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Sample records for agrobacterium radiobacter strain

  1. 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

  2. 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

  3. 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...

  4. 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....

  5. High catalase production by Rhizobium radiobacter strain 2-1.

    Science.gov (United States)

    Nakayama, Mami; Nakajima-Kambe, Toshiaki; Katayama, Hideki; Higuchi, Kazuhiko; Kawasaki, Yoshio; Fuji, Ryujiro

    2008-12-01

    To promote the application of catalase for treating wastewater containing hydrogen peroxide, bacteria exhibiting high catalase activity were screened. A bacterium, designated strain 2-1, with high catalase activity was isolated from the wastewater of a beverage factory that uses hydrogen peroxide. Strain 2-1 was identified as Rhizobium radiobacter (formerly known as Agrobacterium tumefaciens) on the basis of both phenotypic and genotypic characterizations. Although some strains of R. radiobacter are known plant pathogens, polymerase chain reaction (PCR) analysis showed that strain 2-1 has no phytopathogenic factor. Compared with a type strain of R. radiobacter, the specific catalase activity of strain 2-1 was approximately 1000-fold. Moreover, Strain 2-1 grew faster and exhibited considerably higher catalase activity than other microorganisms that have been used for industrial catalase production. Strain 2-1 is harmless to humans and the environment and produces catalase efficiently, suggesting that strain 2-1 is a good resource for the mass production of catalase for the treatment of hydrogen peroxide-containing wastewater. PMID:19134550

  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. 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 ...

  8. 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...

  9. Three cases of post-cataract surgery endophthalmitis due to Rhizobium (Agrobacterium) radiobacter.

    Science.gov (United States)

    Moreau-Gaudry, Viviane; Chiquet, Christophe; Boisset, Sandrine; Croize, Jacques; Benito, Yvonne; Cornut, Pierre Loïc; Bron, Alain; Vandenesch, François; Maurin, Max

    2012-04-01

    We present three unrelated post-cataract surgery endophthalmitis cases caused by Rhizobium radiobacter, hospitalized in three different hospitals. Early diagnosis was obtained in two cases by bacterial DNA detection in vitreous samples. All patients recovered from infection, but pars plana vitrectomy was needed in two patients due to rapid clinical deterioration. PMID:22259203

  10. 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...

  11. 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

  12. Peritonitis due to Rhizobium radiobacter

    OpenAIRE

    Marta, R; Dâmaso, C; Silva, JE; M.De Almeida

    2011-01-01

    Rhizobium radiobacter (Agrobacterium radiobacter) is an aerobic Gram-negative rod belonging to Agrobacterium genus, a group of phytopathogenic bacteria present in the soil that has been implicated in human opportunistic infections. We report a clinical case of bacterial peritonitis in a 5-year-old child with chronic renal disease in peritoneal dialysis, who had a history of direct soil contact identified. The infection was treated with ceftazidime and piperaciline+tazobactam without relapses ...

  13. 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.

  14. 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

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

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    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. 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%。

  17. 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

  18. 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

  19. Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter.

    Science.gov (United States)

    Wang, Xiao-Yu-Zhu; Dong, Jin-Jun; Xu, Guo-Chao; Han, Rui-Zhi; Ni, Ye

    2016-10-01

    Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production. PMID:27312649

  20. 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

  1. 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

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

    Science.gov (United States)

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

    2016-01-01

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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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°

  12. 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.

  13. [Infective endocarditis by Rhizobium radiobacter. A case report].

    Science.gov (United States)

    Piñerúa Gonsálvez, Jean Félix; Zambrano Infantinot, Rosanna del Carmen; Calcaño, Carlos; Montaño, César; Fuenmayor, Zaida; Rodney, Henry; Rodney, Marianela

