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Sample records for agrobacterium tumefaciens c58

  1. Computational prediction of over-annotated protein-coding genes in the genome of Agrobacterium tumefaciens strain C58

    Science.gov (United States)

    Yu, Jia-Feng; Sui, Tian-Xiang; Wang, Hong-Mei; Wang, Chun-Ling; Jing, Li; Wang, Ji-Hua

    2015-12-01

    Agrobacterium tumefaciens strain C58 is a type of pathogen that can cause tumors in some dicotyledonous plants. Ever since the genome of A. tumefaciens strain C58 was sequenced, the quality of annotation of its protein-coding genes has been queried continually, because the annotation varies greatly among different databases. In this paper, the questionable hypothetical genes were re-predicted by integrating the TN curve and Z curve methods. As a result, 30 genes originally annotated as “hypothetical” were discriminated as being non-coding sequences. By testing the re-prediction program 10 times on data sets composed of the function-known genes, the mean accuracy of 99.99% and mean Matthews correlation coefficient value of 0.9999 were obtained. Further sequence analysis and COG analysis showed that the re-annotation results were very reliable. This work can provide an efficient tool and data resources for future studies of A. tumefaciens strain C58. Project supported by the National Natural Science Foundation of China (Grant Nos. 61302186 and 61271378) and the Funding from the State Key Laboratory of Bioelectronics of Southeast University.

  2. The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58.

    Science.gov (United States)

    Hynes, M F; Simon, R; Pühler, A

    1985-03-01

    Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana. PMID:4001194

  3. Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.

    OpenAIRE

    Ashby, A M; Watson, M D; Loake, G J; Shaw, C H

    1988-01-01

    Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak...

  4. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    International Nuclear Information System (INIS)

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P21 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°

  5. Cellulose Synthesis in Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Alan R. White; Ann G. Matthysse

    2004-07-31

    We have cloned the celC gene and its homologue from E. coli, yhjM, in an expression vector and expressed the both genes in E. coli; we have determined that the YhjM protein is able to complement in vitro cellulose synthesis by extracts of A. tumefaciens celC mutants, we have purified the YhjM protein product and are currently examining its enzymatic activity; we have examined whole cell extracts of CelC and various other cellulose mutants and wild type bacteria for the presence of cellulose oligomers and cellulose; we have examined the ability of extracts of wild type and cellulose mutants including CelC to incorporate UDP-14C-glucose into cellulose and into water-soluble, ethanol-insoluble oligosaccharides; we have made mutants which synthesize greater amounts of cellulose than the wild type; and we have examined the role of cellulose in the formation of biofilms by A. tumefaciens. In addition we have examined the ability of a putative cellulose synthase gene from the tunicate Ciona savignyi to complement an A. tumefaciens celA mutant. The greatest difference between our knowledge of bacterial cellulose synthesis when we started this project and current knowledge is that in 1999 when we wrote the original grant very few bacteria were known to synthesize cellulose and genes involved in this synthesis were sequenced only from Acetobacter species, A. tumefaciens and Rhizobium leguminosarum. Currently many bacteria are known to synthesize cellulose and genes that may be involved have been sequenced from more than 10 species of bacteria. This additional information has raised the possibility of attempting to use genes from one bacterium to complement mutants in another bacterium. This will enable us to examine the question of which genes are responsible for the three dimensional structure of cellulose (since this differs among bacterial species) and also to examine the interactions between the various proteins required for cellulose synthesis. We have carried out one

  6. Combined Genetic and Physical Map of the Complex Genome of Agrobacterium tumefaciens

    OpenAIRE

    Goodner, Brad W.; Markelz, Brian P.; Flanagan, M. Casey; Crowell, Chris B.; Racette, Jodi L.; Schilling, Brittany A.; Halfon, Leah M.; Mellors, J. Scott; Grabowski, Gregory

    1999-01-01

    A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which...

  7. 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.

  8. Corn metabolites affect growth and virulence of Agrobacterium tumefaciens.

    OpenAIRE

    Sahi, S. V.; Chilton, M D; Chilton, W S

    1990-01-01

    Homogenates of corn seedlings inhibit both growth of Agrobacterium tumefaciens and induction of its Ti plasmid virulence (vir) genes by acetosyringone (AS). The heat-labile inhibitor has been identified as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), present in 2-week-old seedlings (B73) at a concentration of 1.5 mM or greater. A concentration of 0.3 mM DIMBOA is sufficient to block growth of A. tumefaciens completely for 220 hr. DIMBOA at 0.1 mM concentration completely inhi...

  9. Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens.

    OpenAIRE

    Y. W. Lee; Jin, S.; Sim, W S; Nester, E. W.

    1995-01-01

    The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures o...

  10. 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...

  11. 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...

  12. Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens.

    Science.gov (United States)

    Kemppainen, Minna J; Pardo, Alejandro G

    2011-01-01

    Most boreal and temperate forest trees form a mutualistic symbiosis with soil borne fungi called ectomycorrhiza (ECM). In this association both partners benefit due to nutrient exchange at the symbiotic interface. Laccaria bicolor is the first mycorrhizal fungus with its genome sequenced thus making possible for the first time to analyze genome scale gene expression profiles of a mutualistic fungus. However, in order to be able to take full advantage of the genome sequence, reverse genetic tools are needed. Among them a high throughput transformation system is crucial. Herein we present a detailed protocol for genetic transformation of L. bicolor by means of Agrobacterium tumefaciens with emphasis on critical steps affecting the success and efficiency of the approach. PMID:21636986

  13. 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

  14. 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

  15. Funktion des Lipidtransferproteins 2 (LTP2) und dessen Rolle bei der Bildung von durch Agrobacterium tumefaciens induzierten Wurzelhalsgallen an Arabidopsis thaliana

    OpenAIRE

    Saupe, Stefanie

    2014-01-01

    In Tumoren an Arabidopsis thaliana, induziert über Agrobacterium tumefaciens (Stamm C58), ist von den 49 bekannten Lipidtransferproteinen (LTPs) nur die Expression von LTP2 stark erhöht (Deeken et al., 2006). Mutanten ohne LTP2-Transkripte (ltp2KO) entwickeln deutlich kleinere Tumore als der Wildtyp. Durch die permanenten Zellstreckungs- und Dehnungsprozesse besitzen Tumore keine intakte Epidermis (Efetova et al., 2007). Dies wiederum führt zum Verlust einer vollständigen Cuticula-Schicht, we...

  16. 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

  17. A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

    Directory of Open Access Journals (Sweden)

    Katholiki Skopelitou

    Full Text Available In the present work, we report a novel class of glutathione transferases (GSTs originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701 with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H. This enzyme (designated as AtuGSTH1-1 was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl-glutathione (Nb-GSH. Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34, an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity.

  18. The BlcC (AttM) Lactonase of Agrobacterium tumefaciens Does Not Quench the Quorum-Sensing System That Regulates Ti Plasmid Conjugative Transfer ▿ †

    OpenAIRE

    Khan, Sharik R.; Farrand, Stephen K.

    2008-01-01

    The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the muta...

  19. 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

  20. ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

    Czech Academy of Sciences Publication Activity Database

    Bleho, J.; Obert, B.; Takáč, T.; Petrovská, Beáta; Heym, C.; Menzel, D.; Šamaj, J.

    2012-01-01

    Roč. 249, č. 1 (2012), s. 53-63. ISSN 0033-183X Grant ostatní: GA MŠk(CZ) ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Agrobacterium rhizogenes * Agrobacterium tumefaciens * Endoplasmic reticulum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.855, year: 2012

  1. 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.

  2. Agrobacterium rhizogenes GALLS Protein Substitutes for Agrobacterium tumefaciens Single-Stranded DNA-Binding Protein VirE2

    OpenAIRE

    Hodges, Larry D.; Cuperus, Josh; Ream, Walt

    2004-01-01

    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...

  3. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes

  4. Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots

    OpenAIRE

    Cao, Shi-liang; Masilamany, Pathmalojiny; Li, Wen-bin; Pauls, K. Peter

    2014-01-01

    An Agrobacterium tumefaciens-mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective ...

  5. Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

    OpenAIRE

    Knauf, V C; Panagopoulos, C G; Nester, E. W.

    1983-01-01

    Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homol...

  6. 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

  7. 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.

  8. 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

    Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi has become a common technique for the study of a wide variety of different fungal species over the past 12years. The discovery that the host range of A. tumefaciens could be extended to include fungi provided an efficient transform...... of T-DNA into fungal genomes....

  9. Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

    OpenAIRE

    Wagner, V T; Matthysse, A G

    1992-01-01

    Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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

  15. 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.

  16. [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

  17. Influence of volatile organic compounds emitted by Pseudomonas and Serratia strains on Agrobacterium tumefaciens biofilms.

    Science.gov (United States)

    Plyuta, Vladimir; Lipasova, Valentina; Popova, Alexandra; Koksharova, Olga; Kuznetsov, Alexander; Szegedi, Erno; Chernin, Leonid; Khmel, Inessa

    2016-07-01

    The ability to form biofilms plays an important role in bacteria-host interactions, including plant pathogenicity. In this work, we investigated the action of volatile organic compounds (VOCs) produced by rhizospheric strains of Pseudomonas chlororaphis 449, Pseudomonas fluorescens B-4117, Serratia plymuthica IC1270, as well as Serratia proteamaculans strain 94, isolated from spoiled meat, on biofilms formation by three strains of Agrobacterium tumefaciens which are causative agents of crown-gall disease in a wide range of plants. In dual culture assays, the pool of volatiles emitted by the tested Pseudomonas and Serratia strains suppressed the formation of biofilms of A. tumefaciens strains grown on polycarbonate membrane filters and killed Agrobacterium cells in mature biofilms. The individual VOCs produced by the tested Pseudomonas strains, that is, ketones (2-nonanone, 2-heptanone, 2-undecanone), and dimethyl disulfide (DMDS) produced by Serratia strains, were shown to kill A. tumefaciens cells in mature biofilms and suppress their formation. The data obtained in this study suggest an additional potential of some ketones and DMDS as protectors of plants against A. tumefaciens strains, whose virulence is associated with the formation of biofilms on the infected plants. PMID:27214244

  18. 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

  19. 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

  20. 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...

  1. 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.

  2. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

    OpenAIRE

    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-01-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D11...

  3. 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...

  4. Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts Agrobacterium tumefaciens from Phenotypically Female to Male▿

    OpenAIRE

    Cho, Hongbaek; Pinto, Uelinton M.; Winans, Stephen C.

    2009-01-01

    Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR an...

  5. Behaviour of Prunus cultivars and hybrids towards Agrobacterium tumefaciens estimated from hardwood cuttings

    OpenAIRE

    Pierronnet, A; Salesses, G.

    1996-01-01

    En utilisant la méthode de boutures ligneuses, le comportement de différents Prunus, pouvant servir de porte-greffe, est étudié vis-à-vis de la bactérie tellurique Agrobacterium tumefaciens, microorganisme responsable de la galle du collet. Les résultats montrent que : i) pour les Prunus cerasifera, tous les clones étudiés sont sensibles (parmi eux, le clone P2032 semble le moins sensible) ainsi que les différents hybrides issus de croisements comportant P cerasifera, excepté le P2038 qui est...

  6. Marine algae that display anti-tumorigenic activity against Agrobacterium tumefaciens.

    Science.gov (United States)

    el-Masry, M H; Mostafa, M H; Ibrahim, A M; el-Naggar, M M

    1995-05-01

    Thirty-five extracts representing different seasonal growths of 17 marine algal species collected from the Alexandria coast were tested for anti-tumorigenic activity against Agrobacterium tumefaciens galls on potato discs. Eleven extracts (nine species) displayed > 20% inhibition of tumor initiation, with three of these (Codium tomentosum, winter; Jania rubens, summer; Padina pavonia, winter) displaying relatively high activity. Bacterial viability tests showed that the inhibitory effects were directly due to anti-tumorigenesis rather than an indirect result of anti-bacterial activity. PMID:7750733

  7. A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

    OpenAIRE

    Zhang, L H; Kerr, A.

    1991-01-01

    Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction w...

  8. 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 ...

  9. 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

  10. 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

  11. The BlcC (AttM) lactonase of Agrobacterium tumefaciens does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer.

    Science.gov (United States)

    Khan, Sharik R; Farrand, Stephen K

    2009-02-01

    The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system. PMID:19011037

  12. "Cre/loxP plus BAC": a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens.

    Science.gov (United States)

    Hu, Shengbiao; Liu, Zhengqiang; Zhang, Xu; Zhang, Guoyong; Xie, Yali; Ding, Xuezhi; Mo, Xiangtao; Stewart, A Francis; Fu, Jun; Zhang, Youming; Xia, Liqiu

    2016-01-01

    Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for direct cloning gene clusters. A type three secretion system (T3SS) gene cluster (~32 kb) from Photorhabdus luminescens TT01 and DNA fragment (~78 kb) containing the siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58 have been successfully cloned into pBeloBAC11 with "Cre/loxP plus BAC" strategy. Based on the fact that Cre/loxP system has successfully used for genomic engineering in a wide range of organisms, we believe that this strategy could be widely used for direct cloning of large DNA fragment. PMID:27364376

  13. 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.

  14. EFFECT OF TEMPERATURE AND DETERGENTS ON AGROBACTERIUM TUMEFACIENS, THE CAUSAL PATHOGEN OF CROWN GALL DISEASE OF WALNUT

    Science.gov (United States)

    Crown gall disease caused by the bacterium Agrobacterium tumefaciens causes significant economic losses in commercial walnut orchards and nursery operations in California. In an effort to develop integrated control strategies to ensure pathogen and disease free plant material at nurseries, the effe...

  15. Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts Agrobacterium tumefaciens from Phenotypically Female to Male▿

    Science.gov (United States)

    Cho, Hongbaek; Pinto, Uelinton M.; Winans, Stephen C.

    2009-01-01

    Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors. PMID:19304847

  16. Transsexuality in the rhizosphere: quorum sensing reversibly converts Agrobacterium tumefaciens from phenotypically female to male.

    Science.gov (United States)

    Cho, Hongbaek; Pinto, Uelinton M; Winans, Stephen C

    2009-05-01

    Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors. PMID:19304847

  17. 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.

  18. 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

  19. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å. Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP-354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents

  20. Viral and chloroplastic signals essential for initiation and efficiency of translation in Agrobacterium tumefaciens.

    Science.gov (United States)

    Ahmad, Tauqeer; Venkataraman, Srividhya; Hefferon, Kathleen; AbouHaidar, Mounir G

    2014-09-12

    The construction of high-level protein expression vectors using the CaMV 35S promoter in concert with highly efficient translation initiation signals for Agrobacterium tumefaciens is a relatively less explored field compared to that of Escherichia coli. In the current study, we experimentally investigated the capacity of the CaMV 35S promoter to direct GFP gene expression in A. tumefaciens in the context of different viral and chloroplastic translation initiation signals. GFP expression and concomitant translational efficiency was monitored by confocal microscopy and Western blot analysis. Among all of the constructs, the highest level of translation was observed for the construct containing the phage T7 translation initiation region followed by the chloroplastic Rubisco Large Subunit (rbcL) 58-nucleotide 5' leader region including its SD-like sequence (GGGAGGG). Replacing the SD-like (GGGAGGG) with non SD-like (TTTATTT) or replacing the remaining 52 nucleotides of rbcL with nonspecific sequence completely abolished translation. In addition, this 58 nucleotide region of rbcL serves as a translational enhancer in plants when located within the 5' UTR of mRNA corresponding to GFP. Other constructs, including those containing sequences upstream of the coat proteins of Alfalfa Mosaic Virus, or the GAGG sequence of T4 phage or the chloroplastic atpI and/or PsbA 5' UTR sequence, supported low levels of GFP expression or none at all. From these studies, we propose that we have created high expression vectors in A. tumefaciens and/or plants which contain the CaMV 35S promoter, followed by the translationally strong T7 SD plus RBS translation initiation region or the rbcL 58-nucleotide 5' leader region upstream of the gene of interest. PMID:25117444

  1. Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens.

    Science.gov (United States)

    Sebastianes, Fernanda L S; Lacava, Paulo T; Fávaro, Léia C L; Rodrigues, Maria B C; Araújo, Welington L; Azevedo, João L; Pizzirani-Kleiner, Aline A

    2012-02-01

    We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis. PMID:22210192

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

    Science.gov (United States)

    2012-01-01

    Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that

  3. 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

    Full Text Available Abstract Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus produces many terpenoid indole alkaloids (TIAs, such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS gene and a selectable marker neomycin phosphotransferase II gene (NTPII. The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC

  4. Cloning, sequencing, and transcriptional analysis of the gene coding for the vegetative sigma factor of Agrobacterium tumefaciens.

    OpenAIRE

    Segal, G.; Ron, E. Z.

    1993-01-01

    The sigA gene of Agrobacterium tumefaciens was cloned and sequenced. Comparison with previously analyzed sigA genes revealed a high degree of similarity in nucleotide and amino acid sequences of regions two, three, and four of vegetative sigma factors. However, the upstream regulatory region shows no sequence homology with the Escherichia coli heat shock (sigma 32) promoters. It also does not contain the hairpin-loop structure (inverted repeat sequence) that was found in the upstream region o...

  5. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

    OpenAIRE

    Thomashow, M F; Karlinsey, J E; Marks, J R; Hurlbert, R. E.

    1987-01-01

    We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in...

  6. A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

    OpenAIRE

    Traore Sy; Zhao Bingyu

    2011-01-01

    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 applic...

  7. Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

    OpenAIRE

    Hanif, Mubashir

    2004-01-01

    Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic f...

  8. Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population.

    Science.gov (United States)

    Lang, Julien; Faure, Denis

    2016-05-01

    Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations. PMID:27110651

  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. Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

    Science.gov (United States)

    Hoffmann, Marie-Christine; Ali, Koral; Sonnenschein, Marleen; Robrahn, Laura; Strauss, Daria; Narberhaus, Franz; Masepohl, Bernd

    2016-09-01

    Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators. PMID:27196733

  11. 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

  12. 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

  13. 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.

  14. [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

  15. Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

    2009-06-03

    N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

  16. 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

  17. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    Science.gov (United States)

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. PMID:26686612

  18. Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants.

    Science.gov (United States)

    Czarnecka-Verner, Eva; Salem, Tarek A; Gurley, William B

    2016-02-01

    The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression. PMID:26646288

  19. Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome.

    OpenAIRE

    Rong, L J; Karcher, S J; Gelvin, S B

    1991-01-01

    picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase gen...

  20. 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

  1. Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

    OpenAIRE

    Clarke, Jihong Liu; Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W.; Moe, Roar; Blystad, Dag-Ragnar

    2008-01-01

    Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium i...

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. The role of heterologous nifAc product in the regulation of nif expression in Agrobacterium tumefaciens.

    Science.gov (United States)

    Zhang, J; Zhang, J

    1997-01-01

    The plasmids pCK5, pCK3, pSZ36, and pSZ23-CA, which carried constitutive nifAc gene of Azotobacter chroococcum and Klebsiella pneumoniae were transferred into A. tumefaciens C58/pGV3850 with triparental mating. The growth rate of these transconjugants was similar to the wild type. Nitrogenase synthesis was demonstrated by Western blotting, in the presence of 10 mmol/L NH4+, and the nitrogenase activity was restored to 73%, 24%, 11%, and 62%, respectively. The results showed that the regulative gene of nitrogen fixation in A. chroococcum and K. pneumoniae played a regulative role for the expression of A. tumefaciens nitrogen fixation gene. Among them, the role of A. chroococcum nifAc gene was the strongest, the fusion plasmid pSZ23-CA which carried nifA-ntrC gene of K. pneumoniae was stronger, and the nifAc gene of K. pneumoniae was weak. PMID:9376504

  7. Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase

    OpenAIRE

    Cokesa, Z̆eljko; Knackmuss, Hans-Joachim; Rieger, Paul-Gerhard

    2004-01-01

    Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe2+-, Mg2+-, and Ca2+-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradatio...

  8. Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

    OpenAIRE

    Zupan, John R.; Cameron, Todd A.; Anderson-Furgeson, James; Zambryski, Patricia C.

    2013-01-01

    Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about...

  9. 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.

  10. 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.

  11. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

    Science.gov (United States)

    Thomashow, M F; Karlinsey, J E; Marks, J R; Hurlbert, R E

    1987-07-01

    We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent. PMID:3597321

  12. 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

  13. Theoretical Study of Molecular Determinants Involved in Signal Binding to the TraR Protein of Agrobacterium tumefaciens

    Directory of Open Access Journals (Sweden)

    N. Kumar

    2005-10-01

    Full Text Available N-acylated homoserine lactone (AHL mediated cell-cell communication in bacteria is dependent on the recognition of the cognate signal by its receptor. This interaction allows the receptor-ligand complex to act as a transcriptional activator, controlling the expression of a range of bacterial phenotypes, including virulence factor expression and biofilm formation. One approach to determine the key features of signal- binding is to model the intermolecular interactions between the receptor and ligand using computational-based modeling software (LigandFit. In this communication, we have modeled the crystal structure of the AHL receptor protein TraR and its AHL signal N-(3- oxooctanoyl-homoserine lactone from Agrobacterium tumefaciens and compared it to the previously reported antagonist behaviour of a number of AHL analogues, in an attempt to determine structural constraints for ligand binding. We conclude that (i a common conformation of the AHL in the hydrophobic and hydrophilic region exists for ligand-binding, (ii a tail chain length threshold of 8 carbons is most favourable for ligand-binding affinity, (iii the positive correlation in the docking studies could be used a virtual screening tool.

  14. The effect of selected fungicides on survival of Agrobacterium tumefaciens in various kinds of soils

    Directory of Open Access Journals (Sweden)

    Stanisław Barczyński

    2013-12-01

    Full Text Available The effect of 10 fungicides on survival of A. tumefaciens in various types of soils was studied. In fertile, nonsterile soil Dithane M-45 (mancozeb, Euparen 50 WP (tolyfluanid, Kaptan 50 WP (captan and Ridomil Gold 80 WP (metalaxyl at concentration of 1000 ppm showed the highest antibacterial activity. Similar trends in activity of these fungicides occurred in fertile, sterile soil, however a little lower in case of Kaptan and Euparen. In most of investigated soils Befran 25 SL (imimnoctadyne, Syllit 65 WP (dodine and Thiram Granulfo 80 WG (thiram increased bacteria number. In sandy acidic soil (pH 3,5 all tested fungicides totally eliminated bacteria. On the other hand in sandy neutral soil only Dithane, Euparen, Kaptan and Ridomil showed such activity. Ten fold decrease of fungicides concentration generally did not influence Kaptan and Ridomil effectiveness but it decreased the activity of Dithane and Euparen.

  15. 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

  16. Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

    Science.gov (United States)

    Vinoth, S; Gurusaravanan, P; Jayabalan, N

    2013-02-01

    A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques. PMID:23306888

  17. 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

  18. Rhicadhesin-mediated attachment and virulence of an Agrobacterium tumefaciens chvB mutant can be restored by growth in a highly osmotic medium.

    OpenAIRE

    Swart, S; Lugtenberg, B J; Smit, G.; Kijne, J W

    1994-01-01

    Cyclic beta-1,2-glucan is considered to play a role in osmoadaptation of members of the family Rhizobiaceae in hypotonic media. Agrobacterium tumefaciens chvB mutants, lacking beta-1,2-glucan, exhibit a pleiotropic phenotype, including nonmotility, attachment deficiency, and avirulence. Here we report that by growth of chvB mutant cells in tryptone-yeast extract medium supplemented with 7 mM CaCl2 and 100 mM NaCl, the mutant cells become motile, attach to pea root hair tips, and are virulent ...

  19. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    OpenAIRE

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DN...

  20. Conservation of PcaQ, a transcriptional activator of pca genes for catabolism of phenolic compounds, in Agrobacterium tumefaciens and Rhizobium species.

    OpenAIRE

    Parke, D

    1996-01-01

    In Agrobacterium tumefaciens A348, control of five genes for catabolism of the phenolic compound protocatechuate to beta-ketoadipate is exerted by the gene pcaQ. The product of pcaQ is a transcriptional activator which is distinct from regulators of the beta-ketoadipate pathway characterized in other bacterial groups. An investigation of whether pcaQ is present and conserved in related Rhizobium species employed Southern hybridization and an agrobacterial pcaD::LacZ promoter probe plasmid. Th...

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. 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

    fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the Delta...... infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile...... single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results: The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated...

  4. 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.

  5. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  6. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  7. 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.

  8. Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus.

    Science.gov (United States)

    Clarke, Jihong Liu; Spetz, Carl; Haugslien, Sissel; Xing, Shaochen; Dees, Merete W; Moe, Roar; Blystad, Dag-Ragnar

    2008-06-01

    Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated. PMID:18327592

  9. 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...

  10. Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.

    Science.gov (United States)

    Pantazis, Christopher J; Fisk, Sarah; Mills, Kerri; Flinn, Barry S; Shulaev, Vladimir; Veilleux, Richard E; Dan, Yinghui

    2013-03-01

    KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying. PMID:23160638

  11. Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp.

    OpenAIRE

    Fuchs, R L; Keister, D L

    1980-01-01

    Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium....

  12. 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

  13. An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

    Science.gov (United States)

    Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

    2016-06-01

    The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae. PMID:26780432

  14. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

    Science.gov (United States)

    Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale

    2014-10-01

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438

  17. Isolation of Dibenzothiophene-degrading Bacterium Agrobacterium tumefa UP3%石油生物催化脱硫菌Agrobacterium tumefaciens UP3的分离筛选

    Institute of Scientific and Technical Information of China (English)

    史德青; 赵金生; 侯影飞; 杨金荣; 孔瑛

    2004-01-01

    从胜利油田被原油污染的土壤中筛选到一株能有效降解模型化合物二苯并噻吩(DBT)的菌株.根据常规的形态分析、生理生化性状及16S rDNA序列分析,将其鉴定为根癌土壤杆菌(Agrobacterium tumefaciens UP3).该菌不能以十二烷、十六烷、液体石蜡和萘作为唯一碳源和能源生长,具有工业应用的潜力.对该菌株DBT降解能力的初步研究表明,54h内可将500mg/L的DBT降解至150mg/L.对降解产物的分析表明,根癌土壤杆菌降解DBT的途径与Kodama路线及4-S路线不同.

