which contains pac1 gene, Agrobacterium tumefaciens strain LBA4404 was used to transform the wheat immature embryos precultured 7―10 d. After preregeneration, regeneration and selection culture stage, totally 41 G418 resistant plants were obtained, in which 25 lines were proved to integrate with transgene and express transgene normally by PCR, Dot blot, RT-PCR and ELISA detection. Antivirus test carried out on 25 positive lines with high dose of Barley yellow dwarf virus-GPV revealed that 12 lines had resistance to BVDV-GPV in low level, another 12 lines had resistance to BVDV- GPV in middle level, and 1 line showed resistance to BVDV-GPV in high level. However, both low and middle level of resistance plants showed no symptoms when infected by viruses at low dose, which suggested the dose-dependent effect of the resistance mediated by pac1 to BYDV-GPV.

  18. Opine-based Agrobacterium competitiveness: dual expression control of the agrocinopine catabolism (acc) operon by agrocinopines and phosphate levels.

    Science.gov (United States)

    Kim, H Stanley; Yi, Hyojeong; Myung, Jaehee; Piper, Kevin R; Farrand, Stephen K

    2008-05-01

    Agrobacterium tumefaciens strain C58 can transform plant cells to produce and secrete the sugar-phosphate conjugate opines agrocinopines A and B. The bacterium then moves in response to the opines and utilizes them as exclusive sources of carbon, energy, and phosphate via the functions encoded by the acc operon. These privileged opine-involved activities contribute to the formation of agrobacterial niches in the environment. We found that the expression of the acc operon is induced by agrocinopines and also by limitation of phosphate. The main promoter is present in front of the first gene, accR, which codes for a repressor. This operon structure enables efficient repression when opine levels are low. The promoter contains two putative operators, one overlapping the -10 sequence and the other in the further upstream from it; two partly overlapped putative pho boxes between the two operators; and two consecutive transcription start sites. DNA fragments containing either of the operators bound purified repressor AccR in the absence of agrocinopines but not in the presence of the opines, demonstrating the on-off switch of the promoter. Induction of the acc operon can occur under low-phosphate conditions in the absence of agrocinopines and further increases when the opines also are present. Such opine-phosphate dual regulatory system of the operon may ensure maximum utilization of agrocinopines when available and thereby increase the chances of agrobacterial survival in the highly competitive environment with limited general food sources. PMID:18344359

  19. Bacteremia due to Agrobacterium tumefaciens (radiobacter). Report of infection in a pregnant women and her stillborn fetus.

    Science.gov (United States)

    Southern, P M

    1996-01-01

    Agrobacterium tumefaciens (radiobacter) is usually a plant pathogen, but is isolated occasionally from human clinical specimens, frequently along with other bacteria. Agrobacterium tumefaciens (radiobacter) has been isolated from blood, central intravenous catheters, peritoneal fluid, urine, and cellulitis aspirates, often in immunocompromised individuals. This report details the isolation of A. tumefaciens (radiobacter) from the blood of a pregnant woman, as well as from the blood of her stillborn, premature fetus. It is, to our knowledge, the first report of such an occurrence. PMID:8988763

  20. A genome-wide survey of highly expressed non-coding RNAs and biological validation of selected candidates in Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Keunsub Lee

    Full Text Available Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs, 354 antisense RNAs (asRNAs, 20 5' untranslated region (UTR leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS mapping analysis revealed that about 51% of the mapped mRNAs have 5' UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir gene inducer acetosyringone (AS, a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5' UTR elements: a RNA thermonsensor (C1_109596F that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.

  1. Efficient transformation and artificial miRNA gene silencing in Lemna minor

    OpenAIRE

    Cantó-Pastor, Alex; Mollá-Morales, Almudena; Ernst, Evan; Dahl, William; Zhai, Jixian; Yan, Yiheng; Meyers, Blake; Shanklin, John; Martienssen, Robert

    2014-01-01

    Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally...