  18. [Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

    Science.gov (United States)

    Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

    2006-05-01

    The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement. PMID:16755923

  19. 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.

  20. One out of four: HspL but no other small heat shock protein of Agrobacterium tumefaciens acts as efficient virulence-promoting VirB8 chaperone.

    Directory of Open Access Journals (Sweden)

    Yun-Long Tsai

    Full Text Available Alpha-crystallin-type small heat shock proteins (sHsps are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named hspL, hspC, hspAT1, and hspAT2 were identified in Agrobacterium tumefaciens, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS. HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of hspL but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the hspL deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of hspL and only modest increase of hspC, hspAT1, and hspAT2 at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays in vitro indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated in vivo, in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in A. tumefaciens. Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence

  1. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    OpenAIRE

    J. E. Ward; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-01-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribut...

  2. 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

  3. Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid, pTiC58.

    Science.gov (United States)

    Otten, L; Salomone, J Y; Helfer, A; Schmidt, J; Hammann, P; De Ruffray, P

    1999-12-01

    The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the 'common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c', d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3'. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3' (located on the TR-DNA of octopine strains) is also oncogenic. Although the b-e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c' in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested. PMID:10737141

  4. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    Science.gov (United States)

    Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-09-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. PMID:2203743

  5. 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...

  6. Makarnalık buğdayda (Triticum durum Desf.) Agrobacterium tumefaciens aracılığıyla gen aktarım frekansının artırılmasında gama radyasyondan yararlanma olanakları

    OpenAIRE

    2014-01-01

    Moleküler teknikler kullanılarak herhangi bir organizmadan izole edilen bir gen biyoteknolojik yöntemler aracılığıyla istenen bir organizmaya aktarılabilmektedir. Biyoteknolojik çalışmalarda bitkilere gen aktarımında Agrobacterium tumefaciens, mikroenjeksiyon ve partikül tabancası gibi çok sayıda gen aktarım yöntemi kullanılmasına karşın, Agrobacterium tumefaciens ile gen aktarımının çok önemli üstünlükleri bulunmaktadır. Agrobacter...

  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. Optimisation de la transformation génétique de la pomme de terre par Agrobacterium tumefaciens. Utilisation de la résistance à l'hygromycine comme marqueur sélectif

    Directory of Open Access Journals (Sweden)

    Rouvière C.

    2003-01-01

    Full Text Available Optimization of potato genetic transformation by Agrobacterium tumefaciens using hygromycin resistance as selective marker. The objective of this work is the optimization of a genetic transformation and a regeneration protocol of transgenic plants in Solanum tuberosum cv Désirée using hygromycin resistance as selective marker. The gene Cat2 of Nicotiana plumbaginifolia and the gene SU2 of Gossypium hirsutum, both coding for catalases, have been used. Two antibiotic concentrations (5 and 10 mg . l -1 of culture medium combined with two preculture periods (5 and 20 days on non-selective medium were tested. Coculture medium was supplemented with 10 mg . l -1 of acetosyringone to test its effect on potato transformation. The addition of this phenolic compound to the coculture medium affected positively the regeneration aptitude of transgenic SU2 plants. Only the combination of 5 mg . l -1 of hygromycin and 20 days of preculture without antibiotic allowed the obtention of plants transformed with the gene SU2. Screening of the rooted shoots with PCR showed 45% of transgenic plants for both molecular constructions used.

  9. M-31 mutant (virA::Tn5) of Agrobacterium tumefaciens is capable of transferring its T-DNA into the nucleus of host cell, but incapable of integrating it into the chromosome.

    Science.gov (United States)

    Majumder, P; Yoshida, H; Shioiri, H; Nozue, M; Kojima, M

    2000-01-01

    An avirulent mutant (M-31 strain) was produced by the transposon (Tn5) mutagenesis of Agrobacterium tumefaciens (A-208 strain). A binary vector, pIG121-Hm, containing a kanamycin resistance gene (nptII) and beta-glucuronidase (GUS) gene with an intron, was introduced into M-31 and A-208 strains. The resultant Agrobacteria were inoculated onto leaves of Kalanchoe daigremontiana and to tobacco BY-2 cells to assay GUS activity to monitor the T-DNA transfer into the nuclei of host cells. The results indicated that T-DNA was transferred into the nuclei of cells of both host plants inoculated with the M-31 mutant. The M-31 mutant strain had an insertion of Tn5 in the virA gene on its Ti plasmid. The introduction of the virA gene in the M-31 mutant complemented its avirulent phenotype. No kanamycin-resistant cells were observed when the M-31 mutant harboring the pIG121-Hm was inoculated to tobacco BY-2 cells. The M-31 mutant (virA::Tn5) seems to transfer T-DNA into the nucleus of the host cell, but is unable to integrate it to the chromosome. PMID:16232864

  10. 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.

  11. 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

  12. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    OpenAIRE

    Ye, Fuzhou; Wang, Chao; Fu, Qinqin; Zhang, Lian-Hui; Gao, Yong-Gui

    2015-01-01

    Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These...

  13. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cabeca, Luis Fernando; Pomini, Armando Mateus; Cruz, Pedro Luiz R.; Marsaioli, Anita J. [University of Campinas (UNICAMP), SP (Brazil). Chemistry Inst.

    2011-07-01

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  14. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    International Nuclear Information System (INIS)

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  15. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction

  16. Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Fuzhou [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Wang, Chao [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610 (Singapore); Fu, Qinqin [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Zhang, Lian-hui [Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore); Gao, Yong-gui, E-mail: ygao@ntu.edu.sg [Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673 (Singapore)

    2015-08-25

    The crystallization of the novel virulence factors SghA and SghR is reported. Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen–host interaction.

  17. Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

    International Nuclear Information System (INIS)

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 106 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains

  18. Transformación de Agrobacterium tumefaciens CEPA LBA4404 con el vector binario pNOV022, selección in vitro y caracterización molecular

    Directory of Open Access Journals (Sweden)

    Santos Rodríguez Janneth Fabiola

    2002-06-01

    Full Text Available La caracterización molecular de la infección causada por Agrobacterium tumefaciensy el des-cubrimiento de que una parte del plásmido inductor de tumores (T-DNA es transferido algenoma de las plantas que infecta, han permitido utilizar esta bacteria como sistema detransformación vegetal. La utilización de esta tecnología ha permitido a los científicos resolverproblemas con relación a especies cultivables. En el presente trabajo se realizó la transfor-mación de las cepas LBA4404 de Agrobacterium tumefaciensy DH5a de Escherichia coli. Para tal finse utilizó la técnica de choque térmico incorporando el vector binario pNOV022, que contenía6868Resúmenes la construcción p35S-IP-t35S - pUBQ-PMI-tNos (promotor 35S del CaMV, inhibidor de protea-sas, terminador 35S del CaMV, promotor ubiquitina 3, fosfomanosa isomerasa y terminadorde la nopalinsintetasa. Igualmente se realizó selección de clones bacterianos transformados(basado en la resistencia a espectinomicina y/o estreptomicina y su caracterización molecularpor medio de PCR y un perfil de restricción. Este trabajo apuntó a producir un vector adecuadopara la transformación estable de papa criolla (Solanum phureja y de papa (Solanum tuberosum,un gen que confiere resistencia a insectos. Los resultados de las metodologías empleados evi-denciaron la introducción exitosa del plásmido recombinante en E. coliy A. tumefaciens. Si bienla eficiencia de transformación fue baja (0,032 x 10-8, los resultados de la PCR mostraron laamplificación de una secuencia de 500 pb del gen inhibidor de proteasa aislado a partir de lascepas transformadas. En cuanto al perfil de restricción los resultados no fueron los esperados,sin embargo, permiten diferenciar la cepa de A. tumefacienstransformada. Adicionalmente seestandarizó las condiciones de los medios de selección para diferenciar cepas transformadas.

  19. 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. PMID:21132499

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Targeting Specificity Modification of Agrobacterium tumefaciens VirD2 Protein%农杆菌VirD2蛋白的定位特异性改造

    Institute of Scientific and Technical Information of China (English)

    陈友龙; 陈吉裕; 李翠萍; 苏承刚; 张兴国

    2012-01-01

    Agrobaeterium tumefaciens VirD2 protein performs functions in binding a single-stranded T-DNA (ssT-DNA) covalently, transferring it into plant nuclear, and integrating it efficiently into the nuclear genome. If it can be modified to target plastid, the VirD2 can pilot foreign genes there, and a new method of plastid transformation can be established. In this study, the localization signals of VirD2 were modified, which included the coding sequence of the nuclear localization signal (NLS) at its N terminal was point-mutated and its C terminal containing a bipartite-type NLS was truncated using an overlapping PCR method. This mutated VirD2 (mVirD2) was then fused with an eGFP (enhanced green fluorescent protein) reporter gene at the 3' end, and with or without the coding sequence of plastid-targeting peptide from Arabidopsis thaliana cold-regulated gene (AtCORlSA) at the 5' end, to construct chimeric genes pt-mVirD2-eGFP and mVirD2-eGFP, respectively. The chimeric genes were controlled by CaMV 35S promoter and were integrated into tobacco (Nicotina tabacum) nuclear genome viaAgrobacterium-mediated transformation. The fluorescence was only concentrated in chloroplasts for the transformants expressing pt-mVirD2-eGFP while dispersed among the cytoplasm for those with mVirD2-eGFP, and was not observed in the nuclear for both of them. The results showed that the expressing fused protein pt-mVirD2-eGFP targeted plastid specifically without nuclear localization, which bring forth an idea to explore efficient plastid transformation mediated by protein pt-mVirD2 as a guider of foreign DNA.%农杆菌(Agrobacterium tumefaciens)VirD2蛋白具有与单链T-DNA(ssT-DNA)共价结合并将其转运至植物细胞核,将外源基因高效整合进核基因组的功能;如果将该蛋白改定位于质体,则有可能利用其将外源基因携带进质体,建立质体转化新方法.本研究对VirD2蛋白的定位信号进行了改造,采用重叠延伸PCR突变技术对Vir2的N

  2. 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

  3. 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.

  4. Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos.

    Science.gov (United States)

    Jube, Sandro; Borthakur, Dulal

    2009-01-01

    The tree-legume Leucaena leucocephala (leucaena) is used as a perennial fodder because of its fast-growing foliage, which is high in protein content. The use of leucaena as a fodder is however restricted due to the presence of the toxin mimosine. Improvements in the nutritional contents as well as other agronomic traits of leucaena can be accomplished through genetic transformation. The objective of this research was to develop a transformation protocol for leucaena using phosphinothricin resistance as the plant selectable marker. Explants obtained from immature zygotic embryos infected with the Agrobacterium tumefaciens strain C58C1 containing the binary plasmid pCAMBIA3201 produced four putative transformed leucaena plants. Transformation was con- firmed by PCR, RT-PCR, Southern blot, Western analyses, GUS-specific enzyme activity and herbicide leaf spraying assay. A transformation efficiency of 2% was established using this protocol. PMID:20041041

  5. The spectrum of 3C 58

    International Nuclear Information System (INIS)

    Digital spectra of three faint knots in the SNR 3C 58 have been obtained in the wavelength range lambdalambda4700--7300 with the intensified Reticon spectrometer at the 1.3 m telescope of McGraw-Hill Observatory. Emission lines of [S II], [N II], Hα, and [O III] were detected with radial velocities less than 100 km s-1. Although 3C 58 resembles the Crab Nebula in its radio properties and is thought to be the remnant of the supernova observed in A.D. 1181, the relative line intensities and radial velocities reported here more nearly resemble those of the Cygnus Loop and Kepler's SNR

  6. Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

    Directory of Open Access Journals (Sweden)

    Antonio Orlando Di Mauro

    2000-03-01

    Full Text Available Twenty-five Brazilian soybean cultivars were studied for susceptibility to four strains of Agrobacterium tumefaciens (C58, Ach5, Bo542 and A281 and for their ability to produce somatic embryos. Twelve plants of each cultivar were inoculated in a greenhouse at 4-6 weeks of age, using 12 inoculation sites per plant. The number of galls formed on plants were counted 8-10 weeks after inoculation. To study ability to produce somatic embryos, immature cotyledons, 4-6 mm in length, were plated onto N10 medium for induction of somatic embryogenesis, using four Petri dishes with 20 cotyledons for each cultivar. The embryogenic tissues were transferred onto new N10 medium six times at 15-day intervals and the number of somatic embryos per cultivar determined. Significant interaction between soybean cultivars and A. tumefaciens strains was observed; the most virulent strain was A281. The opine type apparently had no effect on strain virulence, and the most embryogenic cultivars were IAS-5, Cristalina, FT-Cometa, IAC-7 and OC-3.Vinte e cinco cultivares brasileiros de soja foram estudados quanto à suscetibilidade a quatro linhagens de Agrobacterium tumefaciens (C58, Ach5, Bo542 e A281 e quanto à capacidade de produzir embriões somáticos. Doze plantas de cada cultivar foram inoculadas, na casa de vegetação, 4-6 semanas após a semeadura, sendo efetuadas 12 inoculações por planta. O número de galhas derivadas dessas inoculações foi contado 8-10 semanas após as inoculações. Para avaliar a capacidade de produção de embriões somáticos, cotilédones imaturos com 4-6 mm de comprimento foram cultivados em meio N10 para indução de calos embriogênicos, sendo empregadas, para cada cultivar, quatro placas de Petri contendo 20 cotilédones cada. Os tecidos embriogênicos foram transferidos 6 vezes, a cada 15 dias de intervalo, para novo meio N10, sendo contado o número de embriões somáticos por cultivar. Foi observada uma interação significativa

  7. 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

  8. Transformación de Agrobacterium tumefaciens CEPA LBA4404 con el vector binario pNOV022, selección in vitro y caracterización molecular

    OpenAIRE

    Santos Rodríguez Janneth Fabiola

    2002-01-01

    La caracterización molecular de la infección causada por Agrobacterium tumefaciensy el des-cubrimiento de que una parte del plásmido inductor de tumores (T-DNA) es transferido algenoma de las plantas que infecta, han permitido utilizar esta bacteria como sistema detransformación vegetal. La utilización de esta tecnología ha permitido a los científicos resolverproblemas con relación a especies cultivables. En el presente trabajo se realizó la transfor-mación de las cepas LBA4404 de Agrobacteri...

  9. 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...

  10. TRANSFORMACIÓN DE PLANTAS MEDIADA POR AGROBACTERIUM: "INGENIERÍA GENÉTICA NATURAL APLICADA" PLANT TRANSFORMATION MEDIATED BY AGROBACTERIUM: "APPLIED NATURAL GENETIC ENGINEERING"

    OpenAIRE

    Ana Milena Valderrama Fonseca; Rafael Arango Isaza; Lucia Afanador Kafuri

    2005-01-01

    Agrobacterium tumefaciens tiene la capacidad de transferir ADN entre reinos diferentes. El impacto de este hallazgo ha tenido grandes aplicaciones en diversos campos de la biología vegetal, agricultura y biotecnología. En este artículo se describen los procesos por los cuales Agrobacterium realiza la transferencia de ADN a la planta, puntualizando en 7 eventos fundamentales para la interacción A. tumefaciens-planta y esta dirigido a profesionales de las áreas biológicas que estén interesados ...

  11. 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...

  12. NCBI nr-aa BLAST: CBRC-XTRO-01-2352 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-2352 ref|NP_396144.1| hypothetical protein AGR_pAT _298 [Agrobacterium tumefaciens s ... bacterium tumefaciens str. C58] gb|AAK90585.1| AGR_pAT _298p [Agrobacterium tumefaciens str. C58] gb|AAL45 ...

  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. 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...

  15. 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.

  16. Reiterated DNA Sequences in Rhizobium and Agrobacterium spp

    OpenAIRE

    Flores, M.; González, V.; Brom, S; Martínez, E.; Piñero, D; Romero, D.; Dávila, G; Palacios, R

    1988-01-01

    Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens s...

  17. Deviating T-DNA transfer from Agrobacterium tumefaciens to plants

    DEFF Research Database (Denmark)

    van der Graaff, Eric; den Dulk-Ras, A; Hooykaas, P J

    1996-01-01

    . Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T......We analyzed 29 T-DNA inserts in transgenic Arabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data......-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat....

  18. BIOLOGICAL CONTROL OF THE CROWN GALL AGROBACTERIUM TUMEFACIENS

    OpenAIRE

    Lemanova, N.; M. Mager

    2011-01-01

    Genetic date transfer T-DNA from a pathogen to a plant cell occur the process of tumefaction on vineyard and fruit varieties. Treatment of woundings by biologic preparation Paurin (suspension of bacterial cells of Pseudomonas fluorescens cr-330d) deteriorates the interaction of bacteria-pathogen with the cell of host plant. Application of this preparation decrease the quantity of plants with tumors

  19. Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.

    OpenAIRE

    Hooykaas, P J; Den Dulk-Ras, H; Ooms, G.; Schilperoort, R.A.

    1980-01-01

    Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become establis...

  20. TRANSFORMACIÓN DE PLANTAS MEDIADA POR AGROBACTERIUM: "INGENIERÍA GENÉTICA NATURAL APLICADA" PLANT TRANSFORMATION MEDIATED BY AGROBACTERIUM: "APPLIED NATURAL GENETIC ENGINEERING"

    Directory of Open Access Journals (Sweden)

    Ana Milena Valderrama Fonseca

    2005-06-01

    Full Text Available Agrobacterium tumefaciens tiene la capacidad de transferir ADN entre reinos diferentes. El impacto de este hallazgo ha tenido grandes aplicaciones en diversos campos de la biología vegetal, agricultura y biotecnología. En este artículo se describen los procesos por los cuales Agrobacterium realiza la transferencia de ADN a la planta, puntualizando en 7 eventos fundamentales para la interacción A. tumefaciens-planta y esta dirigido a profesionales de las áreas biológicas que estén interesados en actualizarse en el tema. El conocimiento básico sobre el mecanismo de transferencia del ADN ha permitido el desarrollo de vectores para la introducción de genes foráneos, dentro de los cuales se describen los 2 tipos de vectores utilizados en la actualidad: co-integrados y binarios. Así mismo se detallan algunos factores importantes que median la transformación y algunas de las principales aplicaciones de la transformación de plantas y hongos mediada por Agrobacterium.Agrobacterium tumefaciens has the ability to transfer DNA between different kingdoms. This finding has had a great impact in several fields of plant biology, agriculture and biotechnology. This review describe the mechanisms by which Agrobacterium transfer DNA to the plant, emphasizing 7 fundamental steps in the A. tumefaciens-plant interaction, to provide an opportunity for professionals in the biological sciences to familiarize themselves with the topic. Basic knowledge about the DNA transfer mechanism has allowed the development of various vectors for the introduction of foreign genes, among which two classes that are currently used are described: co-integrative and binary vectors. Furthermore, detail some important factors that mediate transformation and some of the principal applications of the transformation of plants and fungi by means of Agrobacterium.

  1. 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.

  2. Stable Agrobacterium-mediated transformation of Norway spruce embryogenic tissues using somatic embryo explants

    Czech Academy of Sciences Publication Activity Database

    Pavingerová, Daniela; Bříza, Jindřich; Niedermeierová, Hana; Vlasák, Josef

    2011-01-01

    Roč. 57, č. 7 (2011), s. 277-280. ISSN 1212-4834 R&D Projects: GA MZe QH71290 Institutional research plan: CEZ:AV0Z50510513 Keywords : Agrobacterium tumefaciens * genetic engineering * GUS activity * Picea abies (L.) Karst Subject RIV: EB - Genetic s ; Molecular Biology

  3. 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...

  4. Transformación de plantas mediada por agrobacterium: “ingeniería genética natural aplicada”

    OpenAIRE

    Valderrama, Ana Milena

    2011-01-01

    Agrobacterium tumefaciens tiene la capacidad de transferir ADN entre reinos diferentes. El impacto de este hallazgo ha tenido grandes aplicaciones en diversos campos de la biología vegetal, agricultura y biotecnología. En este artículo se describen los procesos por los cuales Agrobacterium realiza la transferencia de ADN a la planta, puntualizando en 7 eventos fundamentales para la interacción A. tumefaciens-planta y esta dirigido a profesionales de las áreas biológicas que estén interesados...

  5. NCBI nr-aa BLAST: CBRC-DSIM-04-0023 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DSIM-04-0023 ref|NP_396129.1| hypothetical protein AGR_pAT _277 [Agrobacterium tumefaciens s ... tr. C58] gb|AAK90570.1| AGR_pAT _277p [Agrobacterium tumefaciens str. C58] NP_39612 ...

  6. NCBI nr-aa BLAST: CBRC-DMEL-04-0053 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-04-0053 ref|NP_396129.1| hypothetical protein AGR_pAT _277 [Agrobacterium tumefaciens s ... tr. C58] gb|AAK90570.1| AGR_pAT _277p [Agrobacterium tumefaciens str. C58] NP_39612 ...

  7. Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

    OpenAIRE

    Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

    2014-01-01

    By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structura...

  8. 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.

  9. 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

  10. Agrobacterium infection and plant defense—transformation success hangs by a thread

    OpenAIRE

    Pitzschke, Andrea

    2013-01-01

    The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host de...

  11. Development of Transgenic Papaya through Agrobacterium-Mediated Transformation

    Directory of Open Access Journals (Sweden)

    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.

  12. 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...

  13. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

    Science.gov (United States)

    Agrobacterium rhizogenes and A. tumefaciens are plant pathogenic bacteria causing abnormal tissue growth such as hairy root and crown gall diseases respectively, through the transfer of DNA fragments (T-DNA) bearing functional genes into the host plant genome. This naturally occurring mechanism of g...

  14. 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

  15. Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity

    DEFF Research Database (Denmark)

    Erbs, Gitte; Silipo, Alba; Aslam, Shazia;

    2008-01-01

    , oxidative burst, medium alkalinization, and formation of callose. Highly purified muropeptides from PGNs were more effective elicitors of early defense responses than native PGN. Therefore, PGN and its constituents represent a Microbe-Associated Molecular Pattern (MAMP) in plant-bacterial interactions. PGN...... and muropeptides from aggressive Xanthomonas campestris pv. campestris were significantly more active than those from Agrobacterium tumefaciens, which must maintain host cell viability during infection. The structure of muropeptide components and the distinctive differences are described. Differing...

  16. 农杆菌介导含硫氨基酸γ-zein 转化菊苣的初步研究%Preliminary studies on transgenic chicory using the sulphur-amino acid gene,γ-zein, mediated by Agrobacterium tumefacien

    Institute of Scientific and Technical Information of China (English)

    张玉; 白史且; 李聪

    2015-01-01

    含硫氨基酸具有动物营养与免疫相关的重要生理功能,为提高菊苣中含硫氨基酸含量,采用根癌农杆菌介导法将玉米种子贮藏蛋白含硫氨基酸基因γ-zein 和绿色荧光蛋白 GFP 融合基因转入到菊苣无菌苗叶片中,经过共培养、潮霉素抗性筛选、分化、再生和炼苗,得到抗性植株。对抗性植株进行 PCR、PCR-Southern、斑点杂交和 RT-PCR 分析,结果表明,外源目的基因已经整合到菊苣基因组中并且得到了表达,为提高菊苣含硫氨基酸含量,改善其品质奠定了基础。%Sulfur-containing amino acids have important physiological functions related to animal nutrition and immunity.To improve the sulfur-amino acid content of chicory,leaves of chicory were transformed with the Sulphur-amino acid gene γ-zein,an important prolamin storage protein from Zea mays and a green fluorescent protein (GFP)gene using Agrobacterium mediated transfusion.After co-culture,selective differentiation and regeneration,hygromycin resistant plants were obtained.Resistant plants were detected using PCR,PCR-southern,dot blot hybridization and RT-PCR.The results demonstrated that the γ-zein genes had been inte-grated into the genome of chicory and expressed on a nucleic acid level in the transgenic plants.

  17. Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression.

    Science.gov (United States)

    Gorfer, Markus; Klaubauf, Sylvia; Bandian, Dragana; Strauss, Joseph

    2007-07-01

    Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome. PMID:17662587

  18. 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

  19. Novel object exploration in the C58/J mouse model of autistic-like behavior.

    Science.gov (United States)

    Blick, Mikkal G; Puchalski, Breann H; Bolanos, Veronica J; Wolfe, Kaitlin M; Green, Matthew C; Ryan, Bryce C

    2015-04-01

    Mouse models of autistic like behaviors are a valuable tool to use when studying the causes, symptoms, and potential treatments for autism. The inbred C58/J strain is a strain of interest for this model and has previously been shown to possess face validity for some of the core traits of autism, including low social behavior and elevated motor stereotypies. Higher order repetitive behaviors have not been extensively studied in this strain, or in mice in general. In this study, we looked for evidence of higher-order repetitive behaviors in the C58/J strain using a novel object assay. This assay utilized a mouse's natural exploratory behavior among unfamiliar objects to identify potential sequencing patterns in motor activity. The motor stereotypies displayed by the C58/J strain during testing were consistent with past studies. The C58/J strain also displayed a high preference for a single object in the round arena assays and the females demonstrating elevated sequencing patterns in the round arena. Although the C58/J strain did not show pervasive evidence of higher-order repetitive behaviors across all measures, there was evidence of higher order repetitive behaviors in certain situations. This study further demonstrates the potential of the C58/J mouse strains as a model for lower-order and potentially, higher-order repetitive behaviors. This study also demonstrates that the shape of the novel object arena can change the behavior displayed by the test animals. Further studies utilizing the C58/J strain and further validation of the novel object assay are warranted. PMID:25532914

  20. 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

  1. 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...

  2. On the Distance and Age of the Pulsar Wind Nebula 3C58

    CERN Document Server

    Kothes, Roland

    2010-01-01

    There is a growing community of astronomers presenting evidence that the pulsar wind nebula 3C58 is much older than the connection with the historical supernova of A.D 1181 would indicate. Most of the strong evidence against a young age for 3C58 relies heavily on the assumed distance of 3.2 kpc determined with HI absorption measurements. I have revisited this distance determination based on new HI data from the Canadian Galactic Plane Survey and added newly determined distances to objects in the neighbourhood, which are based on direct measurements by trigonometric parallax. This leads to a new more reliable distance estimate of 2 kpc for 3C58 and makes the connection between the pulsar wind nebula and the historical event from A.D. 1181 once again much more compelling.

  3. 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.

  4. Agrobacterium infection and plant defense-transformation success hangs by a thread.

    Science.gov (United States)

    Pitzschke, Andrea

    2013-01-01

    The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to "yes" or "no." This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655

  5. 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

  6. Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens

    OpenAIRE

    Thomashow, M F; Nutter, R.; Postle, K; Chilton, M.-D.; Blattner, F R; Powell, A.; Gordon, M P; Nester, E. W.

    1980-01-01

    The Ti plasmid sequences (T-DNA) from the octopine-producing crown gall tumor A6S/2 were isolated by molecular cloning, using the bacteriophage λ vector Charon 4A. Analysis of the cloned DNA segments indicates that the Ti plasmid sequences are covalently joined to plant nuclear DNA. These data demonstrate that genetic recombination between a eukaryote and a prokaryote can occur as a natural phenomenon.

  7. 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

  8. 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

  9. Relationship between the limited and wide host range octopine-type Ti plasmids of Agrobacterium tumefaciens.

    OpenAIRE

    Thomashow, M F; Knauf, V C; Nester, E W

    1981-01-01

    The relationship between the limited host range octopine Ti plasmids and the wide host range octopine Ti plasmids pTiB6806 and pTiA6 was studied. The limited host range Ti plasmids shared extensive deoxyribonucleic acid homology; pTiAg63 and pTiAg162 were essentially completely homologous with pTiAg158 while pTiAg57 shared approximately 64% homology with pTiAg158. In contrast, the limited host range octopine Ti plasmids only shared 6 to 15% homology with the wide host range octopine Ti plasmi...

  10. 3C58'S filamentary radial velocities, line intensities, and proper motions

    International Nuclear Information System (INIS)

    Optical spectroscopy on nearly 50 filaments of 3C58 indicates a maximum expansion velocity of 110 ±100 km s-1. A considerable range in radial velocity with projected distance from remnant center is found suggesting that the remnant's optical emission is not confined to a thin shell. Typical filament electron densities are between 200--500 cm-3 with Hα/[N II] ratios in the range 0.2--0.5. Optical extinction to 3C58 is modest with E[B-V] = 0.68 ±0.08. Preliminary radial proper motion measurements for a few outlying filaments suggest values of order 0.05 double-prime--0.07 double-prime yr -1

  11. Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6.

    OpenAIRE

    McBride, K E; Knauf, V C

    1988-01-01

    The virE operon of the Agrobacterium tumefaciens Ti plasmid pTiA6 encodes at least one trans-acting protein involved in the expression of virulence. Two open reading frames designated virE1 and virE2 code for polypeptides of 7 and 60 kilodaltons (kDa), respectively, that can be visualized after expression in Escherichia coli minicells. To determine which virE sequences are required for virulence, a strain deleted for the entire locus [strain KE1(pTiA6 delta E)] was constructed and tested for ...

  12. 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

  13. 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.

  14. Chaxapeptin, a Lasso Peptide from Extremotolerant Streptomyces leeuwenhoekii Strain C58 from the Hyperarid Atacama Desert.

    Science.gov (United States)

    Elsayed, Somayah S; Trusch, Franziska; Deng, Hai; Raab, Andrea; Prokes, Ivan; Busarakam, Kanungnid; Asenjo, Juan A; Andrews, Barbara A; van West, Pieter; Bull, Alan T; Goodfellow, Michael; Yi, Yu; Ebel, Rainer; Jaspars, Marcel; Rateb, Mostafa E

    2015-10-16

    Lasso peptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that possess a unique "lariat knot" structural motif. Genome mining-targeted discovery of new natural products from microbes obtained from extreme environments has led to the identification of a gene cluster directing the biosynthesis of a new lasso peptide, designated as chaxapeptin 1, in the genome of Streptomyces leeuwenhoekii strain C58 isolated from the Atacama Desert. Subsequently, 1 was isolated and characterized using high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance methods. The lasso nature of 1 was confirmed by calculating its nuclear Overhauser effect restraint-based solution structure. Chaxapeptin 1 displayed a significant inhibitory activity in a cell invasion assay with human lung cancer cell line A549. PMID:26402731

  15. 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.

  16. 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.

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

    Science.gov (United States)

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

    2014-05-01

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

  18. Discovery of TeV gamma-ray emission from the pulsar wind nebula 3C 58 by MAGIC

    CERN Document Server

    López-Coto, R; Bednarek, W; Blanch, O; Cortina, J; Wilhelmi, E de Ona; Martín, J; Pérez-Torres, M A

    2015-01-01

    The pulsar wind nebula (PWN) 3C 58 has been proposed as a good candidate for detection at VHE (VHE; E>100 GeV) for many years. It is powered by one of the highest spin-down power pulsars known (5\\% of Crab pulsar) and it has been compared to the Crab Nebula due to its morphology. This object was previously observed by imaging atmospheric Cherenkov telescopes (Whipple, VERITAS and MAGIC), and upper limit of 2.4\\% Crab Unit (C.U.) at VHE. It was detected by Fermi-LAT with a spectrum extending beyond 100 GeV. We analyzed 81 hours of 3C 58 data taken with the MAGIC telescopes and we detected VHE gamma-ray emission with a significance of 5.7 sigma and an integral flux of 0.65\\% C.U. above 1 TeV. We report the first significant detection of PWN 3C 58 at TeV energies. According to our results 3C 58 is the least luminous VHE gamma-ray PWN ever detected at VHE and the one with the lowest flux at VHE to date. We compare our results with the expectations of time-dependent models in which electrons up-scatter photon fiel...

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Biologia molecular do processo de infecção por Agrobacterium spp.

    Directory of Open Access Journals (Sweden)

    Andrade Gisele M. de

    2003-01-01

    Full Text Available Agrobacterium tumefaciens é o agente causal da galha-da-coroa, doença que afeta a maioria das plantas dicotiledôneas e caracteriza-se pelo crescimento de tumores na junção entre o caule e a raiz (coroa. A formação desses tumores é o resultado de um processo natural de transferência de genes de Agrobacterium spp. para o genoma da planta infetada. Esses genes estão contidos em um plasmídio de alto peso molecular (120 a 250 kb, denominado Ti ("tumor inducing", presente em todas as linhagens patogênicas de Agrobacterium spp. Duas regiões do plasmídio Ti estão diretamente envolvidas na indução do tumor: a região-T, que corresponde ao segmento de DNA transferido para a célula vegetal, e a região de virulência (região vir, que contém genes envolvidos na síntese de proteínas responsáveis pelo processo de transferência da região-T. Esta região, uma vez transferida e integrada no genoma da célula vegetal, passa a ser denominada de T-DNA ("transferred DNA". Os genes presentes no T-DNA codificam enzimas envolvidas na via de biossíntese de reguladores de crescimento, auxinas e citocininas. A síntese desses reguladores pelas células transformadas causa um desbalanço hormonal, levando à formação do tumor no local da infecção. Outro grupo de genes presentes no T-DNA codifica enzimas responsáveis pela síntese de opinas, que são catabolisadas especificamente pela bactéria colonizadora, como fonte de nutrientes. O conhecimento preliminar das bases moleculares envolvidas no processo de infecção de uma planta hospedeira por Agrobacterium spp., permitiu a utilização desta bactéria como vetor natural de transformação genética de plantas.

  4. 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

  5. Discovery of TeV gamma-ray emission from the pulsar wind nebula 3C 58 by MAGIC

    Directory of Open Access Journals (Sweden)

    López-Coto Rubén

    2016-01-01

    Full Text Available The pulsar wind nebula (PWN 3C 58 is one of the historical very-high-energy (VHE; E>100 GeV gamma-ray source candidates. It has been compared to the Crab Nebula due to their morphological similarities. This object was detected by Fermi-LAT with a spectrum extending beyond 100 GeV. We analyzed 81 hours of 3C 58 data taken with the MAGIC telescopes and we detected VHE gamma-ray emission for the first time at TeV energies with a significance of 5.7 sigma and an integral flux of 0.65% C.U. above 1 TeV. According to our results 3C 58 is the least luminous PWN ever detected at VHE and the one with the lowest flux at VHE to date. We compare our results with the expectations of time-dependent models in which electrons up-scatter photon fields. The best representation favors a distance to the PWN of 2 kpc and Far Infrared (FIR comparable to CMB photon fields. Hadronic contribution from the hosting supernova remnant (SNR requires unrealistic energy budget given the density of the medium, disfavoring cosmic ray acceleration in the SNR as origin of the VHE gamma-ray emission.

  6. Discovery of TeV gamma-ray emission from the Pulsar Wind Nebula 3C 58 by MAGIC

    CERN Document Server

    Bigas, O Blanch; Carmona, E; Pérez-Torres, M A

    2015-01-01

    The Pulsar Wind Nebula (PWN) 3C 58 is energized by one of the highest spin-down power pulsars known (5% of Crab pulsar) and it has been compared to the Crab Nebula due to their morphological similarities. This object was detected by Fermi-LAT with a spectrum extending beyond 100 GeV. We analyzed 81 hours of 3C 58 data taken with the MAGIC telescopes and we detected VHE gamma-ray emission for the first time at TeV energies with a significance of 5.7 sigma and an integral flux of 0.65% C.U. above 1 TeV. The differential energy spectrum between 400 GeV and 10 TeV is well described by a power-law function $d\\Phi/dE=f_{o}(E/1TeV)^{-\\Gamma}$ with $f_{o}=(2.0\\pm0.4stat\\pm0.6sys) 10^{-13}cm^{-2}s^{-1}TeV^{-1}$ and $\\Gamma=2.4\\pm0.2sta\\pm0.2sys$. This leads 3C 58 to be the least luminous PWN ever detected at VHE and the one with the lowest flux at VHE to date. According to time-dependent models in which electrons up-scatter photon fields, the best representation favors a distance to the PWN of 2 kpc and FIR comparable...

  7. Patogenecidad de agrobacterium tumefaciens en algunas especies de plantas de flóres de exportación

    OpenAIRE

    Ovalle, Germán; Benincore, Germán; Arbeláez, Germán

    2011-01-01

    Tres aislamientos de tumefaelens Agrobaeterlum de rosa de las plantas afectadas con agalla de la corona en la Sabana de Bogotá se han caracterizado e identificado por pruebas de laboratorio y mediante la inoculación en plantas de tomate, zanahoria y remolacha. Patogenicidad fue probado en el campo de rosas, claveles, limonium, chrysanthermum y plantas gipsophila que son especies importantes en el país para la producción de flores de exportación. Su patogenia se ha probado también en las plant...

  8. Estudios orientados a la transformación de papa criolla (Solanum Phureja) mediada por Agrobacterium tumefaciens

    OpenAIRE

    Chaparro Giraldo Alejandro; Carvajal Bernal Diana Angélica

    2004-01-01

    La papa criolla (Solanum phureja Juz. et Buk) es un importante recurso genético colombiano. Fue excluida del Tratado Internacional sobre los Recursos Fitogenéticos de la FAO, y siendo Colombia el principal país que la explota comercialmente y que ha desarrollado un cultivar mejorado tradicionalmente conocido como “Yema de Huevo”, las posibilidades de explotación de este recurso son importantes. Este cultivar presenta problemas de enfermedades y plagas, en particular es atacado por Tecia solan...

  9. 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.

  10. 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.

  11. Transition to quorum sensing in an Agrobacterium population: A stochastic model.

    Directory of Open Access Journals (Sweden)

    Andrew B Goryachev

    2005-09-01

    Full Text Available Understanding of the intracellular molecular machinery that is responsible for the complex collective behavior of multicellular populations is an exigent problem of modern biology. Quorum sensing, which allows bacteria to activate genetic programs cooperatively, provides an instructive and tractable example illuminating the causal relationships between the molecular organization of gene networks and the complex phenotypes they control. In this work we--to our knowledge for the first time--present a detailed model of the population-wide transition to quorum sensing using the example of Agrobacterium tumefaciens. We construct a model describing the Ti plasmid quorum-sensing gene network and demonstrate that it behaves as an "on-off" gene expression switch that is robust to molecular noise and that activates the plasmid conjugation program in response to the increase in autoinducer concentration. This intracellular model is then incorporated into an agent-based stochastic population model that also describes bacterial motion, cell division, and chemical communication. Simulating the transition to quorum sensing in a liquid medium and biofilm, we explain the experimentally observed gradual manifestation of the quorum-sensing phenotype by showing that the transition of individual model cells into the "on" state is spread stochastically over a broad range of autoinducer concentrations. At the same time, the population-averaged values of critical autoinducer concentration and the threshold population density are shown to be robust to variability between individual cells, predictable and specific to particular growth conditions. Our modeling approach connects intracellular and population scales of the quorum-sensing phenomenon and provides plausible answers to the long-standing questions regarding the ecological and evolutionary significance of the phenomenon. Thus, we demonstrate that the transition to quorum sensing requires a much higher threshold

  12. Transition to Quorum Sensing in an Agrobacterium Population: A Stochastic Model.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Understanding of the intracellular molecular machinery that is responsible for the complex collective behavior of multicellular populations is an exigent problem of modern biology. Quorum sensing, which allows bacteria to activate genetic programs cooperatively, provides an instructive and tractable example illuminating the causal relationships between the molecular organization of gene networks and the complex phenotypes they control. In this work we-to our knowledge for the first time-present a detailed model of the population-wide transition to quorum sensing using the example of Agrobacterium tumefaciens. We construct a model describing the Ti plasmid quorum-sensing gene network and demonstrate that it behaves as an "on-off" gene expression switch that is robust to molecular noise and that activates the plasmid conjugation program in response to the increase in autoinducer concentration. This intracellular model is then incorporated into an agent-based stochastic population model that also describes bacterial motion, cell division, and chemical communication. Simulating the transition to quorum sensing in a liquid medium and biofilm, we explain the experimentally observed gradual manifestation of the quorum-sensing phenotype by showing that the transition of individual model cells into the "on" state is spread stochastically over a broad range of autoinducer concentrations. At the same time, the population-averaged values of critical autoinducer concentration and the threshold population density are shown to be robust to variability between individual cells, predictable and specific to particular growth conditions. Our modeling approach connects intracellular and population scales of the quorum-sensing phenomenon and provides plausible answers to the long-standing questions regarding the ecological and evolutionary significance of the phenomenon. Thus, we demonstrate that the transition to quorum sensing requires a much higher threshold cell

  13. Discovery of TeV γ-ray emission from the pulsar wind nebula 3C 58 by MAGIC

    Science.gov (United States)

    Aleksić, J.; Ansoldi, S.; Antonelli, L. A.; Antoranz, P.; Babic, A.; Bangale, P.; Barrio, J. A.; Becerra González, J.; Bednarek, W.; Bernardini, E.; Biasuzzi, B.; Biland, A.; Blanch, O.; Bonnefoy, S.; Bonnoli, G.; Borracci, F.; Bretz, T.; Carmona, E.; Carosi, A.; Colin, P.; Colombo, E.; Contreras, J. L.; Cortina, J.; Covino, S.; Da Vela, P.; Dazzi, F.; De Angelis, A.; De Caneva, G.; De Lotto, B.; de Oña Wilhelmi, E.; Delgado Mendez, C.; Dominis Prester, D.; Dorner, D.; Doro, M.; Einecke, S.; Eisenacher, D.; Elsaesser, D.; Fonseca, M. V.; Font, L.; Frantzen, K.; Fruck, C.; Galindo, D.; García López, R. J.; Garczarczyk, M.; Garrido Terrats, D.; Gaug, M.; Godinović, N.; González Muñoz, A.; Gozzini, S. R.; Hadasch, D.; Hanabata, Y.; Hayashida, M.; Herrera, J.; Hildebrand, D.; Hose, J.; Hrupec, D.; Idec, W.; Kadenius, V.; Kellermann, H.; Kodani, K.; Konno, Y.; Krause, J.; Kubo, H.; Kushida, J.; La Barbera, A.; Lelas, D.; Lewandowska, N.; Lindfors, E.; Lombardi, S.; López, M.; López-Coto, R.; López-Oramas, A.; Lorenz, E.; Lozano, I.; Makariev, M.; Mallot, K.; Maneva, G.; Mankuzhiyil, N.; Mannheim, K.; Maraschi, L.; Marcote, B.; Mariotti, M.; Martínez, M.; Mazin, D.; Menzel, U.; Miranda, J. M.; Mirzoyan, R.; Moralejo, A.; Munar-Adrover, P.; Nakajima, D.; Niedzwiecki, A.; Nilsson, K.; Nishijima, K.; Noda, K.; Orito, R.; Overkemping, A.; Paiano, S.; Palatiello, M.; Paneque, D.; Paoletti, R.; Paredes, J. M.; Paredes-Fortuny, X.; Persic, M.; Prada Moroni, P. G.; Prandini, E.; Puljak, I.; Reinthal, R.; Rhode, W.; Ribó, M.; Rico, J.; Rodriguez Garcia, J.; Rügamer, S.; Saito, T.; Saito, K.; Satalecka, K.; Scalzotto, V.; Scapin, V.; Schultz, C.; Schweizer, T.; Shore, S. N.; Sillanpää, A.; Sitarek, J.; Snidaric, I.; Sobczynska, D.; Spanier, F.; Stamatescu, V.; Stamerra, A.; Steinbring, T.; Storz, J.; Strzys, M.; Takalo, L.; Takami, H.; Tavecchio, F.; Temnikov, P.; Terzić, T.; Tescaro, D.; Teshima, M.; Thaele, J.; Tibolla, O.; Torres, D. F.; Toyama, T.; Treves, A.; Uellenbeck, M.; Vogler, P.; Zanin, R.

    2014-07-01

    Context. The pulsar wind nebula (PWN) 3C 58 is one of the historical very high-energy (VHE; E> 100 GeV) γ-ray source candidates. It is energized by one of the highest spin-down power pulsars known (5% of Crab pulsar) and it has been compared with the Crab nebula because of their morphological similarities. This object was previously observed by imaging atmospheric Cherenkov telescopes (Whipple, VERITAS and MAGIC), although it was not detected, with an upper limit of 2.3% Crab unit (C.U.) at VHE. It was detected by the Fermi Large Area Telescope (LAT) with a spectrum extending beyond 100 GeV. Aims: We aim to extend the spectrum of 3C 58 beyond the energies reported by the Fermi Collaboration and probe acceleration of particles in the PWN up to energies of a few tens of TeV. Methods: We analyzed 81 h of 3C 58 data taken in the period between August 2013 and January 2014 with the MAGIC telescopes. Results: We detected VHE γ-ray emission from 3C 58 with a significance of 5.7σ and an integral flux of 0.65% C.U. above 1 TeV. According to our results, 3C 58 is the least luminous VHE γ-ray PWN ever detected at VHE and has the lowest flux at VHE to date. The differential energy spectrum between 400 GeV and 10 TeV is well described by a power-law function dφ/dE = f0(E/1 TeV)-Γ with f0 = (2.0 ± 0.4stat ± 0.6sys) × 10-13 cm-2 s-1 TeV-1 and Γ = 2.4 ± 0.2stat ± 0.2sys. The skymap is compatible with an unresolved source. Conclusions: We report the first significant detection of PWN 3C 58 at TeV energies. We compare our results with the expectations of time-dependent models in which electrons upscatter photon fields. The best representation favors a distance to the PWN of 2 kpc and far-infrared (FIR) values similar to cosmic microwave background photon fields. If we consider an unexpectedly high FIR density, the data can also be reproduced by models assuming a 3.2 kpc distance. A low magnetic field, far from equipartition, is required to explain the VHE data. Hadronic

  14. 基于STC89C58的PID控制电动车翘翘板%Electric Car Seesaw Controlled by PID Algorithm Based on STC89C58 MCU

    Institute of Scientific and Technical Information of China (English)

    刘潇

    2013-01-01

    本设计采用STC89 C58作为核心处理器,以步进电机为驱动电机。主要功能包括PID算法对步进电机运动控制,角度信号采集与处理,红外循迹。本系统实验最终结果为电动车沿引黑线运动至翘翘板平衡点,经过一定的动态平衡后在翘翘板中间平衡。%This design uses a STC89C58 as the core processor and the stepper motor as the driving motor .The key features include stepper motor motion control with PID algorithm;signal acquisition and processing , and the infrared tracking .The end result of experi-ments on the system for electric motor car shows that when it moves along the black line to the seesaw balance point and after a certain amount of dynamic equilibrium , the motor car will be balanced between the seesaw .

  15. Environment control to improve recombinant protein yields in plants based on Agrobacterium-mediated transient gene expression

    Directory of Open Access Journals (Sweden)

    Naomichi eFujiuchi

    2016-03-01

    Full Text Available Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: 1 recombinant protein content per unit biomass; and 2 recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on those parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and post-inoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Pre-inoculation environmental factors associated with planting density, light quality and nutrient supply affect plant characteristics such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, post-inoculation environmental factors such as temperature, light intensity and humidity have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the post-inoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain

  16. Discovery of TeV gamma-ray emission from the pulsar wind nebula 3C 58 by MAGIC

    CERN Document Server

    Aleksić, J; Antonelli, L A; Antoranz, P; Babic, A; Bangale, P; Barrio, J A; González, J Becerra; Bednarek, W; Bernardini, E; Biasuzzi, B; Biland, A; Blanch, O; Bonnefoy, S; Bonnoli, G; Borracci, F; Bretz, T; Carmona, E; Carosi, A; Colin, P; Colombo, E; Contreras, J L; Cortina, J; Covino, S; Da Vela, P; Dazzi, F; De Angelis, A; De Caneva, G; De Lotto, B; Wilhelmi, E de Oña; Mendez, C Delgado; Prester, D Dominis; Dorner, D; Doro, M; Einecke, S; Eisenacher, D; Elsaesser, D; Fonseca, M V; Font, L; Frantzen, K; Fruck, C; Galindo, D; López, R J García; Garczarczyk, M; Terrats, D Garrido; Gaug, M; Godinović, N; Muñoz, A González; Gozzini, S R; Hadasch, D; Hanabata, Y; Hayashida, M; Herrera, J; Hildebrand, D; Hose, J; Hrupec, D; Idec, W; Kadenius, V; Kellermann, H; Kodani, K; Konno, Y; Krause, J; Kubo, H; Kushida, J; La Barbera, A; Lelas, D; Lewandowska, N; Lindfors, E; Lombardi, S; López, M; López-Coto, R; López-Oramas, A; Lorenz, E; Lozano, I; Makariev, M; Mallot, K; Maneva, G; Mankuzhiyil, N; Mannheim, K; Maraschi, L; Marcote, B; Mariotti, M; Martínez, M; Mazin, D; Menzel, U; Miranda, J M; Mirzoyan, R; Moralejo, A; Munar-Adrover, P; Nakajima, D; Niedzwiecki, A; Nilsson, K; Nishijima, K; Noda, K; Orito, R; Overkemping, A; Paiano, S; Palatiello, M; Paneque, D; Paoletti, R; Paredes, J M; Paredes-Fortuny, X; Persic, M; Moroni, P G Prada; Prandini, E; Puljak, I; Reinthal, R; Rhode, W; Ribó, M; Rico, J; Garcia, J Rodriguez; Rügamer, S; Saito, T; Saito, K; Satalecka, K; Scalzotto, V; Scapin, V; Schultz, C; Schweizer, T; Shore, S N; Sillanpää, A; Sitarek, J; Snidaric, I; Sobczynska, D; Spanier, F; Stamatescu, V; Stamerra, A; Steinbring, T; Storz, J; Strzys, M; Takalo, L; Takami, H; Tavecchio, F; Temnikov, P; Terzić, T; Tescaro, D; Teshima, M; Thaele, J; Tibolla, O; Torres, D F; Toyama, T; Treves, A; Uellenbeck, M; Vogler, P; Zanin, R; Pérez-Torres, M A

    2014-01-01

    The pulsar wind nebula (PWN) 3C 58 is one of the historical very-high-energy (VHE; E>100 GeV) gamma-ray source candidates. It is energized by one of the highest spin-down power pulsars known (5% of Crab pulsar) and it has been compared to the Crab Nebula due to their morphological similarities. This object was previously observed by imaging atmospheric Cherenkov telescopes (Whipple, VERITAS and MAGIC), although not detected, with an upper limit of 2.4% Crab Unit (C.U.) at VHE. It was detected by Fermi-LAT with a spectrum extending beyond 100 GeV. We analyzed 81 hours of 3C 58 data taken with the MAGIC telescopes and we detected VHE gamma-ray emission with a significance of 5.7 sigma and an integral flux of 0.65% C.U. above 1 TeV. The differential energy spectrum between 400 GeV and 10 TeV is well described by a power-law function d\\phi/dE=f_0(E/1TeV)^{-Gamma} with f_0=(2.0\\pm0.4_{stat}\\pm0.6_{sys})\\times10^{-13}cm^{-2}s^{-1}TeV^{-1} and Gamma=2.4\\pm0.2_{stat}\\pm0.2_{sys}. The skymap is compatible with an unre...

  17. Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

    Directory of Open Access Journals (Sweden)

    Julien Lang

    2014-10-01

    Full Text Available By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour, a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D of 0.6 µM greater than that for nopaline (KD of 3.7 µM. Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to

  18. Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

    Science.gov (United States)

    Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

    2014-10-01

    By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche

  19. 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.

  20. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    OpenAIRE

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes...

  1. 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

  2. Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants.

    Directory of Open Access Journals (Sweden)

    Abhilash Padavannil

    2014-03-01

    Full Text Available The Type IV Secretion System (T4SS is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP is a key cytoplasmic protein that recruits the VirD2-T-DNA complex to the VirD4-coupling protein (VirD4 CP of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. Unmasking host and microbial strategies in the Agrobacterium-plant defense tango.