  2. Highly efficient transformation protocol for plum (Prunus domestica L.).

    Science.gov (United States)

    Petri, César; Scorza, Ralph; Srinivasan, Chinnathambi

    2012-01-01

    A high-throughput transformation system for plum has been developed using hypocotyl slices excised from zygotic embryos as the source of explants. The hypocotyl slices are infected in an Agrobacterium tumefaciens suspension and then cocultivated for 3 days in shoot regeneration ¾ MS basal medium supplemented with 9 μM 2,4-dichlorophenoxyacetic acid. Transgenic shoots are regenerated in a medium containing 7.5 μM thidiazuron and elongated in a medium containing 3 μM benzyladenine in the presence of 80 mg/L kanamycin in both media. Transformed shoots are rooted in ½ MS basal medium supplemented with 5 μM NAA and 40 mg/L kanamycin. The transgenic plants are acclimatized in a growth chamber and transferred to a temperature-controlled greenhouse. This protocol has allowed transformation efficiencies up to 42% and enabled the production of self-rooted transgenic plum plants within 6 months of transformation.

  3. Role of Agrobacterium virB genes in transfer of T complexes and RSF1010.

    Science.gov (United States)

    Fullner, K J

    1998-01-01

    Nonpolar virB mutants of Agrobacterium tumefaciens were tested for RSF1010 mobilization and extracellular complementation. virB2 to virB11 were essential for transfer in both assays. virB1 was essential only for high frequency transfer of RSF1010 and VirE2. Coordinated transfer of a preassembled T complex is supported by these data and competition studies. PMID:9440537

  4. Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

    Science.gov (United States)

    Dym, Orly; Albeck, Shira; Unger, Tamar; Jacobovitch, Jossef; Branzburg, Anna; Michael, Yigal; Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Elbaum, Michael

    2008-08-12

    Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners. PMID:18678909

  5. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    Science.gov (United States)

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene. PMID:25857193

  6. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    Science.gov (United States)

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene.

  7. In planta transformation method for T-DNA transfer in orchids

    Science.gov (United States)

    Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

    2014-03-01

    Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

  8. Regeneration of transgenic loblolly pine expressing genes for salt tolerance

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Salinity stress is one of the most serious factors limiting the distribution and productivity of crops and forest trees. The detrimental effects of salt on plants are a consequence of both a water deficit resulting in osmotic stress and the effects of excess sodium ions on critical biochemical process. A novel approach to improve salt tolerance has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBIGM which carrying the mannitol-1-phosphate dehydrogenase (Mt1D) and glucitol-6-phosphate dehydrogenase (GutD). Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15mg/L kanamycin and confirmed by Southern blot analysis of genomic DNA. Salt tolerance assays demonstrated that the salt tolerance of transgenic calli and regenerated plantlets were increased. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

  9. Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

    Directory of Open Access Journals (Sweden)

    Elena García-Cano

    Full Text Available Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.

  10. Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    Martha Liliana Bonilla Betancourt

    2008-10-01

    Full Text Available En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105 con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404 con pCambia 2301. Se usó el medio de infiltración (IM con acetosiringona y se evaluó el tiempo de cocultivo y la densidad óptica de la bacteria al momento de la inducción. Los genotipos mostraron respuesta diferencial con las combinaciones cepa-plásmido: obtuvieron mayor expresión del gen GUS cuando el genotipo CC85-92 se transformó con la cepa AGL-1-pCambia 1305.2. CC84-75 y CC87-505 mostraron mayor expresión cuando se transformaron con la cepa EHA105-pCambia 1305.2. Mayor eficiencia en la expresión se obtuvo cuando la bacteria se indujo en IM después de siete días de cocultivo y cuando la densidad óptica de la bacteria fue de 0.2(600nm al momento de la inducción. Se demostró superioridad de los explantes en la eficiencia de transformación.

  11. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  12. Construction of a gene bank and use of the chromosome walking technique for the detection of new putative agrocin genes in Agrobacterium tumefaciens strain D286

    International Nuclear Information System (INIS)