    Science.gov (United States)

    Hwang, Elizabeth E; Wang, Melinda B; Bravo, Janis E; Banta, Lois M

    2015-01-01

    Coevolutionary forces drive adaptation of both plant-associated microbes and their hosts. Eloquently captured in the Red Queen Hypothesis, the complexity of each plant-pathogen relationship reflects escalating adversarial strategies, but also external biotic and abiotic pressures on both partners. Innate immune responses are triggered by highly conserved pathogen-associated molecular patterns, or PAMPs, that are harbingers of microbial presence. Upon cell surface receptor-mediated recognition of these pathogen-derived molecules, host plants mount a variety of physiological responses to limit pathogen survival and/or invasion. Successful pathogens often rely on secretion systems to translocate host-modulating effectors that subvert plant defenses, thereby increasing virulence. Host plants, in turn, have evolved to recognize these effectors, activating what has typically been characterized as a pathogen-specific form of immunity. Recent data support the notion that PAMP-triggered and effector-triggered defenses are complementary facets of a convergent, albeit differentially regulated, set of immune responses. This review highlights the key players in the plant's recognition and signal transduction pathways, with a focus on the aspects that may limit Agrobacterium tumefaciens infection and the ways it might overcome those defenses. Recent advances in the field include a growing appreciation for the contributions of cytoskeletal dynamics and membrane trafficking to the regulation of these exquisitely tuned defenses. Pathogen counter-defenses frequently manipulate the interwoven hormonal pathways that mediate host responses. Emerging systems-level analyses include host physiological factors such as circadian cycling. The existing literature indicates that varying or even conflicting results from different labs may well be attributable to environmental factors including time of day of infection, temperature, and/or developmental stage of the host plant. PMID:25873923

  9. 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

  10. 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.

  11. 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.

  12. Biodegradation of crystal violet by Agrobacterium radiobacter.

    Science.gov (United States)

    Parshetti, G K; Parshetti, S G; Telke, A A; Kalyani, D C; Doong, R A; Govindwar, S P

    2011-01-01

    Agrobacterium radiobacter MTCC 8161 completely decolorized the Crystal Violet with 8 hr (10 mg/L) at static anoxic conditions. The decreased decolorization capability by A. radiobacter was observed, when the Crystal Violet concentration was increased from 10 to 100 mg/L. Semi-synthetic medium containing 1% yeast extract and 0.1% NH4C1 has shown 100% decolorization of Crystal Violet within 5 hr. A complete degradation of Crystal Violet by A. radiobacter was observed up to 7 cycles of repeated addition (10 mg/L). When the effect of increasing inoculum concentration on decolorization of Crystal Violet (100 mg/L) was studied, maximum decolorization was observed with 15% inoculum concentration. A significant increase in the activities of laccase (184%) and aminopyrine N-demethylase (300%) in cells obtained after decolorization indicated the involvement of these enzymes in decolorization process. The intermediates formed during the degradation of Crystal Violet were analyzed by gas chromatography and mass spectroscopy (GC/MS). It was detected the presence of N,N,N',N"-tetramethylpararosaniline, [N, N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone, N, N-dimethylaminobenzaldehyde, 4-methyl amino phenol and phenol. We proposed the hypothetical metabolic pathway of Crystal Violet biodegradation by A. radiobacter. Phytotoxicity and microbial toxicity study showed that Crystal Violet biodegradation metabolites were less toxic to bacteria (A. radiobacter, P. aurugenosa and A. vinelandii) contributing to soil fertility and for four kinds of plants (Sorghum bicolor Vigna radiata, Lens culinaris and Triticum aestivum) which are most sensitive, fast growing and commonly used in Indian agriculture. PMID:22128547

  13. Characterization of Agrobacterium species by capillary isoelectric focusing

    Czech Academy of Sciences Publication Activity Database

    Süle, S.; Horká, Marie; Matoušková, H.; Kubesová, Anna; Šalplachta, Jiří; Horký, J.

    2012-01-01

    Roč. 132, č. 1 (2012), s. 81-89. ISSN 0929-1873 R&D Projects: GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : capillary isoelectric focusing * Agrobacterium spp. * identification Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.610, year: 2012

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

    Directory of Open Access Journals (Sweden)

    von Arnim Albrecht G

    2009-05-01

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

  15. Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

    OpenAIRE

    Hoshino, Yoichiro; Kashihara, Yukiko; Hirano, Tomonari; MURATA, Naho; Shinoda, Koichi

    2008-01-01

    Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 d after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloro...

  16. 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.

  17. Construction and Application of R Prime Plasmids, Carrying Different Segments of an Octopine Ti Plasmid from Agrobacterium tumefaciens, for Complementation of vir Genes

    NARCIS (Netherlands)

    Hille, Jacques; Klasen, Ina; Schilperoort, Rob

    1982-01-01

    Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with T

  18. 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....

  19. 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...

  20. Synthesis of Methylerythritol Phosphate Analogues and Their Evaluation as Alternate Substrates for IspDF and IspE from Agrobacterium tumefaciens

    OpenAIRE

    Krasutsky, Sergiy G.; Urbansky, Marek; Davis, Chad E.; Lherbet, Christian; Coates, Robert M.; Poulter, C. Dale

    2014-01-01

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. d-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMA...

  1. Hypericin and hyperforin production in St. John’s wort in vitro culture: Influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens

    Czech Academy of Sciences Publication Activity Database

    Pavlík, M.; Vacek, Jan; Klejdus, B.; Kubáň, V.

    2007-01-01

    Roč. 55, č. 15 (2007), s. 6147-6153. ISSN 0021-8561 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hypericin * hyperforin * saccharose Subject RIV: BO - Biophysics Impact factor: 2.532, year: 2007

  2. 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

  3. fRNAdb Summary: FR364279 [

    Lifescience Database Archive (English)

    Full Text Available FR364279 AE007948,AE008167,AE008265,AE008290,AE008980,AE009201,AE009325,AE009348,AF345265,AF5108 ... 0 transfer RNA (tRNA), GAT (Ile/I) Isoleucine tRNA Rhizobium ... galegae,Agrobacterium tumefaciens str. C58,Rhizobi ... um sp. Glm-16,Rhizobium ... sp. Gls-3 RF00005 Rfam v8.1 FR364279.jpg FR364279. ...

  4. 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.

  5. 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.

  6. Recombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure d-Amino Acids▿ †

    OpenAIRE

    Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Clemente-Jiménez, Josefa María; Pozo-Dengra, Joaquín; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

    2007-01-01

    Two recombinant reaction systems for the production of optically pure d-amino acids from different d,l-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were d-hydantoinase and d-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one p...

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  11. DISCOVERY OF PULSATIONS FROM THE PULSAR J0205+6449 IN SNR 3C 58 WITH THE FERMI GAMMA-RAY SPACE TELESCOPE

    International Nuclear Information System (INIS)

    We report the discovery of γ-ray pulsations (≥0.1 GeV) from the young radio and X-ray pulsar PSR J0205 + 6449 located in the Galactic supernova remnant 3C 58. Data in the γ-ray band were acquired by the Large Area Telescope aboard the Fermi Gamma-ray Space Telescope (formerly GLAST), while the radio rotational ephemeris used to fold γ-rays was obtained using both the Green Bank Telescope and the Lovell telescope at Jodrell Bank. The light curve consists of two peaks separated by 0.49 ± 0.01 ± 0.01 cycles which are aligned with the X-ray peaks. The first γ-ray peak trails the radio pulse by 0.08 ± 0.01 ± 0.01, while its amplitude decreases with increasing energy as for the other γ-ray pulsars. Spectral analysis of the pulsed γ-ray emission suggests a simple power law of index -2.1 ± 0.1 ± 0.2 with an exponential cutoff at 3.0+1.1-0.7 ± 0.4 GeV. The first uncertainty is statistical and the second is systematic. The integral γ-ray photon flux above 0.1 GeV is (13.7 ± 1.4 ± 3.0) x 10-8 cm-2 s-1, which implies for a distance of 3.2 kpc and assuming a broad fan-like beam a luminosity of 8.3 x 1034 erg s-1 and an efficiency η of 0.3%. Finally, we report a 95% upper limit on the flux of 1.7 x 10-8 cm-2 s-1 for off-pulse emission from the object.

  12. Discovery of Pulsations from the Pulsar J0205 6449 in SNR 3C 58 with the Fermi Gamma-Ray Space Telescope

    International Nuclear Information System (INIS)

    We report the discovery of γ-ray pulsations ((ge)0.1 GeV) from the young radio and X-ray pulsar PSR J0205 + 6449 located in the Galactic supernova remnant 3C 58. Data in the γ-ray band were acquired by the Large Area Telescope aboard the Fermi Gamma-ray Space Telescope (formerly GLAST), while the radio rotational ephemeris used to fold γ-rays was obtained using both the Green Bank Telescope and the Lovell telescope at Jodrell Bank. The light curve consists of two peaks separated by 0.49 ± 0.01 ± 0.01 cycles which are aligned with the X-ray peaks. The first γ-ray peak trails the radio pulse by 0.08 ± 0.01 ± 0.01, while its amplitude decreases with increasing energy as for the other γ-ray pulsars. Spectral analysis of the pulsed γ-ray emission suggests a simple power law of index -2.1 ± 0.1 ± 0.2 with an exponential cutoff at 3.0-0.7+1.1 ± 0.4 GeV. The first uncertainty is statistical and the second is systematic. The integral γ-ray photon flux above 0.1 GeV is (13.7 ± 1.4 ± 3.0) x 10-8 cm-2 s-1, which implies for a distance of 3.2 kpc and assuming a broad fan-like beam a luminosity of 8.3 x 1034 erg s-1 and an efficiency η of 0.3%. Finally, we report a 95% upper limit on the flux of 1.7 x 10-8 cm-2 s-1 for off-pulse emission from the object.

  13. Effects of Agrobacterium tumefac iens on the Symptoms of Paulownia sp. Plantlet in Vitro Cultured%根癌农杆菌对感染植原体的泡桐组培苗症状的影响

    Institute of Scientific and Technical Information of China (English)

    田国忠; 朱水芳; 罗飞; 李怀方; 裘维蕃

    2001-01-01

    采用含有激素合成相关基因的根癌农杆菌,伤口接种已感染植原体的泡桐丛枝组培苗和健康组培苗,结果发现对丛枝苗的致瘤能力明显低于健康对照苗,且被接种病苗的丛枝症状缓解.从健苗获得的T-DNA转化泡桐瘤组织细胞能在无激素培养基上稳定生长和连续继代培养2年以上,说明瘤组织细胞自身已获得了细胞分裂素和生长素合成能力.根据已报道的根癌农杆菌株系pTil 5955 T-DNA的异戊烯基转移酶基因(ipt)的保守序列,设计了一对引物(CYT和CYT′),用多聚酶链式反应(PCR)扩增了我国杨树致瘤农杆菌ipt基因部分序列(427 bp片段),也从遗传转化的两个泡桐无性系瘤组织At-ZH和At-T35扩增出此特异片段,从而进一步肯定了T-DNA已被整合到泡桐的染色体上,表明泡桐易于通过Ti质粒载体途径进行基因转移操作,但用此引物未能从泡桐、甘薯健株和感染植原体的组培病苗扩增出相应的427 bp特异片段.当用此遗传转化瘤组织嫁接病苗时,可减轻丛枝症状的严重度,延长病苗的存活时间和诱导病株生根,这进一步证实了泡桐在与植原体相互作用过程中激素代谢发生了变化.%By using Agrobacterium tumefac iens isolated from poplar crown gall disease with the hormone-produ cing genes in the T-DNA to inoculate healthy and infected Paulownia plantlets with phytoplasma, it is showed that tumorigensis of dise ased plantlets dropped apparently and the symptoms of witches' broom suppressed to some extent. The T-DNA was transformed into Paulownia resulting in tumor formation independent of exogenous hormone addition and ke eping subculture of tumor tissues for more than 2 years, thus confirming that th e tumor tissues gained the ability to synthesize cytokinin and auxin by itself. Based on the conserved sequence of isopentenyl adenosine transferase gene (ipt) of Agrobacterium tumefaciens Op ine pTil 5955 strain, a pair of DNA

  14. Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6.

    Science.gov (United States)

    McBride, K E; Knauf, V C

    1988-04-01

    The virE operon of the Agrobacterium tumefaciens Ti plasmid pTiA6 encodes at least one trans-acting protein involved in the expression of virulence. Two open reading frames designated virE1 and virE2 code for polypeptides of 7 and 60 kilodaltons (kDa), respectively, that can be visualized after expression in Escherichia coli minicells. To determine which virE sequences are required for virulence, a strain deleted for the entire locus [strain KE1(pTiA6 delta E)] was constructed and tested for the ability to be complemented by subclones with and without site-directed mutations in the virE operon. One subclone containing only virE1 and virE2 as well as upstream promoter sequences was sufficient to restore full virulence on the host plant Kalanchoe daigremontiana. However, some other virulence locus representing a host range determinant appeared to be deleted from strain KE1(pTiA6 delta E), since virE1 and virE2 were not sufficient to fully restore virulence on wounded tomato plants. virE operon constructs with specific lesions in either virE1 or virE2 were impaired for complementation of pTiA6 delta E. Several mutations specific for the promoter-proximal virE1 locus appeared to have a polar effect on expression of the virE2-encoded 60-kDa protein. However, virE2::lacZ fusion constructs suggest that this effect is not at the level of transcription or translation. Collectively, these data indicate that both the 7- and the 60-kDa polypeptides are virulence determinants for the Ti plasmid pTiA6 and suggest that the 60-kDa protein may be less stable in the absence of the 7-kDa protein. PMID:2832362

  15. Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency

    OpenAIRE

    Carlos Henrique S. Carvalho; Usha B. Zehr; Nilupa Gunaratna; Joseph Anderson; Halina H. Kononowicz; Thomas K. Hodges; Axtell, John D.

    2004-01-01

    The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our prot...

  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. 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.

  18. 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 ...

  19. Expressão eficiente do gene reporter beta-glucuronidase nos tecidos vasculares de batata (Solanum tuberosum L. utilizando de um promotor específico (BRA3 de Agrobacterium rhizogenes Efficient expression of beta-glucuronidase reporter gene in vascular tissue of potato (Solanum tuberosum L. utilizing a specific promoter (BRA3 from Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Antonio Carlos Torres

    2003-06-01

    Full Text Available Promotores tecido-específico controlam a transcrição de genes em diferentes tecidos vegetais bem como em diferentes estádios de desenvolvimento da planta, levando à indução de distintos níveis de atividade transiente e/ou estável do gene. Tais promotores podem ser empregados para a expressão seletiva de genes de interesse. O promotor rol A de Agrobacterium rhizogenes, por exemplo, é floema-específico, sugerindo que possa ser empregado em estratégias de defesa de plantas que são infectadas por vírus com replicação restrita ao floema. A expressão do gene marcador da ß-glucuronidase (gus dirigido pelo promotor rol A (pBRA3 foi observada em plantas transgênicas de batata (cvs. Macaca e Baronesa. Entrenós e secções de folhas foram submetidos ao cocultivo com A. tumefaciens. A atividade do gene gus avaliada em brotações resistentes à canamicina não se restringiu ao floema (alto nível de expressão do gene, mas também se manifestou no xilema dos caules. As expressões transiente e estável são, no entanto, tecido-específicas, localizadas sobretudo no sistema vascular de entrenós e ausente em raízes e folhas. As plantas gus positivas foram micropropagadas, plantadas em casa de vegetação e avaliadas por PCR, utilizando-se 'primers' específicos para o gene npt II. Nenhuma alteração fenotípica foi observada em plantas transgênicas, em relação às não transformadas.Tissue-especific promoters allow the modulation of gene transcription in different tissue types as well as in different stages of plant development, leading different levels of transient and stable activity of the gene product. These promoters have been employed for selective gene expression. The Agrobacterium rhizogenes rol A gene promoter (BRA3 controls phloem-specific expression indicating that this promoter might have an important role in plant defense strategies against virus which replicated only in the phloem. The expression of

  20. 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

  1. Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection.

    Science.gov (United States)

    Noda, T; Tanaka, N; Mano, Y; Nabeshima, S; Ohkawa, H; Matsui, C

    1987-07-01

    Surface-sterilized leaf disks of horse-radish (Armoracia lapathifolia) were immersed in a suspension of Agrobacterium rhizogenes harboring the root-inducing plasmid (pRi) and cultured on a solid medium. Within about 10 days after inoculation, adventitious roots (hairy roots) emerged from the leaf disks. No roots emerged from the uninoculated leaf disks. The excised hairy roots grew vigorously in the dark and exhibited extensive lateral branches in the absence of phytohormones. When the hairy roots were moved into the light, numerous adventitious buds thrust out of the roots within about 10 days, and they developed into complete plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of cultured R0 plants were of two types. One had fibrous roots only and the other had both fibrous and tuberous roots Leaf disks of the R0 plants proliferated adventitious roots (R1 generation) on a solid medium after 1-2 weeks of culture. Phenotypical characters of the R1 roots were the same as those observed with the initial hairy roots. The T-DNA sequences of pRi were detected within DNA isolated from the hairy roots and their regenerants. PMID:24248760

  2. Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency

    Directory of Open Access Journals (Sweden)

    Carlos Henrique S. Carvalho

    2004-01-01

    Full Text Available The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. Immature sorghum embryos proved to be very sensitive to Agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving transformation efficiency. The addition of coconut water to the co-cultivation medium, the use of vigorous and actively growing immature embryos and the removal of excess bacteria significantly improved the survival rate of sorghum embryos and was critical for successful transformation. Hygromycin phosphotransferase (hpt proved to be a good selectable marker for sorghum. We also found that b-glucuronidase (GUS activity was low in most of the transgenic plant tissues tested, although it was very high in immature inflorescences. Although promising, the overall transformation efficiency of the protocol is still low and further optimization will require particular attention to be given to the number of Agrobacterium in the inoculum and the selection of sorghum genotypes and explants less sensitive to Agrobacterium infection.

  3. 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;

    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...

  4. 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

    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......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...

  5. 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

  6. Producción de plantas transgénicas de Solanum phureja variedad yema de huevo clon 1 mediada por Agrobacteriun tumefaciens

    Directory of Open Access Journals (Sweden)

    Chaparro Giraldo Alejandro

    2006-12-01

    Full Text Available El objetivo general de este proyecto fue la producción de plantas transformadas genéticamente de S. phureja
    variedad Yema de Huevo Clon 1 que sean potencialmente tolerantes a insectos, rasgo conferido por la inserción
    en su genoma del gen mirl2 derivado del pomelo (Citrus paradisi que codifica para un inhibidor de proteasas de
    tipo Serina. Se trabajó con el sistema de transformación mediado por Agrobacterium tumefaciens utilizando la cepa
    LBA4404 que contiene el plásmido residente pAL4404 y el vector binario pNOV022 el cual contiene el gen de interés mirl2. Se utilizaron como explantes entrenudos con una longitud de 0,5 a 1 cm provenientes de material in
    vitro entre cuatro a cinco semanas de edad, los cuales fueron co-cultivados en una concentración bacteriana de
    1/50, para posteriormente ser colocados en medio de regeneración sin agente de selección. Se encontró que el
    medio de regeneración más apropiado esta compuesto por Zeatina Ribósido (ZR 2 mg/L, ácido naftalenacético
    (ANA 0,04 mg/L y ácido giberélico (AG3 0,1 mg/L en el cual se logró obtener un 87,6% de callogénesis y un
    48,6% de regeneración. Posteriormente se estableció una población de individuos potencialmente transgénicos,
    a partir de los cuales se realizó una extracción de DNA, organizando los extractos en pools de diez individuos,
    obteniéndose un total de 52 pools, a partir de 520 individuos. Se utilizaron cuatro diferentes cantidades de DNA para un volumen final de 50 μl en la PCR: 150 ng; 300 ng; 600 ng; 1.200 ng y se realizó PCR para los dos genes mirl2 (IP y manA (PMI. Tras el análisis por electrofóresis de los resultados de la PCR para cada uno de los pools no se observó la señal de amplificación específica de los genes de interés, en tanto que se observó la señal para los genes endógenos de control. Finalmente se analizaron 62 clones provenientes de un segundo ensayo de transformaci

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Yuying Jia

    2015-08-01

    Full Text Available 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.

  9. 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.

  10. Organogênese e transformação genética de maracujá amarelo (Passiflora edulis f. flavicarpa Deg.) com os genes CMe-ACO1 AS e nptll via Agrobacterium tumefaciens

    OpenAIRE

    Winkler, Larissa Macedo

    2013-01-01

    O Brasil é o principal produtor mundial de maracujá-amarelo (Passiflora edulis f. flavicarpa Deg.). Seus frutos, de grande valor nutricional e farmacêutico são comercializados in natura e industrializados, garantindo o abastecimento do mercado interno e exportação. Contudo, seus frutos perdem grande quantidade de água durante o processo de maturação, conferindo à eles um aspecto de murcha em poucos dias, além de aumentar a fragilidade durante o transporte. Assim, o presente trabalho teve como...

  11. 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.

  12. 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.

  13. Tumorogênese em plantas causadas por espécies de Agrobacterium Tumorigenesis in plants induced by species of Agrobacterium

    OpenAIRE

    Reginaldo da Silva Romeiro; José Roberto Vieira Júnior; Sérgio Hermínio Brommonschenkel

    2007-01-01

    Tumores - sintomas hiperplásicos em plantas - incitados por espécies de Agrobacterium sp. sempre exerceram fascínio sobre fitopatologistas desde o início do Século XX, quando Erwin Smith e colaboradores demonstraram serem eles de etiologia bacteriana. No início, imaginava-se que os tumores eram decorrentes de alterações hormonais na planta provocadas pela bactéria. Contudo, até recentemente, a microbiologia e a biologia molecular não eram suficientemente avançadas para que os cientistas pudes...

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Agrobacterium mediated transformation in Rubia cordifolia for obtaining hairy root producing Anthraquinone dye

    OpenAIRE

    Siva R; Sairam V; Saleel Y; Promit B; Abhishek A.; Sarang H; Abhishek K; Alifiya P; Anshul G; Siddharth D

    2015-01-01

    Rubia cordifolia is known to contain substantial amount of secondary metabolites such as anthraquinones and dye, especially in the roots. This plant was infected with Agrobacterium rhizogenes to transfer the genes rolA, rolB, rolC into the plant genome. It causes tumour formation and hairy root disease in the plant. Thus, it was found that the production of anthraquinones was increased after transfection. In further studies, anthraquinones can be isolated from these roots. There are many appl...

  19. Degradation of the Ferric Chelate of EDTA by a Pure Culture of an Agrobacterium sp

    OpenAIRE

    Lauff, John J.; Steele, D. Bernie; Coogan, Louise A.; Breitfeller, James M.

    1990-01-01

    A pure culture of an Agrobacterium sp. (deposited as ATCC 55002) that mineralizes the ferric chelate of EDTA (ferric-EDTA) was isolated by selective enrichment from a treatment facility receiving industrial waste containing ferric-EDTA. The isolate grew on ferric-EDTA as the sole carbon source at concentrations exceeding 100 mM. As the degradation proceeded, carbon dioxide, ammonia, and an unidentified metabolite(s) were produced; the pH increased, and iron was precipitated from solution. The...

  20. 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

  1. AGROBACTERIUM-MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

    Science.gov (United States)

    Kathiresan, S; Chandrashekar, A; Ravishankar, G A; Sarada, R

    2009-06-01

    The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed β-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries. PMID:27034041

  2. 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

  3. Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum.

    Science.gov (United States)

    Ooi, Chai Theam; Syahida, Ahmad; Stanslas, Johnson; Maziah, Mahmood

    2013-03-01

    This article presents the abilities and efficiencies of five different strains of Agrobacterium rhizogenes (strain ATCC 31798, ATCC 43057, AR12, A4 and A13) to induce hairy roots on Solanum mammosum through genetic transformation. There is significant difference in the transformation efficiency (average number of days of hairy root induction) and transformation frequency for all strains of A. rhizogenes (P ammonium nitrate and potassium nitrate and 5 % (w/v) sucrose, had exhibited improvement in growth index, that is, the fresh biomass was almost double as compared to its initial growth in unmodified half-strength MS medium. PMID:23090845

  4. 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

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. Agrobacterium mediated transformation in Rubia cordifolia for obtaining hairy root producing Anthraquinone dye

    Directory of Open Access Journals (Sweden)

    Siva R

    2015-05-01

    Full Text Available Rubia cordifolia is known to contain substantial amount of secondary metabolites such as anthraquinones and dye, especially in the roots. This plant was infected with Agrobacterium rhizogenes to transfer the genes rolA, rolB, rolC into the plant genome. It causes tumour formation and hairy root disease in the plant. Thus, it was found that the production of anthraquinones was increased after transfection. In further studies, anthraquinones can be isolated from these roots. There are many applications of the secondary metabolites produced by plants.

  10. Tumor induction by Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome

    OpenAIRE

    White, Frank F; Ghidossi, Gina; Gordon, Milton P.; Nester, Eugene W.

    1982-01-01

    The DNA from tumors of Nicotiana glauca initiated by strains of Agrobacterium rhizogenes was shown to contain sequences that are homologous to the root-inducing (Ri) plasmid of the bacterium. Two independently established tumor lines contained a similar portion of the Ri-plasmid. The Ri-plasmid also hybridized to DNA fragments from uninfected N. glauca. A cosmid clone of the Ri-plasmid encompassing the region containing the Ri-plasmid sequences that are stably transferred to the plant also hy...

  11. Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

    OpenAIRE

    Rink, R; Fennema, M.; Smids, M; Dehmel, U; Janssen, DB

    1997-01-01

    The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme, The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa, An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A, radiobacte...

  12. 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.

  13. 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.

  14. 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%.

  15. Tumorogênese em plantas causadas por espécies de Agrobacterium Tumorigenesis in plants induced by species of Agrobacterium

    Directory of Open Access Journals (Sweden)

    Reginaldo da Silva Romeiro

    2007-03-01

    Full Text Available Tumores - sintomas hiperplásicos em plantas - incitados por espécies de Agrobacterium sp. sempre exerceram fascínio sobre fitopatologistas desde o início do Século XX, quando Erwin Smith e colaboradores demonstraram serem eles de etiologia bacteriana. No início, imaginava-se que os tumores eram decorrentes de alterações hormonais na planta provocadas pela bactéria. Contudo, até recentemente, a microbiologia e a biologia molecular não eram suficientemente avançadas para que os cientistas pudessem compreender e deduzir a forma através da qual o patógeno incitava os tumores. Demorou quase um século para que se deslindassem os complexos mecanismos bioquímicos, genéticos e fi­siológicos através dos quais o patógeno transforma a planta, inserindo no genoma desta uma região de seu megaplasmídeo de modo a criar para si mesmo um nicho ecológico espe­cífico. Neste trabalho é apresentada uma súmula histórica da evolução do conhecimento a respeito, das características genômicas do plasmídeo Ti, dos eventos e requerimentos ati­nentes ao processo infectivo bem como é discutida a dinâmica da transformação da planta pelo patógeno.Tumors - the plant hyperplasia symptoms- - induced by species of Agrobacterium sp. have deeply impressed plant pathologists since early 20th Century when Erwin Smith and his co-workers demonstrated that such tumors had a bacterial etiology. Nevertheless, until recently the state of art of Microbiology and Molecular Biology was not developed enough for scientists to realize and to elucidate the complexes biochemical, genetic and physiologic mechanisms by which the pathogen transforms the plant by inserting a region of its own plasmid into the genome of the latter, creating an specific ecological niche for itself. In this paper its is showed a historical brief on the evolution of knowledge about the genomic characteristics of the Ti Plasmid, events and requirements needed for infection to take

  16. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  17. 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.

  18. 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.

  19. 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.

  20. Agrobacterium phytochrome as an enzyme for the production of ZZE bilins.

    Science.gov (United States)

    Lamparter, Tilman; Michael, Norbert

    2005-06-14

    Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2. PMID:15938635

  1. Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids.

    Science.gov (United States)

    Boulanger, F; Berkaloff, A; Richaud, F

    1986-07-01

    Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the TL-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the TL-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the TL-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the TL-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the TL-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114. PMID:24307326

  2. Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

    Directory of Open Access Journals (Sweden)

    Ruffing Anne M

    2012-02-01

    Full Text Available Abstract Background The ability to synthesize exopolysaccharides (EPS is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery. Results We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis. Conclusions This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

  3. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol

    Directory of Open Access Journals (Sweden)

    Ülker Bekir

    2006-10-01

    Full Text Available Abstract Background The Agrobacterium vacuum (Bechtold et al 1993 and floral-dip (Clough and Bent 1998 are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown. Results To avoid problems associated with large bacterial liquid cultures, we investigated whether bacteria grown on plates are also suitable for plant transformation. We demonstrate here that bacteria grown on plates can be used with similar efficiency for transforming plants even after one week of storage at 4°C. This makes it much easier to synchronize Agrobacterium and plants for transformation. DNA gel blot analysis was carried out on the T1 plants surviving the herbicide selection and demonstrated that the surviving plants are indeed transgenic. Conclusion The simplified method works as efficiently as the previously reported protocols and significantly reduces the workload, cost and time. Additionally, the protocol reduces the risk of large scale contaminations involving GMOs. Most importantly, many more independent transformations per day can be performed using this modified protocol.

  4. 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。

  5. An efficient protocol for Agrobacterium-mediated transformation of the biofuel plant Jatropha curcas by optimizing kanamycin concentration and duration of delayed selection

    OpenAIRE

    Fu, Qiantang; Li, Chaoqiong; Tang, Mingyong; Tao, Yan-Bin; Pan, Bang-Zhen; Zhang, Lu; Niu, Longjian; He, Huiying; Wang, Xiulan; Xu, Zeng-Fu

    2015-01-01

    Jatropha curcas is considered a potential biodiesel feedstock crop. Currently, the value of J. curcas is limited because its seed yield is generally low. Transgenic modification is a promising approach to improve the seed yield of J. curcas. Although Agrobacterium-mediated genetic transformation of J. curcas has been pursued for several years, the transformation efficiency remains unsatisfying. Therefore, a highly efficient and simple Agrobacterium-mediated genetic transformation method for J...

  6. Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation.

    Science.gov (United States)

    Polito, V S; McGranahan, G; Pinney, K; Leslie, C

    1989-04-01

    Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants. PMID:24233141

  7. Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

    Czech Academy of Sciences Publication Activity Database

    Konieczny, R.; Obert, B.; Bleho, J.; Novák, Ondřej; Heym, C.; Tuleja, M.; Mueller, J.; Strnad, Miroslav; Menzel, D.; Šamaj, J.

    2011-01-01

    Roč. 168, č. 7 (2011), s. 722-729. ISSN 0176-1617 R&D Projects: GA ČR GA301/08/1649; GA MŠk ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Agrobacterium * Callus * Common ice plant * Cytokinin content * GFP Subject RIV: EF - Botanics Impact factor: 2.791, year: 2011

  8. 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...

  9. 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

  10. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

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    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.

  11. 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)

  12. An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes.

    Science.gov (United States)

    Park, Nam Il; Tuan, Pham Anh; Li, Xiaohua; Kim, Yong Kyoung; Yang, Tae Jin; Park, Sang Un

    2011-04-01

    The balloon flower (Platycodon grandiflorum) is a popular traditional medicinal plant used in Korea to treat conditions such as bronchitis, asthma, tuberculosis, diabetes, and inflammatory diseases. Recently, immunopharmacological research identified triterpenoid and saponin as important active compounds in P. grandiflorum. To study and extract these compounds and other metabolites from P. grandiflorum, a technique was developed for producing hairy root cultures, which are a reliable source of plant compounds. To achieve this, the activity of Agrobacterium rhizogenes was exploited, which can transfer DNA segments into plant genomes after infecting them. In this study, the A. rhizogenes strain R1000 was determined that had the highest infection frequency (87.5%) and induced the most hairy roots per plant, and the concentration of antibiotics (75 mg/l kanamycin) was elucidated for selection after transformation. Wild-type and transgenic hairy roots contained various phenolic compounds, although both of them had similar concentrations of phenolic compounds. In the future, the protocols described here should be useful for studying and extracting valuable metabolites such as phenolic compounds from P. grandiflorum hairy root cultures. PMID:21052843

  13. Effective Immobilization of Agrobacterium sp. IFO 13140 Cells in Loofa Sponge for Curdlan Biosynthesis

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    Camila Ortiz Martinez

    2015-05-01

    Full Text Available Curdlan production by Agrobacterium sp. IFO13140 immobilized on loofa sponge, alginate and loofa sponge with alginate was investigated. There was no statistically-significant difference in curdlan production when the microorganism was immobilized in different matrices. The loofa sponge was chosen because of its practical application and economy and because it provides a high stability through its continued use. The best conditions for immobilization on loofa sponge were 50 mg of cell, 200 rpm and 72 h of incubation, which provided a curdlan production 1.50-times higher than that obtained by free cells. The higher volumetric productivity was achieved by immobilized cells (0.09 g/L/h at 150 rpm. The operating stability was evaluated, and until the fourth cycle, immobilized cells retained 87.40% of the production of the first cycle. The immobilized cells remained active after 300 days of storage at 4 °C. The results of this study demonstrate success in immobilizing cells for curdlan biosynthesis, making the process potentially suitable for industrial scale-up. Additional studies may show a possible contribution to the reduction of operating costs.

  14. A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

    Science.gov (United States)

    Warren, Jeremy G; Kasun, George W; Leonard, Takara; Kirkpatrick, Bruce C

    2016-05-01

    Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity. PMID:26177065

  15. Key Amino Acids in the Bacterial (6-4 Photolyase PhrB from Agrobacterium fabrum.

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    Dominik Graf

    Full Text Available Photolyases can repair pyrimidine dimers on the DNA that are formed during UV irradiation. PhrB from Agrobacterium fabrum represents a new group of prokaryotic (6-4 photolyases which contain an iron-sulfur cluster and a DMRL chromophore. We performed site-directed mutagenesis in order to assess the role of particular amino acid residues in photorepair and photoreduction, during which the FAD chromophore converts from the oxidized to the enzymatically active, reduced form. Our study showed that Trp342 and Trp390 serve as electron transmitters. In the H366A mutant repair activity was lost, which points to a significant role of His366 in the protonation of the lesion, as discussed for the homolog in eukaryotic (6-4 photolyases. Mutants on cysteines that coordinate the Fe-S cluster of PhrB were either insoluble or not expressed. The same result was found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines was mutated and for expression in minimal medium with limited Fe concentrations. We therefore assume that the Fe-S cluster is required for protein stability. We further mutated conserved tyrosines that are located between the DNA lesion and the Fe-S cluster. Mutagenesis results showed that Tyr424 was essential for lesion binding and repair, and Tyr430 was required for efficient repair. The results point to an important function of highly conserved tyrosines in prokaryotic (6-4 photolyases.

  16. Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Lu, Chaofu; Kang, Jinling

    2008-02-01

    Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products. PMID:17899095

  17. In vitro and in vivo toxicity studies of Agrobacterium radiobacter k84 biopolymer (ARB)
    Estudos in vitro e in vivo de toxicidade de biopolímero de Agrobacterium radiobacter k84 (ARB)

    OpenAIRE

    Aparecida Donizette Malvezi; Pedro Sebastião Dionízio Filho; Alexandre Ykuio Saito; Marciane Magnani; Caroline Maria Calliari; Raúl Hernan Castro Gómez

    2011-01-01

    Sugar cane molasses is a cheaper carbon source alternative than glucose traditionally used in fermentation processes. In the present study a biopolymer soluble from Agrobacterium radiobacter k84 (ARB) was obtained by fermentation using sugar cane molasses as a carbon source in a process with yield of 10.0 g.L-1. The ARB is composed by minerals (40%), carbohydrate (35%) and protein (15%). In vitro test of the cytotoxic effect of ARB at concentrations 2.5 mg/mL, 5.0 mg/mL and 10.0 mg/mL in LLC ...

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

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    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.

  19. 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.

  20. Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.

    Science.gov (United States)

    Gao, Xiquan; Shan, Libo

    2013-01-01

    Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses. PMID:23386302

  1. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

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    Praveen Guleria

    Full Text Available BACKGROUND: Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. METHODOLOGY/PRINCIPAL FINDINGS: RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. CONCLUSIONS: SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1996-02-01

    Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

  10. Avaliação da mutagenicidade e antimutagenicidade de um biopolímero extraído do microorganismo Agrobacterium radiobacter em camundongos Swiss Assessment of mutagenicity and antimutagenicity of a biopolymer extracted from the microorganism Agrobacterium radiobacter in mice

    OpenAIRE

    Milka Selestina Primo; Caroline Maria Calliari; Raúl Jorge Hernan Castro-Gómez; Mariana de Oliveira Mauro; Mário Sérgio Mantovani; Rodrigo Juliano Oliveira

    2010-01-01

    A presente pesquisa avaliou a ação mutagênica e antimutagênica de um biopolímero de glucose extraído da Agrobacterium radiobacter (Biopolímero de Agrobacterium radiobacter). O experimento foi realizado com camundongos Swiss machos divididos em oito grupos. O tratamento com o biopolímero foi realizado por gavage em dose única concomitante a uma dose de solução tampão fosfato nos grupos de avaliação da mutagenicidade, ou ao agente indutor de danos no DNA, ciclofosfamida, na concentração de 50 m...

  11. 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.

  12. 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.

  13. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM. PMID:27125317

  14. 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

  15. 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.

  16. 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

  17. Inheritance of rol-genes from Agrobacterium rhizogenes through two generations in Kalanchoë

    DEFF Research Database (Denmark)

    Lütken, Henrik Vlk; Wallström, Saba Victoria; Jensen, Erik Bjørn;

    2012-01-01

    Transformation of Kalanchoë blossfeldiana ‘Molly’ using the naturally occurring bacterium Agrobacterium rhizogenes is a non-GMO strategy to breed compact plants. In the present study, crosses resembling a commercial breeding strategy were made to determine if the improved ornamental quality obser....... Compact potted plants and lines without delayed flowering and with improved ethylene tolerance were obtained and are valuable in commercial breeding programmes without using recombinant DNA technology....

  18. 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...

  19. Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock microbial identification system

    OpenAIRE

    Tighe, S.W.; de Lajudie, Philippe; Dipietro, K.; Lindström, K.; Nick, G; Jarvis, B. D. W.

    2000-01-01

    Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera #Agrobacterium$, #Bradyrhizobium$, #Mesorhizobium$, #Rhizobium$ and #Sinorhizobium$ were evaluated using the gas-chromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that ...

  20. 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

  1. Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

    Science.gov (United States)

    Tavizi, Ashkan; Javaran, Mokhtar Jalali; Moieni, Ahmad; Mohammadi-Dehcheshmeh, Manijeh; Mohebodini, Mehdi; Ebrahimie, Esmaeil

    2015-05-01

    Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry. PMID:25403333

  2. Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.

    Science.gov (United States)

    Bukovinszki, Agnes; Divéki, Zoltán; Csányi, Márta; Palkovics, László; Balázs, Ervin

    2007-04-01

    In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species. PMID:17103215

  3. Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

    Science.gov (United States)

    Shaw; Lehner; Fuhrmann; Kulla; Brass; Birch; Tinschert; Venetz; Venetz; Sanchez; Tonella; Hochstrasser

    1999-06-01

    The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions. PMID:10455485

  4. Crystallization, X-ray diffraction analysis and preliminary structure determination of the polygalacturonase PehA from Agrobacterium vitis

    International Nuclear Information System (INIS)

    The acidic polygalacturonase PehA from A. vitis has been crystallized. A molecular-replacement solution indicated a right-handed parallel β-helix fold. Polygalacturonases are pectate-degrading enzymes that belong to glycoside hydrolase family 28 and hydrolyze the α-1,4 glycosidic bond between neighboring galacturonasyl residues of the homogalacturonan substrate. The acidic polygalacturonase PehA from Agrobacterium vitis was overexpressed in Escherichia coli, where it accumulated in the periplasmic fraction. It was purified to homogeneity via a two-step chromatography procedure and crystallized using the hanging-drop vapour-diffusion technique. PehA crystals belonged to space group P21, with unit-cell parameters a = 52.387, b = 62.738, c = 149.165 Å, β = 89.98°. Crystals diffracted to 1.59 Å resolution and contained two molecules per asymmetric unit. An initial structure determination by molecular replacement indicated a right-handed parallel β-helix fold

  5. In vitro and in vivo toxicity studies of Agrobacterium radiobacter k84 biopolymer (ARBEstudos in vitro e in vivo de toxicidade de biopolímero de Agrobacterium radiobacter k84 (ARB

    Directory of Open Access Journals (Sweden)

    Aparecida Donizette Malvezi

    2011-12-01

    Full Text Available Sugar cane molasses is a cheaper carbon source alternative than glucose traditionally used in fermentation processes. In the present study a biopolymer soluble from Agrobacterium radiobacter k84 (ARB was obtained by fermentation using sugar cane molasses as a carbon source in a process with yield of 10.0 g.L-1. The ARB is composed by minerals (40%, carbohydrate (35% and protein (15%. In vitro test of the cytotoxic effect of ARB at concentrations 2.5 mg/mL, 5.0 mg/mL and 10.0 mg/mL in LLC MK2 (Rhesus Monkey Kidney cells revealed a 50% cytotoxic concentration (CC50 of 9.32 mg/mL. In a 30-day in vivo oral toxicity study, Swiss mice were administered ARB by gavage at 5 mg/mL, 15 mg/mL, 50 mg/mL and 150 mg/mL (approximately 25 mg/kg/day, 75 mg/kg/day, 250 mg/kg/day and 750 mg/kg/day. The results did not present any hematological or histopathological signs of adverse effects, leading us to define the no observed adverse effect level (NOAEL as 150 mg/mL (approximately 750 mg/kg/day.O melaço de cana-de-açúcar é uma fonte de carbono alternativa de menor custo que a glicose tradicionalmente utilizada em processos fermentativos. No presente estudo, um biopolímero solúvel de Agrobacterium radiobacter k84 (ARB foi obtido por fermentação utilizando melaço de cana de açúcar como fonte de carbono em um processo com rendimento de 10,0 g.L-1. O ARB é composto de minerais (40%, carboidratos (35% e proteínas (15%. O teste do efeito citotóxico do ARB in vitro nas concentrações de 2,5 mg/mL, 5,0 mg/mL e 10,0 mg/mL em células LLC MK2 (Rim de Macaco Rhesus revelou uma concentração citotóxica 50% (CC50 de 9,32 mg/mL. Em estudo in vivo de toxicidade oral durante 30 dias, camundongos Swiss receberam por gavagem soluções de ARB nas concentrações de 5 mg/mL, 15 mg/mL, 50 mg/mL e 150 mg/mL (aproximadamente 25 mg/kg/dia, 75 mg/kg/dia, 250 mg/ kg/dia e 750 mg/kg/dia. Os resultados não apresentaram sinais hematológicos ou histopatológicos de efeitos

  6. Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids.

    Science.gov (United States)

    Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Clemente-Jiménez, Josefa María; Pozo-Dengra, Joaquín; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier

    2007-03-01

    Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2. PMID:17220246

  7. Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium

    Science.gov (United States)

    We determined the antibacterial activity of clove oil against seven different genera of plant pathogenic bacteria including Gram-negative Agrobacterium tumefaciens, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv. syringae, Ralstonia solanacearum, and Xanthomonas campestris pv. pelargonii...

  8. Production, characterization and technological properties of biopolymer produced by Agrobacterium radiobacter k84
    Produção, caracterização e propriedades tecnológicas de um biopolímero produzido por Agrobacterium radiobacter k84

    OpenAIRE

    Raúl Jorge Hernan Castro Gómez; Marciane Magnani; Caroline Maria Calliari

    2011-01-01

    In this study, a biopolymer composed of carbohydrates (35%), protein (15%) and minerals (40%) was obtained through fermentation using sugar cane molasses as the sole carbon source for Agrobacterium radiobacter k84. The process yield was 10 gL-1 of biopolymer, which showed high solubility in water, neutral pH in aqueous solution and low water activity (0.52). The analysis in Scanning Electron Microscopy revealed microstructure characteristic of an amorphous solid, with particles of irregular s...

  9. Genome and proteome analysis of 7-7-1, a flagellotropic phage infecting Agrobacterium sp H13-3

    Directory of Open Access Journals (Sweden)

    Kropinski Andrew M

    2012-05-01

    Full Text Available Abstract Background The flagellotropic phage 7-7-1 infects motile cells of Agrobacterium sp H13-3 by attaching to and traveling along the rotating flagellar filament to the secondary receptor at the base, where it injects its DNA into the host cell. Here we describe the complete genomic sequence of 69,391 base pairs of this unusual bacteriophage. Methods The sequence of the 7-7-1 genome was determined by pyro(454sequencing to a coverage of 378-fold. It was annotated using MyRAST and a variety of internet resources. The structural proteome was analyzed by SDS-PAGE coupled electrospray ionization-tandem mass spectrometry (MS/MS. Results Sequence annotation and a structural proteome analysis revealed 127 open reading frames, 84 of which are unique. In six cases 7-7-1 proteins showed sequence similarity to proteins from the virulent Burkholderia myovirus BcepB1A. Unique features of the 7-7-1 genome are the physical separation of the genes encoding the small (orf100 and large (orf112 subunits of the DNA packaging complex and the apparent lack of a holin-lysin cassette. Proteomic analysis revealed the presence of 24 structural proteins, five of which were identified as baseplate (orf7, putative tail fibre (orf102, portal (orf113, major capsid (orf115 and tail sheath (orf126 proteins. In the latter case, the N-terminus was removed during capsid maturation, probably by a putative prohead protease (orf114.

  10. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

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    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.

  11. 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...

  12. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene.

    Science.gov (United States)

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-06-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  13. Genetic transformation of extremophilic fungi Acidea extrema and Acidothrix acidophila

    Czech Academy of Sciences Publication Activity Database

    Hršelová, Hana; Hujslová, Martina; Gryndler, Milan

    2015-01-01

    Roč. 60, č. 4 (2015), s. 365-371. ISSN 0015-5632 R&D Projects: GA ČR(CZ) GAP504/11/0484 Institutional support: RVO:61388971 Keywords : TUMEFACIENS-MEDIATED TRANSFORMATION * AGROBACTERIUM-TUMEFACIENS * FILAMENTOUS FUNGI Subject RIV: EE - Microbiology, Virology Impact factor: 1.000, year: 2014

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

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    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. Inducible expression of p50 from TMV for increased resistance to bacterial crown gall disease in tobacco.

    Science.gov (United States)

    Niemeyer, Julia; Ruhe, Jonas; Machens, Fabian; Stahl, Dietmar J; Hehl, Reinhard

    2014-01-01

    The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements. These promoter::p50 constructs induce local necrotic lesions on NN tobacco plants in an Agrobacterium tumefaciens infiltration assay. A tobacco genotype void of N (nn) was transformed with the promoter::p50 constructs and subsequently crossed to NN plants. Nn F1 offspring selected for the T-DNA develop normally under sterile conditions. After transfer to soil, some of the F1 plants expressing the 2S2D::p50 constructs develop spontaneous necrosis. Transgenic Nn F1 plants with 4D::p50 and 2S2D::p50 expressing constructs upregulate p50 transcription and induce local necrotic lesions in an A. tumefaciens infiltration assay. When leaves and stems of Nn F1 offspring harbouring promoter::p50 constructs are infected with oncogenic A. tumefaciens C58, transgenic lines harbouring the 2S2D::p50 construct induce necrosis and completely lack tumor development. These results demonstrate a successful reprogramming of the viral N gene response against bacterial crown gall disease and highlight the importance of achieving tight regulation of avirulence gene expression and the control of necrosis in the presence of the corresponding resistance gene. PMID:23955710

  16. Production, characterization and technological properties of biopolymer produced by Agrobacterium radiobacter k84Produção, caracterização e propriedades tecnológicas de um biopolímero produzido por Agrobacterium radiobacter k84

    Directory of Open Access Journals (Sweden)

    Raúl Jorge Hernan Castro Gómez

    2011-07-01

    Full Text Available In this study, a biopolymer composed of carbohydrates (35%, protein (15% and minerals (40% was obtained through fermentation using sugar cane molasses as the sole carbon source for Agrobacterium radiobacter k84. The process yield was 10 gL-1 of biopolymer, which showed high solubility in water, neutral pH in aqueous solution and low water activity (0.52. The analysis in Scanning Electron Microscopy revealed microstructure characteristic of an amorphous solid, with particles of irregular shapes and sizes. In the evaluation of technological properties, the biopolymer showed formation of viscous solutions at room temperature from concentration of 0.5% in aqueous solution, gelling activity in solution at 2%, emulsifying (56.11±1.39% and stabilizing activity (98.02±0.78%. The results suggest that the biopolymer from Agrobacterium radiobacter k84 is a promising candidate for industrial use.No presente estudo, utilizando melaço de cana-de-açúcar como única fonte de carbono para Agrobacterium radiobacter k84 foi obtido, em processo fermentativo, um biopolímero composto por carboidratos (35%, proteínas (15% e minerais (40%. O rendimento do processo foi de 10 g.L-1 do biopolímero que apresentou elevada solubilidade em água, pH neutro em solução aquosa e baixa atividade de água (0.52. As análises em Microscopia Eletrônica de Varredura revelaram microestrutura característica de um sólido amorfo, com partículas de formas irregulares e tamanhos variáveis. Na avaliação das propriedades tecnológicas, o biopolímero mostrou viscosidade à temperatura ambiente a partir da concentração 0.5% em solução aquosa, atividade geleificante em solução a 2%, atividade emulsificante (56.11±0.78% e estabilizante (98.02±1.39%. Os resultados sugerem o biopolímero de Agrobacterium radiobacter k84 como um candidato promissor para uso industrial.

  17. 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...

  18. Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation.

    Science.gov (United States)

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

    1989-04-01

    Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of alpha-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis. PMID:16666706

  19. Morfogênese in vitro de variedades brasileiras de cana-de-açúcar In vitro morphogenesis of Brazilian sugarcane varieties

    Directory of Open Access Journals (Sweden)

    Daniela Anhel de Paula Cidade

    2006-03-01

    Full Text Available O objetivo deste trabalho foi estabelecer sistemas de multiplicação de plantas de cana-de-açúcar in vitro e avaliar sua utilização, como material inicial, para a indução de regeneração a partir de ápices caulinares. Três métodos de cultivo foram avaliados: cultura em meio semi-sólido, cultura líquida estacionária e cultura líquida sob agitação. A taxa de multiplicação mais elevada foi alcançada por meio da cultura líquida sob agitação. Ápices caulinares, excisados dessas plantas, apresentaram taxas de regeneração in vitro compatíveis com sua utilização em protocolos de transformação. Calos resistentes a PPT e GUS-positivos foram obtidos de explantes da variedade Chunnee com inoculação de Agrobacterium tumefaciens C58C1 (pMP90 (pDUBarA9. O protocolo estabelecido a partir de cultivo in vitro pode ser utilizado para a produção de plantas transgênicas de cana-de-açúcar, visando à realização de estudos de regulação da expressão gênica, assim como à introdução de características de interesse agronômico.The objective of this work was to establish in vitro systems for sugarcane plant multiplication and for regeneration from shoot apices excised from these plants. Three methods were analyzed: culture on semi-solid medium and liquid culture with or without agitation. The highest regeneration rates were obtained from cultures in liquid-medium with agitation. Shoot tips derived from these plants presented regeneration rates suitable for utilization in transformation protocols. PPT-resistant and GUS positive calluses were obtained from explants of in vitro plants of Chunnee variety inoculated with Agrobacterium tumefaciens C58C1 (pMP90 (pDUBarA9. The established in vitro culture system can be applied for transgenic sugarcane production, aiming at gene regulation studies and introduction of agronomical traits.

  20. 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

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    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.