    A gene bank of Agrobacterium tumefaciens D286 wt has been constructed by cloning D286 wt DNA partially digested with EcoRI in the cosmid vector pLAFRI. The library; composed of 1750 members with a 27.7 kb average insert size was probed with pCDTn5-3, a cosmid vector carrying a D286:: Tn5 insert from the strain D286:: Tn5 Ag-. One recombinant cosmid of the library, pCDO932, was detected. The insert DNA of pCDO932 has sequences homologous to the D286:: Tn5 insert of pCDTn5-3, therefore it carries putative wt agrocin D286 genes. The insert DNA of pCDO932 was isolated and used to probe the D286 wt gene library. Chromosome walking resulted in the detection of pCD2375. EcoRI restriction digestions and DNA homology studies of pCDO932 and pCD2375 showed that their D286 wt inserts are both composed of 4 EcoRI DNA sub-fragments totalling 21.8 and 24.8 kb respectively, with an overlapping sequence extending 3.5 kb. In order to overcome the failure to detect A. tumefaciens cells transformed with pCDO932. Vectors pSUP204-1 was constructed. Such vector has been derived from pSUP204 which were slightly altered by cloning into it a 700 bp λ DNA SalI fragmet. This resulted in insertion inactivation of the Tcr gene, allows the use of pSUP204-1 as a subcloning vector in conjugations and transformations involving pCDO932 or pCD2375 and strains D286:: Tn5 Ag- and C58 C1G. Two recombinant cosmids bearing D286 wt DNA inserts, at least one of which (pCDO932) contains DNA sequences putatively affecting agrocin D286 production, are now available for further genetic manipulations. pSUP204-1 should prove useful as a subcloning vector for transformations and conjugations involving recombinant cosmids from the D286 wt gene bank and Agrobacterium strains. Future work on the molecular biology of agrocin D286 production is discussed. The DNA probe used in this study was labelled with phosphorus 32

  13. Visualizing Transformation

    DEFF Research Database (Denmark)

    Pedersen, Pia

    2012-01-01

    is not preformed, but formed through the process of transformation; this means that the purpose of transformation is not the styling of charts with pictograms but rather creating a meaningful message. The contribution of this paper is an elaborated understanding of the process of transformation and a demonstration...

  14. Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens Transient gene expression in sugarcane using Agrobacterium tumefaciens

    OpenAIRE

    Martha Liliana Bonilla Betancourt; Jaime Eduardo Muñoz Flórez; Fernando ángel Sánchez

    2008-01-01

    En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105) con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404) con pCambia 2301. Se usó el medio de infiltración (IM) con acetosiringona...

  15. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    Science.gov (United States)

    Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind. PMID:23745358

  16. A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

    Directory of Open Access Journals (Sweden)

    Gray John C

    2006-11-01

    Full Text Available Abstract Background The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants. Results A method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4–6 h light, 48 h dark and 24 h light (3.25 d. Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8–1.0 cm whereas non-resistant seedlings have short hypocotyls (0.2–0.4 cm. Conclusion The method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7–10 d required for selection using current protocols.

  17. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)

    2012-09-05

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  18. Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens.

    OpenAIRE

    Ooms, G.; Klapwijk, P M; Poulis, J A; Schilperoort, R A

    1980-01-01

    Seven Tn904 insertion mutants of pTi Ach5 affecting Agrobacterium tumefaciens virulence were studied. The mutant character was shown to be plasmid borne. Four of these mutants were avirulent and carried an insertion in restriction endonuclease HpaI fragment 12, a 3.3-megadalton fragment, which therefore appears to be a Ti plasmid region essential for virulence. Two mutants were attenuated in virulence. The inserts mapped close to HpaI fragment 12. One mutant giving rise to small tumors with e...

  19. Low-intensity microwave radiation and the virulence of Agrobacterium tumefaciens strain B6.

    Science.gov (United States)

    Moore, H A; Raymond, R; Fox, M; Galsky, A G

    1979-01-01

    When virulent cells of Agrobacterium tumefaciens strain B6 were exposed to low-level microwave radiation at a frequency of 10,000 MHz and an intensity of 0.58 mW/cm2 for 30 to 120 min, a 30 to 60% decrease in their ability to produce tumors on potato and turnip disks was observed. This microwave exposure did not affect the viability of these bacteria or their ability to attach to a tumor-binding site nor did it induce thermal shock. This loss of virulence was reversible within 12 h.

  20. Agrobacterium-mediated inoculation of chrysanthemum (Chrysanthemum morifolium) plants with chrysanthemum stunt viroid.

    Science.gov (United States)

    Nabeshima, Tomoyuki; Doi, Motoaki; Hosokawa, Munetaka

    2016-08-01

    Agroinfiltration was tested as a method of inoculation of chrysanthemum plants with chrysanthemum stunt viroid (CSVd). Binary vectors harboring dimeric CSVd sequences in sense and antisense orientations were constructed, and Agrobacterium transfected with these binary vectors was infiltrated into chrysanthemum leaves. Northern blotting and reverse transcription polymerase chain reaction analysis showed that local infection was established within 7 days and systemic infection within 20 days. CSVd polarities showed no difference in infectivity. This study showed that agroinfiltration of chrysanthemum plants is an easy, rapid, and cost-effective method for CSVd inoculation. PMID:27155239