  1. Avaliação da mutagenicidade e antimutagenicidade de um biopolímero extraído do microorganismo Agrobacterium radiobacter em camundongos Swiss Assessment of mutagenicity and antimutagenicity of a biopolymer extracted from the microorganism Agrobacterium radiobacter in mice

    Directory of Open Access Journals (Sweden)

    Milka Selestina Primo

    2010-07-01

    Full Text Available A presente pesquisa avaliou a ação mutagênica e antimutagênica de um biopolímero de glucose extraído da Agrobacterium radiobacter (Biopolímero de Agrobacterium radiobacter. O experimento foi realizado com camundongos Swiss machos divididos em oito grupos. O tratamento com o biopolímero foi realizado por gavage em dose única concomitante a uma dose de solução tampão fosfato nos grupos de avaliação da mutagenicidade, ou ao agente indutor de danos no DNA, ciclofosfamida, na concentração de 50 mg/kg (peso corpóreo - p.c., nos grupos de avaliação da antimutagenicidade. Utilizou-se o teste de micronúcleo em sangue periférico e a coleta de sangue foi realizada 24 e 48 h após a aplicação das substâncias-teste. A análise estatística demonstrou que o biopolímero não possui atividade mutagênica e que é efetivo em prevenir danos no DNA. As porcentagens de redução de danos nos grupos de antimutagenicidade foram de 83,9%, 89,1% e 103,1% em 24 h e 101,24%, 98,14% e 120,64% em 48 h para as doses de 75, 150 e 300mg/kg (p.c., respectivamente. A alta porcentagem de redução de danos associada à ausência de efeitos mutagênicos indica, além da atividade quimioprotetora, a possibilidade do biopolímero ser um alimento funcional candidato à utilização como co-adjuvante na quimioterapia para prevenir efeitos colaterais.This study evaluated the mutagenic and ant mutagenic action of a biopolymer of glucose extracted from Agrobacterium radiobacter (Biopolymer of Agrobacterium radiobacter. The experiment was conducted with Swiss male mice divided into eight groups. Treatment with the biopolymer was performed in a single dose by gavage at a dose of concomitant phosphate buffer groups in the evaluation of mutagenicity, or the agent of inducing DNA damage, cyclophosphamide, the concentration of 50 mg/kg (body weight --b.w., in groups of assessment ant mutagenic. We used the micronucleus test in peripheral blood. The blood sample was

  2. 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.

  3. 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

  4. Utilization of Trihalogenated Propanes by Agrobacterium radiobacter AD1 through Heterologous Expression of the Haloalkane Dehalogenase from Rhodococcus sp. Strain m15-3

    Science.gov (United States)

    Bosma, Tjibbe; Kruizinga, Edwin; de Bruin, Erik J.; Poelarends, Gerrit J.; Janssen, Dick B.

    1999-01-01

    Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters Plac, PdhlA, and Ptrc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes. PMID:10508091

  5. Resistance to rice blast(Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The gene of trichosanthin has been transferred into rice plants through agrobacterium method.The single copy insertion and the expression of foreign gene have been proved in regenerated plants.In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressing GUS gene as control have been evaluated.The differences such as the time of disease symptom observed,the number of infected plants and damaged leaves,the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant.The transgenic plants with trichosanthin gene grew faster than the plants with GUS gene,even when humidity environment was removed.The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control.In addition,no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.

  6. Enzymological Characterization of Atm, the First Laccase from Agrobacterium sp. S5-1, with the Ability to Enhance In Vitro digestibility of Maize Straw.

    Directory of Open Access Journals (Sweden)

    Wei Si

    Full Text Available Laccase is an enzyme that catalyzes oxidation of phenolic compounds, diamines and aromatic amines. In this study, a novel laccase-like gene (atm in a ligninolyitic isolate Agrobacterium sp. S5-1 from soil humus was identified and heterologously expressed in Escherichia coli. Atm exhibited its maximal activity at pH 4.5 and at 50°C. This enzyme was tolerant to high temperature, a broad range of pH, heavy metal ions (Co3+, Mn2+, Cu2+ and Ni2+, 20 mM and all tested organic solvents. Furthermore, Atm significantly (p<0.05 increased dry matter digestibility of maize straw from 23.44% to 27.96% and from 29.53% to 37.10% after 8 or 24 h of digestion and improved acid detergent fiber digestibility from 5.81% to 10.33% and from 12.80% to 19.07% after 8 or 24 h of digestion, respectively. The combination of Atm and fibrolytic enzymes significantly (p<0.05 enhanced neutral detergent fiber digestibility from 19.02% to 24.55% after 24 h of digestion respectively. Results showed treatment with Atm effectively improved in vitro digestibility of maize straw, thus suggesting that Atm has an application potential for bioconversion of lignin rich agricultural byproducts into animal feed and cellulosic ethanol.

  7. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    Science.gov (United States)

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  8. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants. PMID:26575143

  9. Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa.

    Science.gov (United States)

    Plyuta, Vladimir; Zaitseva, Julia; Lobakova, Elena; Zagoskina, Natalia; Kuznetsov, Alexander; Khmel, Inessa

    2013-11-01

    In the natural environment, bacteria predominantly exist in matrix-enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3- to 7-fold under the action of vanillin and epicatechin, and 2- to 2.5-fold in the presence of 4-hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4-hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N-3-oxo-dodecanoyl-homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N-acyl-homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs. PMID:23594262

  10. Inhibitory effect of different product analogues on {beta}-alanine synthase: A thermodynamic and fluorescence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Andujar-Sanchez, Montserrat; Martinez-Gomez, Ana Isabel; Martinez-Rodriguez, Sergio; Clemente-Jimenez, Josefa Maria; Heras-Vazquez, Francisco Javier Las; Rodriguez-Vico, Felipe [Departamento de Quimica Fisica, Bioquimica y Quimica Inorganica, Facultad de Ciencias Experimentales, Universidad de Almeria, Carretera de Sacramento s/n, La Canada de San Urbano, Almeria 04120 (Spain); Jara-Perez, Vicente [Departamento de Quimica Fisica, Bioquimica y Quimica Inorganica, Facultad de Ciencias Experimentales, Universidad de Almeria, Carretera de Sacramento s/n, La Canada de San Urbano, Almeria 04120 (Spain)], E-mail: vjara@ual.es

    2009-02-15

    The enzyme N-carbamoyl-{beta}-alanine amidohydrolase catalyse the hydrolysis of N-carbamoyl-{beta}-alanine or N-carbamoyl-{beta}-aminoisobutyric acid to {beta}-alanine or 3-aminoisobutyric acid, under the release of carbon-dioxide and ammonia. This work studies the inhibition of N-carbamoyl-{beta}-alanine amidohydrolase from Agrobacterium tumefaciens C58 (At{beta}car) by different carboxylic acid compounds that differ in number of carbons, and position and size of ramification, while the binding thermodynamics of the inhibitors are studied by isothermal titration calorimetry (ITC) and fluorescence. From the binding constants and inhibition studies, we conclude that propionate is the most efficient inhibitor among those tested. Substitution of the linear alkyl acids in positions 2 and 3 resulted in a drastic decrease of the affinity. The thermodynamic parameters show that a conformational change is triggered upon ligand binding. Binding enthalpy {delta}H{sub b} is negative in all cases for all ligands, and thus, Van der Waals interactions and hydrogen bonding are most probably the major sources for this term. The process is entropically favoured at all temperatures and pH studied, most probably due to the liberation of water molecules accompanying the conformational change of the enzyme.

  11. Molecular Cloning and Characterization of cgt, the Brucella abortus Cyclic β-1,2-Glucan Transporter Gene, and Its Role in Virulence

    OpenAIRE

    Roset, Mara S.; Ciocchini, Andrés E.; Ugalde, Rodolfo A.; Iñón de Iannino, Nora

    2004-01-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic β-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic β-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt cont...

  12. 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.

  13. 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.

  14. Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

    OpenAIRE

    Osterås, M; Stanley, J; Finan, T. M.

    1995-01-01

    Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the...

  15. Rhizogenic Induction in Adult Juglans regia L. cv. Serr Tissue Induced by Indole Butyric Acid and Agrobacterium rhizogenes Inducción Rizogénica en Tejido Adulto de Juglans regia L. cv. Serr Mediada por Ácido Indol Butírico y Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Manuel Sánchez-Olate

    2009-06-01

    Full Text Available The in vitro introduction of adult walnut (Juglans regia L. tissue represents an opportunity to clone elite genotypes whose selection occurs in advanced ontogenic states. With the purpose of developing a protocol to allow mass propagation of valuable genotypes from adult material, a comparison was made between two root induction systems of walnut microshoots of the fourth subculture of adult walnut tissue of an in vitro introduction program previously reinvigorated through traditional grafting. Rhizogenic induction by indole-3-butyric acid (IBA and Agrobacterium rhizogenes was used. The rhizogenic process was analyzed in two phases for both auxinic (T1: 3 mg L-1 IBA; T2: 5 mg L-1 IBA and A. rhizogenes inductions (T3: A-477; T4: A-478. The first phase of root induction was during 3 days in the dark while the second phase, root manifestation, was 27 days. Rooting percentage was evaluated and the induced root systems characterized (number, length, diameter, and root insertion zone in all the procedures. The best rooting results were obtained in T2, although the response obtained with A. rhizogenes didn’t differ from the T1 response. This appears to be an increasingly interesting methodology for adventitious rhizogenesis in this species.La introducción in vitro de tejido adulto de nogal (Juglans regia L. representa una oportunidad de clonación de genotipos elite, cuya selección ocurre en estados ontogénicos avanzados. Así, con el objeto de desarrollar un protocolo que permita la propagación masiva de genotipos valiosos a partir de material adulto, se compararon dos sistemas de inducción rizogénica de microtallos de nogal provenientes del cuarto subcultivo de un programa de introducción in vitro de tejido adulto de nogal, previamente revigorizado mediante injerto tradicional. Se utilizó la inducción rizogénica por ácido indol-3-butírico (AIB y Agrobacterium rhizogenes. El proceso rizogénico se analizó tanto para inducción aux

  16. 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

  17. Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae

    OpenAIRE

    Liu, C.-M.; McLean, P A; Sookdeo, C. C.; Cannon, F C

    1991-01-01

    Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not a...

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

    OpenAIRE

    Tuttle John; Haigler Candace H; Robertson Dominique

    2012-01-01

    Abstract Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost pa...

  19. Evaluación y selección de un protocolo vía Agrobacterium para la incorporación de resistencia al cogollero en la variedad de tomate Unapal-Arreboles

    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.

  20. Influence of Barley Seeds Germination and Seedling Growth with Agrobacterium%农杆菌对大麦种子萌发及幼苗生长发育的影响

    Institute of Scientific and Technical Information of China (English)

    徐开杰; 孟敏; 史丽丽; 刘曙东; 奚亚军

    2009-01-01

    以大麦品种(系)为主区('云引大麦Ⅰ'、'云引大麦Ⅱ'和'U008'),农杆菌浸种时间为副区(0.5、1.5和2.5 h),农杆菌菌液浓度为副副区(0.5、1.5和2.5 OD),采用再裂区试验研究了农杆菌浸种处理对大麦种子萌发和幼苗生长发育的影响.结果表明:品种、农杆菌菌液浓度、浸种时间对大麦的种子发芽率、幼苗高度、幼苗鲜重、叶绿素含量、丙二醛(MDA)含量影响无显著的互作效应,而对幼苗POD活性的影响存在显著互作效应;随着浸种时间的延长和菌液浓度的增加,各大麦品种(系)的种子发芽率、幼苗高度、幼苗鲜重、叶绿素含量均呈逐渐降低趋势,幼苗MDA含量则逐渐增加,并以'U008'变化幅度最大;在菌液浓度为0~1.5 OD、浸种时间为0~1.5 h范围内,幼苗POD活性随着菌液浓度的增加和浸种时间的延长而增强,超过该范围则均呈下降趋势,并以'U008'下降最为明显.可见,农杆菌处理对大麦种子萌发和幼苗生长发育有抑制作用,并在菌液浓度超过1.5 OD、浸种时间大于1.5 h时达极显著水平,且大麦品种间存在一定差异.%The barley varieties (lines) of 'YunyinⅠ','YunyinⅡ' and 'U008' as the dominated factors,agrobacterium soaking time including 0.5,1.5 and 2.5 h as the deputy factors,the agribacteria density of 0.5,1.5 and 2.5 OD as the deputy vice-factors,by adopting the resplit-plot design,the germination of seeds treated with agrobacterium and seedling growth and development of barley were studied.The results showed that the interaction effects of species,agribacteria density and soaking time for the barley seed germination rate,seedling height,seedling fresh weight,chlorophyll content,MDA content were not significant and the interaction effects for seedling POD activity existed.With the soaking time and agribacteria density increased,the barley seed germination rate,seedling height,seedling fresh weight,chlorophyll content in different

  1. Bleomycin resistance : a new dominant selectable marker for plant cell transformation

    NARCIS (Netherlands)

    Hille, Jacques; Verheggen, Frank; Roelvink, Peter; Franssen, Henk; Kammen, Ab van; Zabel, Pim

    1986-01-01

    Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. Th

  2. 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

  3. Tolerance of Norway spruce (Picea abies [L.] Karst.) embryogenic tissue to penicillin, carbapenem and aminoglycoside antibiotics

    Czech Academy of Sciences Publication Activity Database

    Malá, J.; Pavingerová, Daniela; Cvrčková, H.; Bříza, Jindřich; Dostál, J.; Šíma, P.

    2009-01-01

    Roč. 55, č. 4 (2009), s. 156-161. ISSN 1212-4834 R&D Projects: GA MZe QH71290 Institutional research plan: CEZ:AV0Z50510513 Keywords : somatic embryogenesis * Norway spruce * penicillin antibiotics * Agrobacterium tumefaciens * carbapenem antibiotics Subject RIV: EB - Genetics ; Molecular Biology

  4. 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...

  5. ANTIBACTERIAL ACTIVITY OF THREE MEDICINAL PLANTS OF KUMAUN HIMALAYA AGAINST SOME PATHOGENIC BACTERIA

    OpenAIRE

    Sati, S. C.; POONAM TAKULI; Kumar, P.; K. KHULBE

    2015-01-01

    The antibacterial property of methanol, ethanol and hexane extracts of Berberis aristata, Chenopodium ambrosioides and Tinospora cordifolia grown in Kumaun Himalayan were investigated against some pathogenic gram positive and gram negative bacterial strains (Bacillus subtilis, Agrobacterium tumefaciens, Escherichia coli, Xanthomonas phaseoli and Erwinia chrysanthemi) using disc diffusion method. Methanol extract of B. aristata was found with highest inhibitory activity against E. chrysanth...

  6. Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis

    Czech Academy of Sciences Publication Activity Database

    Bříza, Jindřich; Pavingerová, Daniela; Vlasák, Josef; Niedermeierová, Hana

    2013-01-01

    Roč. 60, č. 3 (2013), s. 395-400. ISSN 0001-527X R&D Projects: GA MZe QH71290; GA ČR(CZ) GAP502/11/1471 Institutional support: RVO:60077344 Keywords : Cry3A gene modification * Picea abies * Agrobacterium tumefaciens Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.389, year: 2013

  7. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  8. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  9. Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn

    Czech Academy of Sciences Publication Activity Database

    Trifunovic, M.; Cingel, A.; Simonovic, A.; Jevremovic, S.; Petric, M.; Dragicevic, I.; Motyka, Václav; Dobrev, Petre; Zahajská, Lenka; Subotic, A.

    2013-01-01

    Roč. 115, č. 2 (2013), s. 139-150. ISSN 0167-6857 R&D Projects: GA ČR(CZ) GAP506/11/0774 Institutional research plan: CEZ:AV0Z50380511 Keywords : Centaurium erythraea Rafn. * Agrobacterium tumefaciens * Genetic transformation Subject RIV: EF - Botanics Impact factor: 2.612, year: 2013

  10. Main: ASF1ATNOS [PLACE

    Lifescience Database Archive (English)

    Full Text Available ASF1ATNOS S000073 7-Sep-2000 (last modified) seki Tobacco ASF-1 binding site in nopaline synthas ... e (NOS) promoter of Ti -plasmid of Agrobacterium tumefaciens (A.t.); From ... 131 to -111; See S000312; Containing two hexamer moti fs; Essenti al for the nos promoter acti vity; nopali ...

  11. fRNAdb Summary: FR390147 [

    Lifescience Database Archive (English)

    Full Text Available FR390147 AB102732,AB102733,AB102734,AB102735,AF271644,AF271645,AF271648,AF321872,AF345269,AF3452 ... (Ile/I) Isoleucine tRNA Agrobacterium tumefaciens,Rhizobium ... leguminosarum,Rhizobium ... leguminosarum bv. phaseoli ... ,Rhizobium ... leguminosarum bv. viciae,Rhizobium ... tropici,Rhizobi ...

  12. Evaluación y selección de un protocolo vía Agrobacterium para la incorporación de resistencia al cogollero en la variedad de tomate Unapal-Arreboles

    Directory of Open Access Journals (Sweden)

    Vallejo Cabrera Franco Alirio

    2009-06-01

    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.

  13. 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。

  14. 放射型根瘤杆菌植酸酶对麸皮中植酸的脱磷作用%Dephosphorization of Phytic Acid in Wheat Bran by Phytase from Agrobacterium radiobacter

    Institute of Scientific and Technical Information of China (English)

    李文; 王陶; 杨克

    2011-01-01

    Crude phytase solution separated from Agrobacterium radiobacter fermentation broth was used to hydrolyze wheat bran for phytic acid dephosphorization.Using one-factor-at-a-time combined with orthogonal array design method,such process conditions as enzyme amount,substrate concentration,pH,temperature and hydrolysis time were optimized to be 750 U/g,40 mg/mL,7,45 ℃,and 5 h,respectively.Under the optimized conditions,the hydrolysis rate of phytic acid was up to 81.50%.%采用放射型根瘤杆菌植酸酶粗酶液水解麸皮中的植酸,研究其脱磷的最适条件。依据单因素试验和正交试验获得脱磷的最优条件为加酶量750U/g、底物质量浓度40mg/mL、温度45℃、pH7、时间5h。最优条件下,麸皮中植酸的水解率在5h达最大,为81.50%。

  15. Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica.

    Science.gov (United States)

    Glaeser, Stefanie P; Imani, Jafargholi; Alabid, Ibrahim; Guo, Huijuan; Kumar, Neelendra; Kämpfer, Peter; Hardt, Martin; Blom, Jochen; Goesmann, Alexander; Rothballer, Michael; Hartmann, Anton; Kogel, Karl-Heinz

    2016-04-01

    The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4's genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4's pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant's jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4- and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium. PMID:26495996

  16. 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.

  17. 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

  18. Agrobacterium-mediated gene transfer to Chrysanthemum.

    OpenAIRE

    Wordragen, van, M.F.

    1991-01-01

    Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is a random process in which two complete genomes are mixed and the desired phenotype has to be regained by repeated back crossing with the cultivated parent line. Despite these differences, both pro...

  19. Biodegradation of Crystal Violet by Agrobacterium radiobacter

    DEFF Research Database (Denmark)

    Parshetti, G.K.; Parshetti, S.G.; Telke, A.A.;

    2011-01-01

    containing 1% yeast extract and 0.1% NH4C1 has shown 100% decolorization of Crystal Violet within 5 hr. A complete degradation of Crystal Violet by A. radiobacter was observed up to 7 cycles of repeated addition (10 mg/L). When the effect of increasing inoculum concentration on decolorization of Crystal......) contributing to soil fertility and for four kinds of plants (Sorghum bicolor, Vigna radiata, Lens culinaris and Triticum aestivum) which are most sensitive, fast growing and commonly used in Indian agriculture....

  20. Agrobacterium-mediated gene transfer to Chrysanthemum.

    NARCIS (Netherlands)

    Wordragen, van M.F.

    1991-01-01

    Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is

  1. Jatropha (Jatropha curcas L.).

    Science.gov (United States)

    Maravi, Devendra Kumar; Mazumdar, Purabi; Alam, Shamsher; Goud, Vaibhav V; Sahoo, Lingaraj

    2015-01-01

    The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance. Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by histochemical GUS assay, polymerase chain reaction, and Southern hybridization. PMID:25416246

  2. Plant-Produced Cottontail Rabbit Papillomavirus L1 Protein Protects against Tumor Challenge: a Proof-of-Concept Study

    OpenAIRE

    Kohl, T.; Hitzeroth, I. I.; Stewart, D.; Varsani, A.; Govan, V. A.; Christensen, N. D.; Williamson, A.-L.; Rybicki, E. P.

    2006-01-01

    The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive p...

  3. 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...

  4. Cell Suspension Culture-Mediated Incorporation of the Rice Bel Gene into Transgenic Cotton

    OpenAIRE

    Liping Ke; RuiE Liu; Bijue Chu; Xiushuang Yu; Jie Sun; Brian Jones; Gang Pan; Xiaofei Cheng; Huizhong Wang; Shuijin Zhu; Yuqiang Sun

    2012-01-01

    Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liqu...

  5. Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucuronidase gene fusion in transgenic tobacco plants.

    OpenAIRE

    Liang, X W; Dron, M; J. Schmid; Dixon, R. A.; Lamb, C J

    1989-01-01

    A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, ...

  6. 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...

  7. 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...

  8. Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

    OpenAIRE

    Chao Xu; Liang Li; Wujun Jin; Yusong Wan

    2014-01-01

    Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed t...

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

    OpenAIRE

    Li Li; Li Jian-Feng; Sheen Jen

    2010-01-01

    Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with s...

  10. Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro

    OpenAIRE

    Stickler, David J.; Morris, Nicola S.; McLean, Robert J. C.; Fuqua, Clay

    1998-01-01

    Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive Agrobacterium tumefaciens reporter strain. Positive reactions were obtained from 14 isolates of Pseudomonas aeruginosa; negative or weakly positive reactions were recorded for isolat...

  11. Initial in vitro evaluations of antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria

    OpenAIRE

    Aires, A.; Mota, V.R.; Saavedra, M.J.; Monteiro, A.A.; Simões, M; Rosa, E.A.S.; Bennett, R.N.

    2009-01-01

    Aims: The aim of the study was to evaluate the in vitro antibacterial effects of glucosinolate hydrolysis products (GHP) against plant pathogenic micro-organisms namely Agrobacterium tumefaciens, Erwinia chrysanthemi, Pseudomonas cichorii, Pseudomonas tomato, Xanthomonas campestris and Xanthomonas juglandis. Methods and Results: Using a disc diffusion assay, seven different doses of 10 GHP were tested against each bacteria. The results showed that the isothiocyanates were...

  12. Researchers develop high-efficiency transformation of strawberry

    OpenAIRE

    Whyte, Barry James

    2006-01-01

    Researchers at the Virginia Bioinformatics Institute (VBI) and the Department of Horticulture in the College of Agriculture and Life Sciences at Virginia Tech have developed a new procedure for the efficient transfer of specific DNA sequences into the genome of strawberry. The scientists have used Agrobacterium tumefaciens, nature's genetic engineer, to introduce DNA into the woodland or alpine strawberry Fragaria vesca. The work was funded by a Virginia Tech ASPIRES grant (A Support Program ...

  13. 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...

  14. Scientific Opinion on application (EFSA-GMO-UK-2009-76) for the placing on the market of soybean MON 87769 genetically modified to contain stearidonic acid, 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

    Soybean MON 87769 was developed using Agrobacterium tumefaciens transformation and was intended to modify the lipid profile of the extracted oil. Soybean MON 87769 contains a single insert consisting of the Pj.D6D gene encoding the Δ6 desaturase protein from Primula juliae and the Nc.Fad3 gene encoding the Δ15 desaturase protein from Neurospora crassa, both involved in the desaturation of endogenous fatty acids into stearidonic acid. The molecular characterisation of soybean MON 87769 does no...

  15. Effect of N-hydroxyurea, mitomycin C and actinomycin D on tumour formation on the leaves of Kalanchoe daigremontiana

    OpenAIRE

    Józef Koawalczyk

    2014-01-01

    The leaves of Kalanchoe daigremontiana wounded and infected with Agrobacterium tumefaciens were treated with single doses of inhibitors (hydroxyurea - 190, mitomycin - 0.5, actinomycin - 2 µ,g per leaf). After delaying the time' of dosage of inhibitors during five days after inoculation, changes in susceptibility of the system to antitumorous activity of analysed compounds were observed. In several hours after inoculation (period of the bacteria metabolic activity in wounds) all the inhibitor...

  16. Phases of crown-gall transformation susceptible to hydroxyurea

    OpenAIRE

    Aldona Rennert; Józef Kowalczyk; Halina Pajkowska

    2014-01-01

    With the use of bacterial strains, both sensitive and resistant to hydroxyurea the action of this inhibitor on tumour formation on the leaves of Kalanchoe daigremontiana infected with Agrobacterium tumefaciens was tested for five days after inoculation. The results are in agreement with the opinion that the anti-tumour effect of hydroxyurea applied in the induction phase (between 18 and 60 h after inoculation) is the result of its direct action on plant cells, whereas inhibition of tumour for...

  17. Difference between resistant and susceptible maize to systematic colonization as revealed by DsRed-labeled Fusarium verticillioides

    OpenAIRE

    Lei Wu; Xiaoming Wang; Rongqi Xu; Hongjie Li

    2013-01-01

    Fusarium verticillioides was labeled with DsRed via Agrobacterium tumefaciens-mediated transformation to examine differences in colonization and reactions of resistant and susceptible inbred lines of maize (Zea mays L.). The extent of systemic colonization of F. verticillioides in roots from maize lines either resistant or susceptible to the fungus was studied by visualizing the red fluorescence produced by the fungus expressing DsRed. The difference in quantities of colony forming units (CFU...

  18. Isolation and Molecular Characterization of Biofouling Bacteria and Profiling of Quorum Sensing Signal Molecules from Membrane Bioreactor Activated Sludge

    OpenAIRE

    Harshad Lade; Diby Paul; Ji Hyang Kweon

    2014-01-01

    The formation of biofilm in a membrane bioreactor depends on the production of various signaling molecules like N-acyl homoserine lactones (AHLs). In the present study, a total of 200 bacterial strains were isolated from membrane bioreactor activated sludge and screened for AHLs production using two biosensor systems, Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136. A correlation between AHLs production and biofilm formation has been made among screened AHLs producing strai...

  19. Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

    Czech Academy of Sciences Publication Activity Database

    Zhang, P.; Wang, W.Q.; Zhang, G.L.; Kamínek, Miroslav; Dobrev, Petre

    2010-01-01

    Roč. 52, č. 7 (2010), s. 653-669. ISSN 1672-9072 R&D Projects: GA MŠk 1M06030; GA AV ČR IAA600380805 Institutional research plan: CEZ:AV0Z50380511 Keywords : MANIHOT-ESCULENTA CRANTZ * TRANSGENIC CASSAVA * AGROBACTERIUM-TUMEFACIENS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.603, year: 2010

  20. Type IVB Secretion Systems of Legionella and Other Gram-Negative Bacteria

    OpenAIRE

    Nagai, Hiroki; Kubori, Tomoko

    2011-01-01

    Type IV secretion systems (T4SSs) play a central role in the pathogenicity of many important pathogens, including Agrobacterium tumefaciens, Helicobacter pylori, and Legionella pneumophila. The T4SSs are related to bacterial conjugation systems, and are classified into two subgroups, type IVA (T4ASS) and type IVB (T4BSS). The T4BSS, which is closely related to conjugation systems of IncI plasmids, was originally found in human pathogen L. pneumophila; pathogenesis by L. pneumophila infection ...

  1. 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.

  2. Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Tarkowská, Danuše; Doležal, Karel; Tarkowski, Petr; Astot, C.; Holub, Jan; Fuksová, K.; Schmülling, T.; Sandberg, G.; Strnad, Miroslav

    2003-01-01

    Roč. 117, č. 4 (2003), s. 579-590. ISSN 0031-9317 R&D Projects: GA ČR GA522/01/0275 Grant ostatní: Volkswagen Stiftung(DE) I/76 865 Institutional research plan: CEZ:AV0Z5038910; CEZ:MSM 153100008 Keywords : 9--D-ribofuranosyl derivatives * Agrobacterium tumefaciens * bombardment-mass spectrometry Subject RIV: CE - Biochemistry Impact factor: 1.767, year: 2003

  3. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

    OpenAIRE

    Zhong-Tao Ding; Zhi Zhang; Di Luo; Jin-Yan Zhou; Juan Zhong; Jie Yang; Liang Xiao; Dan Shu; Hong Tan

    2015-01-01

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, w...

  4. TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis

    OpenAIRE

    Huang, Quanjun; Wang, Yan; Li, Bin; Chang, Junli; Chen, Mingjie; Li, Kexiu; Yang, Guangxiao; He, Guangyuan

    2015-01-01

    Background NAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses. Methods TaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and ...

  5. Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other gram-negative bacterial species.

    OpenAIRE

    Mutharia, L M; Crockford, G; Bogard, W C; Hancock, R E

    1984-01-01

    Four monoclonal antibodies against Escherichia coli J5 were studied. Each of these monoclonal antibodies reacted with purified lipopolysaccharides from E. coli J5, the deep rough mutant Salmonella minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa PAO1 as well as with the purified lipid A of P. aeruginosa. Enzyme-linked immunosorbent assays using the outer membranes from a variety of gram-negative bacteria demonstrated that these lipid A-specific monoclonal antibodies inte...

  6. 农杆菌介导虎杖芪合酶基因遗传转化壶瓶枣的研究%Agrobacterium-Mediated Transformation of Resveratrol Synthase Gene (PcPKS5) into Huping Jujube (Zizyphus jujuba)

    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

  7. 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

  8. Effects of bacteria on cadmium bioaccumulation in the cadmium hyperaccumulator plant Beta vulgaris var. cicla L.

    Science.gov (United States)

    Chen, Su; Chao, Lei; Sun, Lina; Sun, Tieheng

    2013-01-01

    To investigate the effects of two cadmium-tolerant bacteria, Staphylococcus pasteuri (S. pasteuri X1) and Agrobacterium tumefaciens (A. tumefaciens X2), on cadmium uptake by the cadmium hyperaccumulator plant Beta vulgaris var. cicla L., a pot experiment with artificially contaminated soil was conducted. The results demonstrated that both cadmium-tolerant bacteria enhanced the dry weight of Beta vulgaris var. cicla L. The total dry weights of plants in the control CK20, S. pasteuri X1 and A. tumefaciens X2 treatments were 0.85, 1.13, and 1.38 g/pot, respectively. Compared with the control CK20 findings, the total dry weight of plants was increased by 32.8 and 61.1% after inoculation with S. pasteuri X1 and A. tumefaciens X2, respectively, indicating that A. tumefaciens X2 more strongly promoted the growth of Beta vulgaris var. cicla L. than S. pasteuri X1. In addition, inoculation with S. pasteuri X1 and A. tumefaciens X2 significantly (p < 0.05) promoted cadmium uptake by plants and improved the bioaccumulation of cadmium by the plants from the soil. Moreover, the inoculation of S. pasteuri X1 and A. tumefaciens X2 effectively facilitated the transfer of cadmium in the soil from the Fe-Mn oxide and residual fractions to the soluble plus exchangeable and weakly specially adsorbed fractions in the rhizosphere soils of plants. The bacterial enhancement of cadmium phytoavailability might provide a potential and promising method to increase the efficiency of phytoextraction. PMID:23488173

  9. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

    Science.gov (United States)

    Bondage, Devanand D; Lin, Jer-Sheng; Ma, Lay-Sun; Kuo, Chih-Horng; Lai, Erh-Min

    2016-07-01

    Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity. PMID:27313214

  10. Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence.

    Science.gov (United States)

    Roset, Mara S; Ciocchini, Andrés E; Ugalde, Rodolfo A; Iñón de Iannino, Nora

    2004-04-01

    The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor. PMID:15039351

  11. 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.

  12. Construction of two-dimensional electrophoresis (2-DE) proteomic map of Agrobacterium species ATCC31749%产热凝胶土壤杆菌ATCC31749蛋白质组双向电泳图谱的构建

    Institute of Scientific and Technical Information of China (English)

    刘佳; 詹晓北; 吴剑荣; 郑志永; 于丽珺

    2011-01-01

    建立了热凝胶生产茵土壤杆茵茵体总蛋白的蛋白质提取方法和双向电泳方案,确定了使用蛋白质裂解液(7 mol/L尿素,2 mol/L硫脲,1%ASB-14去垢剂,40 mmol/L Tris,0.001%溴酚蓝,1 mmol/L EDTA,1%TBP和1%两性电解质)结合超声破碎法来提取茵体总蛋白的方案为最佳,选择17 cm pH 4~7的IPG预制干胶条和适宜的等电聚焦程序进行第一向等电聚焦电泳.最后比较了考马斯亮蓝和硝酸银两种染色方法对双向电泳图谱的影响,确立了蛋白分辨率高并且重复性较好的土壤杆茵双向电泳图谱构建的可靠方法.%The method for extraction total cellular protein of Agrobacterium species for 2-DE was constructed and optimized. The optimum process was the following: cells suspended in 1 mL of a lysis buffer (7 mol/L urea, 2 mol/L thiourea, 1% ASB-14 detergent, 40 mmol/L Tris base, 0.001% bromophenol blue, 1 mmol/L EDTA, 1%TBP and 1 % carrier ampholyte), and then sonication for 3 min on ice. After centrifugation, the supernatant was reserved in -80℃ for two-dimensional electrophoresis (2-DE). And the 2-DE conditions were also constructed. The immobilized pH gradient (IPG) gel strips (17 cm) with linear pH 4 ~ 7 were used for the first dimension electrophoresis with suitable electrophoresis parameters. After compared two staining methods (coomassie blue staining and silver staining), it was found that the silver staining could get more and clearly protein spots on 2-DE map. Finally, the method for constructing 2-DE protein map with high resolution rate and good repeatability was established.

  13. Cloning, purification, crystallization and preliminary X-ray analysis of a bacterial GABA receptor with a Venus flytrap fold

    International Nuclear Information System (INIS)

    A 1.35 Å resolution data set was collected from a crystal of the periplasmic GABA receptor Atu2422 from A. tumefaciens. Atu2422 adopts a closed Venus flytrap conformation. In response to infection by the pathogen Agrobacterium tumefaciens, plants synthesize several stress amino acids, including γ-aminobutyric acid (GABA), which modulates the expression of bacterial virulence factors. GABA penetrates into the bacterial cytoplasm via an ABC transporter that is associated with the periplasmic receptor Atu2422. Mature receptor Atu2422 (without its signal peptide) was overexpressed in Escherichia coli, purified and crystallized. A complete data set was collected to 1.35 Å resolution at 100 K. The crystals belonged to the monoclinic space group C2 and contained one molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed a closed form of this Venus flytrap (VFT) receptor, suggesting the presence of an endogenous E. coli ligand

  14. GM FOODS - A CONTROVERSY BETWEEN TRADITION AND MODERNITY

    Directory of Open Access Journals (Sweden)

    Venugopal Rao V, Meena Kumari and K Anthonamma

    2013-04-01

    Full Text Available Genetically engineered plants are developed in a laboratory by altering their genetic makeup and are evaluated in the laboratory for desired qualities. This is accomplished by adding one or few genes to a native or a local genome using genetic engineering techniques. Genetically modified plants are generated by the biolistic particle gun method or by Agrobacterium tumefaciens mediated transformation. When plants of desired quality are produced, sufficient seeds are multiplied and the companies producing them have to apply for regulatory approval for field trials.

  15. Caracterização morfocultural, biossíntese de autoindutor e formação de biofilme por rizobactérias de hortaliças Morphocultural characterization, autoinducer biosynthesis and biofilm formation in rhizobacteria isolated from vegetable crops

    OpenAIRE

    Rachel Pinton; Anelise Dias; Terezinha Ferreira Xavier; Luc Felicianus Marie Rouws; Gustavo Ribeiro Xavier; Norma Gouvêa Rumjanek; Raul de Lucena Duarte Ribeiro

    2010-01-01

    O objetivo deste trabalho foi caracterizar e agrupar rizobactérias, isoladas de hortaliças, quanto à morfologia cultural, riqueza e diversidade e avaliar a biossíntese de autoindutores N-acil lactonas homoserinas (ALH) e a capacidade de formação de biofilmes. Sete estirpes também foram avaliadas quanto ao potencial de promoção de crescimento de Brassica oleraceae var. acephala em casa de vegetação. Para verificar a produção de ALH, foram realizados ensaios com Agrobacterium tumefaciens estirp...

  16. TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in Escherichia coli

    OpenAIRE

    Schmidt-Eisenlohr, Heike; Domke, Natalie; Baron, Christian

    1999-01-01

    Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper...

  17. Collective biology of neoplastic disease in dicotyledonous plants

    International Nuclear Information System (INIS)

    We discuss the two different responses from the angiosperms to the specific molecular mechanisms of the tumor-inducing agent contained in the bacterium Agrobacterium tumefaciens. This is done in terms of the collective variables for expressing genetic response to a continuously varying supply of energy from metabolic pathways. We are led to the conjecture that the expression of the recessive oncogenes may not be restricted to humans (retinoblastoma and osteosarcoma), but may also occur in plants (crown gall), and be expressed through a heat-shock. (author). 11 refs

  18. Effect of rolB transgene on Prunus cerasus × P. canescens and Cydonia oblonga microshoots rhizogenesis

    OpenAIRE

    Stanienė, G.; Rugienius, R.; Gelvonauskienė, D.; Stanys, V.

    2007-01-01

    With the aim to improve rooting ability, the dwarfing sweet cherry hybrid rootstock (Prunus cerasus × P. canescens) and pear rootstock (Cydonia oblonga P. Mill) were transformed with the rolB gene using Agrobacterium tumefaciens mediated gene transfer. The binary vectors with rolB gene (cloned from A. rhizogenes plasmid pRiA4), driven by their own and constitutive CaMV promoter, were used for transformation. More than 400 regenerants of both rootstocks were obtained in vitro within seven mont...

  19. Phases of crown-gall transformation susceptible to hydroxyurea

    Directory of Open Access Journals (Sweden)

    Aldona Rennert

    2014-02-01

    Full Text Available With the use of bacterial strains, both sensitive and resistant to hydroxyurea the action of this inhibitor on tumour formation on the leaves of Kalanchoe daigremontiana infected with Agrobacterium tumefaciens was tested for five days after inoculation. The results are in agreement with the opinion that the anti-tumour effect of hydroxyurea applied in the induction phase (between 18 and 60 h after inoculation is the result of its direct action on plant cells, whereas inhibition of tumour formation by the inhibitor in the inoculation period depends on its action on the pathogenic bacteria.

  20. Actividad antitumoral y toxicidad de (+)-curcufenol y de curcudiol aislados de la esponja marina didisus oxeata

    OpenAIRE

    Salama, Ahmed M.; Fonseca, Claudia; Renjifo, Laura

    2009-01-01

    El extracto metanólico, el extracto clorofórmico, el curcufenol y el curcudiol obtenido de la esponja fueron evaluados frente a Artemia salina obteniéndose una concentración letal media de 0.54, 5.57, 3.10 y 0.70 m g/mL, respectivamente. También se probó su actividad antitumoral mediante el bioensayo de tumorogénesis inducida por Agrobacterium tumefaciens en disco de zanahoria, produciéndose inhibición en concentraciones mayores de 60 m g/mL.

  1. 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.

  2. ANTIBACTERIAL ACTIVITY OF THREE MEDICINAL PLANTS OF KUMAUN HIMALAYA AGAINST SOME PATHOGENIC BACTERIA

    Directory of Open Access Journals (Sweden)

    S. C. SATI

    2015-11-01

    Full Text Available The antibacterial property of methanol, ethanol and hexane extracts of Berberis aristata, Chenopodium ambrosioides and Tinospora cordifolia grown in Kumaun Himalayan were investigated against some pathogenic gram positive and gram negative bacterial strains (Bacillus subtilis, Agrobacterium tumefaciens, Escherichia coli, Xanthomonas phaseoli and Erwinia chrysanthemi using disc diffusion method. Methanol extract of B. aristata was found with highest inhibitory activity against E. chrysanthemi (ZOI, 11±0.3mm. Whereas lowest inhibition was recorded in ethanolic extract of B. aristata against E. coli. The hexane extract of B. aristata and methanolic extract of C. ambrosioides were found totally inactive against all the pathogens tested.

  3. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    Energy Technology Data Exchange (ETDEWEB)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Quimica]. E-mail: anita@iqm.unicamp.br; Grognux, Johann; Reymond, Jean-Louis [University of Berne (Switzerland). Dept. of Chemistry and Biochemistry

    2004-12-01

    Biocatalysis reactions were performed on microtiter plates (200 {mu}L) aiming at the utilization of fluorogenic substrates (100 {mu}mol L{sup -1}) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  4. Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

    DEFF Research Database (Denmark)

    Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn;

    2014-01-01

    1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was......, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function...

  5. In vitro synergistic antibacterial activity of Salvia officinalis L. and some preservatives

    Directory of Open Access Journals (Sweden)

    Stanojević Dragana

    2010-01-01

    Full Text Available The aim of this work was to investigate the antibacterial activity of aqueous extracts of the species Salvia officinalis L. and its synergistic action with the preservatives sodium nitrite, sodium benzoate and potassium sorbate in vitro against selected food spoiling bacteria. Synergism was assessed by the checkerboard assay method and quantitatively represented by the FIC index. Synergistic action was established for aqueous extract/sodium benzoate, aqueous extract/potassium sorbate, aqueous extract/sodium nitrite combinations. Synergism was detected in relation to: Agrobacterium tumefaciens, Bacillus subtilis and Proteus sp. Synergism was established at plant extract and preservative concentrations corresponding up to 1/8 MIC values.

  6. Plant Male Sterility Induced by Anti-Gene CYP86MFin Brassica oleracea var. Italica

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    An anti-gene CYP86MF was introduced into hypocotyls of broccoli (Brassica oleracea L.var. italica Plenck) with Agrobacterium tumefaciens, and the transgenic plants were obtained by kanamycin selection. The results of PCR, Southern blot and Northern blot indicated that the anti-CYP86MF has been integrated into chromosome of the transgenic plant.And also, plants with hypogenetic stamina or ungerminated pollen were observed. The transgenic male sterility plant could fructify via artificial pollination with normal pollen. Thus it was proved that the pistil of male sterility plant was normally developed, and the sterility originated from anti-CYP86MF.

  7. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L-1) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  8. AcEST: DK954222 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST39A01NGRL0019_N23 614 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0019_N23. 5' end seq ... ibrio vulnificus... 38 0.41 tr|Q7D226|Q7D226_AGRT5 Biopolymer ... transport protein OS=Agrobacte... 36 1.6 tr|B3MJ56 ... Sbjct: 450 -IHMAEQNGL 458 >tr|Q7D226|Q7D226_AGRT5 Biopolymer ... transport protein OS=Agrobacterium tumefaciens (st ...

  9. Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

    Directory of Open Access Journals (Sweden)

    Jackson Marcondes

    2008-12-01

    Full Text Available The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L. using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety "Vitória de Verão" genetically modified.

  10. Expression and detection of the FMDV VP1 transgene and expressed structural protein in Arabidopsis thaliana

    OpenAIRE

    Pan, Li; Zhang, Yongguang; Wang, Yonglu; Lv, Jianliang; Zhou, Peng; Zhang, Zhongwang

    2011-01-01

    To explore the feasibility of developing a new type of plantderived foot-and-mouth disease virus (FMDV) oral vaccine, the plant seed-specific expression vector p7SBin438/VP1 carrying the VP1 gene of the FMDV strain O/China/99 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. This strain was used for transformation of Arabidopsis thaliana via the floral-dip method. The kanamycin-resistant transgenic plants were selected, and the VP1 gene and protein expressions were...

  11. Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences*

    OpenAIRE

    Yang, Funmei; Simpson, Robert B.

    1981-01-01

    BT37 is a crown gall teratoma incited on tobacco by Agrobacterium tumefaciens containing pTi-T37, a nopaline-type Ti plasmid. Treatment of this cloned tumor tissue with kinetin at 1 mg/liter results in the formation of relatively normal-appearing shoots. These shoots can be induced to root and set viable seed. In contrast to BT37 tissue, the derived tissues are not phytohormone independent and do not produce nopaline. The reverted plants, like normal tobacco plants, are susceptible to infecti...

  12. 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...

  13. Development of Cotton leaf curl virus resistant transgenic cotton using antisense ßC1 gene.

    Science.gov (United States)

    Sohrab, Sayed Sartaj; Kamal, Mohammad A; Ilah, Abdul; Husen, Azamal; Bhattacharya, P S; Rana, D

    2016-05-01

    Cotton leaf curl virus (CLCuV) is a serious pathogen causing leaf curl disease and affecting the cotton production in major growing areas. The transgenic cotton (Gossypium hirsutum cv. Coker 310) plants were developed by using βC1 gene in antisense orientation gene driven by Cauliflower mosaic virus-35S promoter and nos (nopaline synthase) terminator and mediated by Agrobacterium tumefaciens transformation and somatic embryogenesis system. Molecular confirmation of the transformants was carried out by polymerase chain reaction (PCR) and Southern blot hybridization. The developed transgenic and inoculated plants remained symptomless till their growth period. In conclusion, the plants were observed as resistant to CLCuV. PMID:27081361

  14. Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch).

    Science.gov (United States)

    Islam, M Ashraful; Thorstensen, Tage; Clarke, Jihong Liu

    2015-01-01

    Genetic engineering is an important tool for introducing desired genes into poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch). We describe in this chapter an Agrobacterium tumefaciens-mediated transformation protocol for poinsettia. A detailed description of genetic transformation, antibiotic selection, subsequent regeneration via somatic embryogenesis, and rooting as well as molecular and morphological analyses is included. The methodology described here could facilitate the future engineering of poinsettia for research purpose as well as commercial production of poinsettia plants with improved resistance or novel traits. PMID:25416270

  15. Transgenic Rape with hrf2 Gene Encoding HarpinXooc Resistant to Sclerotinia sclerotinorium

    Institute of Scientific and Technical Information of China (English)

    MA Ling-li; HUO Rong; GAO Xue-wen; HE Dan; SHAO Min; WANG Qi

    2008-01-01

    The objective of this study was to increase the resistance of rape using the method of transformation. The gene hrf2 derived from Xathomonas oryzae pv. oryzicola encoding harpinXooc protein was constructed into transgenic vector pCAMBIA1301. The cotyledonal petiole segments from rapeseed variety Yangyou 4 were infected by Agrobacterium tumefaciens strain LBA4404/pCAMBIA1301-hrf2. Hygromycin-resistant green shoots were obtained. Successful integration of the foreign gene into the genome of the rapeseed variety Yangyou 4 was confirmed by PCR, RT-PCR, and β-glucuronidase analyses. Disease bioassays of transgenic plants revealed an improved resistance of transgenic plants to Rape sclerotiniose. In brief, the hrf2 gene can be transferred into rape using the method of Agrobacterium-medmted transformation, which increased the resistance to Sclerotinia sclerotinorium in the transgenic plant.

  16. Transient Expression of P-type ATPases in Tobacco Epidermal Cells.

    Science.gov (United States)

    Poulsen, Lisbeth R; Palmgren, Michael G; López-Marqués, Rosa L

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellular space between leaf epidermal cells, which results in DNA transfer from the bacteria to the plant and expression of the corresponding proteins. By injecting mixes of Agrobacterium strains, this system offers the possibility to co-express a number of target proteins simultaneously, thus allowing for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits. PMID:26695049

  17. Agrobacterium mediated construction of rice mutant population with transposon

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Transposon tagging is a powerful tool for gene study, especially for genes among different plants, such as Arabidopsis, tobacco, and tomato, etc. This effective strategy for gene isolation has been applied to rice and progress has been made in recent years.

  18. 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%.

  19. Mungbean plants expressing BjNPR1 exhibit enhanced resistance against the seedling rot pathogen, Rhizoctonia solani.

    Science.gov (United States)

    Vijayan, S; Kirti, P B

    2012-02-01

    Mungbean, Vigna radiata (L.) Wilczek is an important pulse crop that is widely cultivated in semi- arid tropics. The crop is attacked by various soil-borne pathogens like Rhizoctonia solani, which causes dry rot disease and seriously affects its productivity. Earlier we characterized the non-expressor of pathogenesis related gene-1(BjNPR1) of mustard, Brassica juncea, the counterpart of AtNPR1 of Arabidopsis thaliana. Here, we transformed mungbean with BjNPR1 via Agrobacterium tumefaciens. Because of the recalcitrant nature of mungbean, the effect of some factors like Agrobacterium tumefaciens strains (GV2260 and LBA4404), pH, L: -cysteine and tobacco leaf extract was tested in transformation. The transgenic status of 15 plants was confirmed by PCR using primers for nptII. The independent integration of T-DNA in transgenic plants was analyzed by Southern hybridization with an nptII probe and the expression of BjNPR1 was confirmed by RT-PCR. Some of the T(0) plants were selected for detached leaf anti-fungal bioassay using the fungus Rhizoctonia solani, which showed moderate to high level of resistance depending on the level of expression of BjNPR1. The seedling bioassay of transgenic T(2) plants indicated resistance against dry rot disease caused by R. solani. PMID:21584838

  20. Natural Transgenic“Engineer”and Its Implications to Genetically Modified Food%天然的转基因“工程师”及其对转基因食品的意蕴

    Institute of Scientific and Technical Information of China (English)

    邓心安; 许冰茹

    2015-01-01

    Under the background that genetically modified foods(GM foods)have been highly controversial and even demonized at present in China,to know the transgenic process of agrobacterium tumefaciens is just like to find the key to understand transgenic technologies and GM foods fundamentally,which contributes to eliminate the mystery and people's fear of GM foods. The natural transgenic process of agrobacterium tumefaciens proves that DNA is really safe,transgenic technologies are not as terrible as some people thought,and GM foods have the substantial equivalence in security to the traditional foods.%在转基因食品之于中国当前倍受争议甚至被“妖魔化”的背景下,了解根瘤农杆菌的转基因过程,就好比找到从根本上理解转基因技术和转基因食品的钥匙,有助于消除对转基因食品的神秘感与恐惧。根瘤农杆菌的天然转基因过程证明:DNA真的很安全,转基因技术并不可怕,转基因食品具有与传统食品实质等同的安全性。

  1. NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

    Science.gov (United States)

    Bayliss, Richard; Harris, Richard; Coutte, Loic; Monier, Amy; Fronzes, Remi; Christie, Peter J; Driscoll, Paul C; Waksman, Gabriel

    2007-01-30

    Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system. PMID:17244707

  2. Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Millar Andrew J

    2009-02-01

    Full Text Available Abstract Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination.

  3. NARINGENIN ENHANCED EFFICIENCY OF GUS ACTIVITY IN Passiflora mollissima (H.B.K. Bailey

    Directory of Open Access Journals (Sweden)

    G.O. Cancino

    2004-06-01

    Full Text Available The flavonoid naringenin has been investigated as a possible vir gene inducer in Agrobacterium-mediated transformation in Passiflora mollissima, P. giberti and Nicotiana tabacum cv. Xanthi. The transformation efficiency percentage of explants showing blue GUS expression and the extent of staining following inoculation with Agrobacterium tumefaciens strains EHA 105 and 1065, carrying gus and nptII genes was enhanced with the supplementation of the co-cultivation medium with naringenin. Supplementation of medium with 100µM (strain EHA 105 and 300 µM (strain 1065 naringenin was most effective at enhancing mean (±s.e.m., n=3 GUS activity in leaf explants (20.3 ± 2.4%, strain EHA; 105; 6.0 ± 0.57%, strain 1065 and nodal segments (16.7 ± 2.4% strain EHA 105; 8.3 ± 0.57% strain 1065 of P. mollissima. In P. giberti and N. tabacum maximum GUS activity was obtained in leaf and root explants with 100µM naringenin for both strains analysed. Additionally, when naringenin was added to Luria Bertani (LB medium, both bacterial growth via optical density and colony forming units were higher when compared to control. This is the first report of the use of naringenin to enhance gene transfer from Agrobacterium to plants. These findings suggest that naringenin can be used as an alternative to acetosyringone for vir gene induction in Agrobacterium. This approach may be especially useful in plants that are generally recalcitrant to Agrobacterium-mediatedtransformation.

  4. 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

  5. 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

  6. Direct Gene Transfer into Plant Mature Seeds via Electroporation After Vacuum Treatment

    Science.gov (United States)

    Hagio, Takashi

    A number of direct gene transfer methods have been used successfully in plant genetic engineering, providing powerful tools to investigate fundamental and applied problems in plant biology (Chowrira et al., 1996; D'halluin et al., 1992; Morandini and Salamini, 2003; Rakoczy-Trojanowska, 2002; Songstad et al., 1995). In cereals, several methods have been found to be suitable for obtaining transgenic plant; these include bombardment of scutellum (Hagio et al., 1995) and inflorescence cultures (He et al., 2001), and silicon carbide fiber-mediated DNA delivery (Asano et al., 1991) and Agrobacterium tumefaciens transformation (Potrykus, 1990). Electroporation of cereal protoplasts also has proved successful but it involves prolonged cell treatments and generally is limited by the difficulties of regeneration from cereal protoplast cultures (Fromm et al., 1987). Many laboratories worldwide are now using Agrobacterium as a vehicle for routine production of transgenic crop plants. The primary application of the particle system (Klein et al., 1987) has been for transformation of species recalcitrant to conventional Agrobacterium (Binns, 1990) or protoplast methods. But these conventional methods can be applied to the species and varieties that are amenable to tissue culture (Machii et al., 1998). Mature seeds are readily available and free from the seasonal limits that immature embryo, inflorescence, and anther have. This method enables us to produce transgenic plants without time-consuming tissue culture process.

  7. Quantitative transient GUS expression in J-104 rice calli through manipulation of in vitro culture conditions.

    Directory of Open Access Journals (Sweden)

    Maylin Pérez Bernal

    2009-10-01

    This paper purposes suitable conditions for callus induction and co-cultivation with Agrobacterium tumefaciens of J-104 rice cultivar. It was evaluated the effect of different concentrations of 2.4-D and agar, and the inclusion of L-proline and L-glutamine in callus culture medium. The use of 2.5 mg/L 2.4-D and 0.8% agar allowed the highest percentage of embryogenic calli. Callus formation was improved considerably with 500 mg/L of L-proline and L-glutamine in the culture medium. Different factors were studied throughout co-cultivation of calli with A. tumefaciens: inoculation time, co-cultivation temperature, concentration of acetosyringone and co-cultivation period. Transient GUS expression was quantified by fluorometry in all co-cultivated calli. The best results were obtained with the following conditions: 10 min as inoculation time, 100µM acetosyringone in co-cultivation medium, temperature of 20ºC, and 3 days as co-cultivation period. Key words: Agar; callus; co-cultivation; fluorometric GUS activity. Resumen Se describen las condiciones óptimas para la callogénesis y cocultivo de callos con Agrobacterium tume-faciens de la variedad de arroz J-104. Se determinó el efecto de diferentes concentraciones de 2.4-D, agar y de L-prolina y L-glutamina en el medio de cultivo de callos. El uso de 2,5 mg/L de 2.4-D y 0,8% de agar permitió lograr el porcentaje más alto de callos embriogénicos. La formación de callos fue mejorada considerablemente con la adición de 500 mg/L de L-prolina e igual concentración de L-glutamina en el medio de cultivo. Se estudiaron diferentes factores en el cocultivo de los callos con A. tumefaciens: tiempo de inoculación, concentración de acetosiringona, temperatura y tiempo de cocultivo. Para comparar el efecto de cada factor sobre la expresión GUS se cuantificó la actividad transitoria mediante fluorimetría. Los valores más altos de actividad fluorimétrica fueron obtenidos con las siguientes condiciones: 10 min de

  8. N-hydroxyurea, mitomycin C and actinomycin D activity in the process of tumour formation on the primary leaves of the 'Pinto' bean

    Directory of Open Access Journals (Sweden)

    Aldona Rennert

    2014-02-01

    Full Text Available Mitomycin C (MC, N-hydroxyurea (HU and actinomycin D (AD inhibit tumour formation on the primary leaves of Pinto beans. Agrobacterium tumefaciens was inoculated into bean leaves with application of the above named inhibitors at various times. It was found that MC action is strongest during inoculation and immediately after it, the maximal effect of HU take place within 12 h after inoculation, whereas the antitumour action of AD starts as late as 12 h after leaf inoculation. In view of the different degree of susceptibility of bacteria and plant cells to the inhibitors applied, the above described results allowed to distinguish three critical periods in the process of tumour formation in the tested host-pathogen system.

  9. Transgenic Paulownia Expressing shiva-1 Gene Has Increased Resistance to Paulownia Witches' Broom Disease

    Institute of Scientific and Technical Information of China (English)

    Tao DU; Yao WANG; Qin-Xue HU; Jie CHEN; Sheng LIU; Wen-Jin HUANG; Mu-Lan LIN

    2005-01-01

    Stem segments from diseased Paulownia tomentosa×P. fortunei and leaves from healthy control were transformed with the expression vector p438PRSI via Agrobacterium tumefaciens. The p438PRSI vector contained shiva-1 gene, which encodes an antibacterial peptide under the control of a CaMV35S promoter. The regenerated plants from transformed explants were planted in a greenhouse and nursery. PCR and Southern blotting analysis showed that the shiva-1 gene was successfully integrated into the Paulownia genome. Transcription of the integrated shiva-1 gene was confirmed by RT-PCR. Bioassay in the green house and phytoplasma DNA-dot blotting demonstrated that resistance to Paulownia witch's broom disease (PWB) increased significantly in shiva-1-transgenic Paulownia. Further investigations indicated that higher Shiva-1 expression correlated with fewer phytoplasma and less symptoms in diseased transgenic Paulownia. Together, our findings strongly suggest that breeding shiva-1-Paulownia is an effective strategy to control PWB disease.

  10. 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.

  11. Quorum sensing in plant-pathogenic bacteria.

    Science.gov (United States)

    Von Bodman, Susanne B; Bauer, W Dietz; Coplin, David L

    2003-01-01

    Quorum sensing (QS) allows bacteria to assess their local population density and/or physical confinement via the secretion and detection of small, diffusible signal molecules. This review describes how phytopathogenic bacteria have incorporated QS mechanisms into complex regulatory cascades that control genes for pathogenicity and colonization of host surfaces. Traits regulated by QS include the production of extracellular polysaccharides, degradative enzymes, antibiotics, siderophores, and pigments, as well as Hrp protein secretion, Ti plasmid transfer, motility, biofilm formation, and epiphytic fitness. Since QS regulatory systems are often required for pathogenesis, interference with QS signaling may offer a means of controlling bacterial diseases of plants. Several bacterial pathogens of plants that have been intensively studied and have revealed information of both fundamental and practical importance are reviewed here: Agrobacterium tumefaciens, Pantoea stewartii, Erwinia carotovora, Ralstonia solanacearum, Pseudomonas syringae, Pseudomonas aeruginosa, and Xanthomonas campestris. PMID:12730390

  12. 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...

  13. Lignin reduction in transgenic poplars by expressing antisense CCoAOMT gene

    Institute of Scientific and Technical Information of China (English)

    LU Jing; ZHAO Huayan; WEI Jianhua; HE Yikun; SHI Chao; WANG Hongzhi; SONG Yanru

    2004-01-01

    The antisense Caffeoyl CoA O-methyltransferase (CCoAOMT) cDNA was transformed into Chinese white poplar (Populus tomentosa) mediated by Agrobacterium tumefaciens. Many factors affecting the transformation efficiency were studied and a stable transformation system was established. PCR-Southern blot analysis indicated that antisense CCoAOMT cDNA had been integrated into the genome of the transgenic poplars. RT-PCR and Western blot analyses demonstrated that the endogenous CCoAOMT gene was suppressed at both transcriptional and translational levels. Klason lignin content assay exhibited the lignin reduction to different degrees in transgenic poplars. The stems of partial transgenic poplars with the remarkable lignin reduction turned red, and the color distribution was stripped or spotted. Taken together, these results suggested that CCoAOMT gene would be a potential useful gene in altering lignin biosynthesis by biotechnology for improving wood properties.

  14. Transgene directionally integrated into C-genome of Brassica napus

    Institute of Scientific and Technical Information of China (English)

    FANG Xiaoping; WANG Zhuan; LI Jun; LUO Lixia; HU Qiong

    2006-01-01

    Integration of a transgene into a C-genome chromosome plays an important role in reducing ecological risk of transgenic Brassica napus.To obtain C-genome transgenic B. napus, herbicide-resistant bar gene was firstly transferred into B.oleracea var. a/bog/abra mediated by Agrobacterium tumefaciens strain LBA4404. Then using the transgenic B. oleracea as paternal plants and 8 nontransgenic varieties of B. rapa as maternal plants, Cgenome transgenic B. napus with bar gene was artificially resynthesized by means of ovary culture and chromosome doubling. Among 67 lines of the resynthesized B. napus, 31 were positive, and 36 were negative according to PCR test for bar gene. At least 2 plants from each line were kept for PPT spray confirmation. The result was in consistence with the PCR test. Genomic Southern blotting of three randomly chosen lines also showed that bar gene had been integrated into the genome of resynthesized B. napus lines.

  15. 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.

  16. Gene Insertion Patterns and Sites

    Science.gov (United States)

    Vain, Philippe; Thole, Vera

    During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.

  17. A Common Catalytic Mechanism for Proteins of the HutI Family

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi,R.; Eswaramoorthy, S.; Burley, S.; Raushel, F.; Swaminathan, S.

    2008-01-01

    Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-l-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [N-formimino-l-glutamate (NIG)] and an inhibitor [3-(2, 5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.

  18. Dicty_cDB: Contig-U06540-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available bda K H 1.37 0.711 1.31 Matrix: blastn matrix:1 -3 Gap Penaltie...ia K2... 192 4e-61 CP000826_1568( CP000826 |pid:none) Serratia proteamaculans 568...7869_694( AE007869 |pid:none) Agrobacterium tumefaciens str. C... 215 1e-71 CP000774_2443( CP000774 |pid:none) Parvibaculum lavam...CP000108_1625( CP000108 |pid:none) Chlorobium chlorochromatii CaD3... 187 2e-61 CP000057_1650( CP000057 |pid:non...3e-55 CP001277_1623( CP001277 |pid:none) Candidatus Hamiltonella defensa... 168 4e-55 CP000786_2293( CP000786 |pid:non

  19. The Antibacterial Activity of Chitosan Products Blended with Monoterpenes and Their Biofilms against Plant Pathogenic Bacteria

    Science.gov (United States)

    Badawy, Mohamed E. I.; Rabea, Entsar I.; Taktak, Nehad E. M.; El-Nouby, Mahmoud A. M.

    2016-01-01

    This study focuses on the biological activities of eleven chitosan products with a viscosity-average molecular weight ranging from 22 to 846 kDa in combination with the most active monoterpenes (geraniol and thymol), out of 10 tested, against four plant pathogenic bacteria, Agrobacterium tumefaciens, Erwinia carotovora, Corynebacterium fascians, and Pseudomonas solanacearum. The antibacterial activity was evaluated in vitro by the agar dilution technique as a minimum inhibitory concentration (MIC) that was found to be dependent on the type of the microorganism tested. The most active product of chitosan was used for biofilm production enriched with geraniol and thymol (0.1 and 0.5%) and the films were also evaluated against the tested bacteria. The biological bioactivities summarized here may provide novel insights into the functions of chitosan and some monoterpenes and potentially allow their use for food protection from microbial attack.

  20. NMR Structure of the hypothetical protein encoded by the YjbJ gene from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Pineda-Lucena, Antonio; Liao, Jack; Wu, Bin; Yee, Adelinda; Cort, John R.; Kennedy, Michael A.; Edwards, Aled M.; Arrowsmith, Cheryl H.

    2002-06-01

    Here we describe the solution structure of YjbJ (gil418541) as part of a structural proteomics project on the feasibility of the high-throughput generation of samples from Escherichia coli for structural studies. YjbJ is a hypothetical protein from Escherichia coli protein of unknown function. It is conserved, showing significant sequence identity to four predicted prokaryotic proteins, also of unknown function (Figure 1A). These include gil16762921 from Salmonella enterica (S. typhi), gil17938413 from Agrobacterium tumefaciens, gil16265654 from Sinorizhobium meliloti, and gil15599932 from Pseudomona aeruginosa. The structure of YjbJ reveals a new variation of a common motif (four-helix bundle) that could not be predicted from the protein sequence. Although the biochemical function is unknown, the existence of patterns of conserved residues on the protein surface suggest that the fold and function of all these proteins could be similar.

  1. 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.

  2. Species-specific cell mobility of bacteria-feeding myxamoebae in plasmodial slime molds.

    Science.gov (United States)

    Hoppe, Thomas; Kutschera, Ulrich

    2015-01-01

    On decaying wood or litter in forests, plasmodial slime molds (myxomycetes) represent a large fraction of eukaryotic protists that feed on bacteria. In his seminal book Experimental Physiology of Plants (1865), Julius Sachs referred to the multinucleate plasmodium of myxomycetes, which were considered at that time as primitive plants (or fungi). Today it is well established that myxomycetes are members of the Amoebozoa (Protista). In this study we compare the mobility of myxamoebae of 3 European species, Lycogala epidendrum (order Liceales), Tubulifera arachnoidea, and Trichia decipiens (order Trichiales). Using agar plates, on which 3 separate bacterial species were cultivated as prey organisms (Methylobacterium mesophilicum, Escherichia coli, Agrobacterium tumefaciens), we document large differences in cell motility between the myxomycetes investigated. In addition, we show that the 3 species of myxamoebae can be distinguished based on their average cell size. These data shed light on the mode of co-occurrence via differential substrate utilization in these members of the Amoebozoa. PMID:26357877

  3. Transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.

  4. The role of plant bio-technologies with P4502E1 gene to remove radioactive contamination

    International Nuclear Information System (INIS)

    The aim of this research is to attempt removing pollution caused by some radioactive materials in polluted soils as a result exposure to radioactive particularly radioactive bombardments or misuse of atomic reactors, causing soil, water and air pollution. Two plant species were used in this study namely, sabins grandiflora and Arabidopsis thaliana. The bacterium victor Agrobacterium tumefaciens was used to transform the two plant species with used to transform the two plant species with the cytochrome gene P4502E1 which has been isolated from rabbit liver. The presence of the gene was investigated in plants using polymers chain reaction (PCR) after design of primers for this purpose. Transformation was proved after gene expression of P4502E1. Results showed that the plants were examined after transformation for their ability in removing the uranium, cesium and strontium in soils polluted with the three pollutants. (Author)

  5. Microspore culture of winter oilseed rape (Brassica napus L.) in conjunction with other in vitro technologies

    International Nuclear Information System (INIS)

    Microspore culture in conjunction with other technologies such as selection, mutagenesis and transformation has been used for the production of novel genotypes of Brassica napus L. for crop improvement. The example of in vitro selection of microspore - derived embryos includes: a) ploidy level, b) seed oil composition (for example: high level of erucic acid), c) genotypes with restorer gene for CMS-ogura system (by means of isozyme marker PGI-2 ), d) herbicide resistant forms. Efficiency of microspore mutagenesis has been tested by the treatment of freshly isolated microspores with UV and MNU. Direct delivery of foreign gene to the microspores (microprojectile bombardment) combined with the use of Agrobacterium tumefaciens to microspore derived embryos seems to be a promising way of oilseed rape transformation. (author)

  6. Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.

    Science.gov (United States)

    Schripsema, J; de Rudder, K E; van Vliet, T B; Lankhorst, P P; de Vroom, E; Kijne, J W; van Brussel, A A

    1996-01-01

    Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum. PMID:8550454

  7. The radioprotective effect of a new aminothiol (20-PRA

    Directory of Open Access Journals (Sweden)

    M.F. Dolabela

    1998-08-01

    Full Text Available We examined the radioprotective effect of aminothiol 2-N-propylamine-cyclo-hexanethiol (20-PRA on a human leukemic cell line (K562 following various radiation doses (5, 7.5 and 20 Gy using a source of 60Co g-rays. At 5 Gy and 1 nM 20-PRA, a substantial protective effect (58% was seen 24 h after irradiation, followed by a decrease at 48 h (11%. At the high radiation dose (20 Gy a low protective effect was also seen (35%. In addition, the antitumorigenic potential of 10 nM 20-PRA was shown by the inhibition of crown gall formation induced by Agrobacterium tumefaciens. The radioprotective potency of 20-PRA is 105-106 times higher than that of the aminothiol WR-1065 (N-(2-mercaptoethyl-1,3-diaminopropane whose protective effect is in the 0.1 to 1.0 mM range.

  8. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov;

    2008-01-01

    coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...... technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...... tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step with an...

  9. Cloning and transformation analysis of isoflavone synthase gene into Minshan Trifolium pratense.

    Science.gov (United States)

    Hu, H H; Jing, C Q; Liu, R; Li, W D; Feng, H G

    2015-01-01

    The aim of this study was to clone the isoflavone synthase (IFS) gene and establish the recombinant Minshan Trifolium pratense. The IFS gene was cloned from the callus of Minshan T. pratense using reverse transcription-polymerase chain reaction. The plant expression vector pRI101-AN-IFS was constructed and introduced into Agrobacterium tumefaciens strain LBA4404, and then screened under cephalosporin. IFS expression was detected by reverse transcription-polymerase chain reaction. The IFS gene was cloned successfully. Sequence analysis indicated that IFS gene had high homology with similar genes from other plants. The IFS-overexpressing callus was obtained by introducing the LBA4404-harboring IFS-pRI101-AN-IFS vector into T. pratense calluses. PMID:26345862

  10. Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

    DEFF Research Database (Denmark)

    Flodgaard, Lars; Dalgaard, Paw; Andersen, Jens Bo; Nielsen, Kristian Fog; Givskov, Michael Christian; Gram, Lone

    2005-01-01

    Bioluminescence is a common phenotype in marine bacteria, such As Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets......, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the......) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non...

  11. Transgenic tetraploid Isatis indigotica expressing Bt Cry1Ac and Pinellia ternata agglutinin showed enhanced resistance to moths and aphids.

    Science.gov (United States)

    Xiao, Ying; Wang, Kai; Ding, Ruxian; Zhang, Hanming; Di, Peng; Chen, Junfeng; Zhang, Lei; Chen, Wansheng

    2012-01-01

    Co-expression of multiple genes encoding different kinds of insect resistant proteins has been developed to confer a broader spectrum of pest control. Tetraploid Isatis indigotica Fort was transformed with a plasmid, p3300BP, containing Bacillus thuringiensis Cry1Ac gene (Bt) and Pinellia ternata agglutinin gene (Pta) and the selectable marker herbicide resistance gene (Bar) driven by the CaMV35S promoter via Agrobacterium tumefaciens-mediated transformation. The integration and expression of introduced genes in regenerated transgenic plants were confirmed by PCR and Western blot assays. Insect bioassay test demonstrated transgenic lines had significant inhibition to diamondback moths (Plutella xylostella L.) and peach potato aphids (Myzus persicae Sulzer) simultaneously. Our study reported here would be a great motivation for field culture of tetraploid I. indigotica, also providing an efficient molecular breeding strategy to provide insect tolerant plants. PMID:21559837

  12. A Plant-Based Transient Expression System for the Rapid Production of Malaria Vaccine Candidates.

    Science.gov (United States)

    Boes, Alexander; Reimann, Andreas; Twyman, Richard M; Fischer, Rainer; Schillberg, Stefan; Spiegel, Holger

    2016-01-01

    There are currently no vaccines that provide sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates, but none of them have fulfilled expectations. Therefore, combinations of key antigens from different stages of the parasites life cycle may be essential for the development of efficacious malaria vaccines. Following the identification of promising antigens using bioinformatics, proteomics, and/or immunological approaches, it is necessary to express, purify, and characterize these proteins and explore the potential of fusion constructs combining different antigens or antigen domains before committing to expensive and time-consuming clinical development. Here, using malaria vaccine candidates as an example, we describe how Agrobacterium tumefaciens-based transient expression in plants can be combined with a modular and flexible cloning strategy as a robust and versatile tool for the rapid production of candidate antigens during research and development. PMID:27076325

  13. Breeding of Coenzyme Q10 Produced Strain by Low-Energy Ion Implantation and Optimization of Coenzyme Q10 Fermentation

    Institute of Scientific and Technical Information of China (English)

    XU Dejun; ZHENG Zhiming; WANG Peng; WANG Li; YUAN Hang; YU Zengliang

    2008-01-01

    In order to increase the production efficiency of coenzyme Q10, the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents of coenzyme Q10 was selected. Subsequently, the conditions such as carbohydrate concentration, nitrogen source concentration, inoculum's size, seed age, aeration and temperature which might affect the production of CoQ10 were investigated in detail. Under optimal conditions, the maximum concentration of the intracellular CoQ10 reached 200.3 mg/L after 80 h fed-batch fermentation, about 245% increasing in CoQ10 production after ion implantation, compared to the original strain.

  14. Structural elucidation and biological activity of acyl-homoserine lactones from the phytopathogen Pantoea ananatis Serrano 1928.

    Science.gov (United States)

    Pomini, Armando M; Araújo, Welington L; Marsaioli, Anita J

    2006-08-01

    In Gram-negative bacteria, the acyl-homoserine lactones (acyl-HSLs) are the main signaling substances employed in cell-to-cell communication systems. This paper describes the chemical characterization of acyl-HSLs produced by the worldwide-spread phytopathogen Pantoea ananatis (Serrano 1928) by using gas chromatography-electron impact mass spectrometry. The absolute configuration of the major identified substance, (S)-(--)-N-hexanoyl-HSL, was determined with gas chromatography-flame ionization detection with a chiral capillary column. Biological activities of extracts, fractions, and synthetic products were evaluated with the specific reporter Agrobacterium tumefaciensNTL4(pZLR4) in beta-galactosidase expression assays. PMID:16900431

  15. Extraction estimation and gas chromatographic mass spectrometric analysis of the non polar fraction of the pistia stratiotes

    International Nuclear Information System (INIS)

    The non-polar compounds of the Pistia stratiotes were extracted using n-hexane as solvent. The extraction yields were determined both for the cold and hot extraction procedure as 8.50 +- 0.05% and 12.00 +- 0.05%, respectively. The extract was analyzed and separated into its components using GC equipped with FID and GC mass in separate experiments. The most important compounds identified in n-hexane extract of leaves of P. stratiotes are long chain compound of the nitrogenous nature and oxygenated compounds of mixed functional groups. The antibacterial activity of this fraction was investigated against eight pathogenic bacteria using disc diffusion method. Larger zones of inhibition were observed for Bacillus subtilis, Pseudomonas aeruginosa and Agrobacterium tumefaciens as compared to Klebsiella pneumoniaee and Staphylococcus aureus where the activity was relatively less. No activity was observed against Escherichia coli, Salmonella typhi and Bacillus atrophaeus. (author)

  16. Dicty_cDB: Contig-U13851-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6 |pid:none) Chlamydomonas reinhardtii CR051 pr... 115 6e-24 CP000091_1281( CP000091 |pid:none) Ralstonia eutr...703( CP000091 |pid:none) Ralstonia eutropha JMP134 chromo... 106 2e-21 CU928158_1763( CU928158 |pid:none) Escheric...91 |pid:none) Shewanella baltica OS195, compl... 102 4e-20 (Q9WYG0) RecName: Full=Uncharacterized oxidoreduc...E007869_2701( AE007869 |pid:none) Agrobacterium tumefaciens str. ... 102 5e-20 >T16638( T16638 ) hypothetical...tonia eutropha H16 chromoso... 176 2e-42 CP000699_360( CP000699 |pid:none) Sphingomonas wittic

  17. Dicty_cDB: Contig-U12666-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available pha-xylo... 161 3e-38 AB090361_1( AB090361 |pid:none) Mortierella alliacea agdA mRNA for... 160 3e-38 ( Q09901 ) RecName: Full... Agrobacterium tumefaciens str. C... 59 2e-07 AP006841_1248( AP006841 |pid:none) Bacteroides fragilis YCH46 ...us oryzae RIB40 genomic... 57 9e-07 CR626927_476( CR626927 |pid:none) Bacteroides fragili...s NCTC 9343, ... 56 1e-06 AP006841_551( AP006841 |pid:none) Bacteroides fragilis ...5... 55 3e-06 CR626927_1013( CR626927 |pid:none) Bacteroides fragilis NCTC 9343,... 55 3e-06 AE015928_3657( AE015928 |pid

  18. Combinatorial synthesis, lead identification, and antitumor study of a chalcone-based positional-scanning library.

    Science.gov (United States)

    Ullah, Ahsan; Ansari, Farzana Latif; Nazir, Samina; Mirza, Bushra

    2007-02-01

    A 175-member chalcone library was designed and synthesized from seven differently substituted acetophenones (A(1)-A(7)) and 25 differently substituted aryl or heteroaryl aldehydes (B(1)-B(25)). Potential lead compounds were identified by deconvolution of a two-dimensional library matrix via positional scanning, and the members of the most-active sub-libraries were synthesized and screened against crown-gall tumors with the aid of the potato-disc assay. The resulting hits gave rise to significant antitumor activities, with no antibacterial effect on the tumor-producing bacterium Agrobacterium tumefaciens. Two identified lead structures, (2E)-3-(2-chlorophenyl)-1-phenylprop-2-en-1-one (A(1)B(9)) and the hydroxy analogue (2E)-3-(2-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (A(2)B(9)), are promising candidates to be developed into highly effective anticancer chemotherapeutics. PMID:17311221

  19. Rapid identification of cytokinins by an immunological method

    Energy Technology Data Exchange (ETDEWEB)

    Morris, R.O.; Jameson, P.E.; Morris, J.W. (Univ. of Missouri-Columbia (USA)); Laloue, M. (Centre Nationale de la Recherche Scientifique, Gif-sur-Yvette (France))

    1991-04-01

    A method for rapid identification of bacterial cytokinins has been developed in which cultures are fed ({sup 3}H)adenine, the cytokinins (including, {sup 3}H-labeled cytokinins) are isolated by immunoaffinity chromatography, and analyzed by HPLC with on-line scintillation counting. Analysis of Agrobacterium tumefaciens strains showed that some produced primarily trans-zeatin, whereas others produced primarily trans-zeatin riboside. Pseudomonas syringae pv savastanoi produced mixtures of transzeatin, dihydrozeatin, 1{double prime}-methyl-trans-zeatin riboside, and other unknown cytokinin-like substances. Corynebacterium fascians, produced cis-zeatin, isopentenyladenine and isopentenyladenosine. The technique is designed for qualitative rather than quantitative studies and allows ready identification of bacterial cytokinins. It may also have utility in the study of plant cytokinins if adequate incorporation of label into cytokinin precursor pools can be achieved.

  20. Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

    DEFF Research Database (Denmark)

    Flodgaard, Lars; Dalgaard, Paw; Andersen, Jens Bo;

    2005-01-01

    Bioluminescence is a common phenotype in marine bacteria, such As Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets......, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction......) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non...