    2013-03-01

    Rhizobium radiobacter is a Gram-negative, nitrogen-fixing bacterium, which is found mainly on the ground. It rarely causes infections in humans. It has been associated with bacteremia, secondary to colonization of intravascular catheters, in immunocompromised patients. The aim of this paper was to report the case of an infective endocarditis caused by R. radiobacter, in a 47-year-old male, diagnosed with chronic kidney disease stage 5, on replacement therapy with hemodialysis and who attended the medical center with fever of two weeks duration. The patient was hospitalized and samples of peripheral blood were taken for culture. Empirical antibiotic therapy was started with cefotaxime plus vancomycin. The transthoracic echocardiogram revealed fusiform vegetation on the tricuspid valve, with grade III-IV/IV regurgitation. On the seventh day after the start of antibiotic therapy, the patient had a clinical and paraclinical improvement. The bacterium identified by blood culture was Rhizobium radiobacter, ceftriaxone-resistant and sensitive to imipenem, amikacin, ampicillin and ampicillin/sulbactam. Because of the clinical improvement, it was decided to continue treatment with vancomycin and additionally, with imipenem. At 14 days after the start of antibiotic therapy, the patient was discharged with outpatient treatment with imipenem up to six weeks of treatment. The control echocardiogram showed the absence of vegetation on the tricuspid valve. This case suggests that R. radiobacter can cause endocarditis in patients with intravascular catheters. PMID:23781714

  14. 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

  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. 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

  17. 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...

  18. 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

  19. Pseudo-Outbreak of Rhizobium radiobacter Infection Resulting from Laboratory Contamination of Saline Solution▿

    OpenAIRE

    Pereira, Lynette A.; Chan, Douglas Su Gin; Ng, Toon Mae; Lin, Raymond; Jureen, Roland; Fisher, Dale A; Tambyah, Paul A.

    2009-01-01

    We report a pseudo-outbreak of Rhizobium radiobacter infections resulting from contamination by a saline dispenser in the microbiology laboratory. Isolates from clinical specimens had identical antimicrobial susceptibilities and electrophoretic fingerprints. The episode resolved with autoclaving of the dispenser. This demonstrates the importance of timely, thorough investigation of unusual organisms, particularly when they appear as a cluster.

  20. Rhizobium Radiobacter Infection in a 27-Year-Old African American Woman With Munchausen Syndrome.

    Science.gov (United States)

    Sawhney, Sameer; Naab, Tammey; Oneal, Partricia

    2016-08-01

    Rhizobium radiobacter is an opportunistic, usually saprophytic, gram-negative bacillus found in agricultural soil. Isolation from blood has been reported most often in hospitalized patients harboring malignant neoplasms or human immunodeficiency virus (HIV) associated immunosuppression, who have catheter or medical device-related febrile neutropenia; treatment involves removal of the catheter or implanted medical device.(1)Herein, we report a case of a 27-year-old African American woman with sickle cell anemia who sought treatment of generalized body pain, shaking, chills, dyspnea, and fever, suggestive of sickle cell crisis. As part of her work up, routine blood cultures were drawn, revealing the presence of a Gram negative bacillus that was identified as the nonfermenter bacillus R. radiobacter The patient displayed a unique infection with R. radiobacter sepsis in a patient secondary to self-injection of organic material into a peripheral line during hospitalization. The growth of an unusual organism in the blood of a patient, without the usual risk factors of R. radiobacter, raised suspicion of a factitious psychiatric disorder known as Munchausen syndrome, which was confirmed when we discovered self-injection of feces and dirt into a central intravenous (IV) line. PMID:27107290

  1. 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 ...

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

    Directory of Open Access Journals (Sweden)

    Marisol Medina Sierra

    2011-12-01

    Full Text Available Kudzú (P. phaseoloides and carrot (D. carota roots were transformed in this survey into different kinds of culture medium by using five different A. rhizogenes strains. These presented different behaviour both in carrot transformation by A. rhizogenes 15834, A.r.8196 and A.r.2659 strains as well as kudzu transformation by A.r.15834 and A.r.1724 strains. Transformed carrot root growth was increased in WM culture medium, whilst transformed kudzu root growth did not increase in either the same medium or in modified MS medium. Transformed carrot roots were used for G. intrarradices increase and sporulation; however, wild AMF strains, isolated from a mining area (the lower Cauca area of Antioquia, did not grow either in roots from this specie or those from kudzu, in spite of this plant having great affinity for wild AMF strains. The results represent an advance in the procedure for DNA isolation and keeping AMF collections, required for other research.

  3. 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...

  4. 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.

  5. 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...

  6. 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.

  7. 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...

  8. 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...

  9. Biosynthesis of monobactam compounds: origin of the carbon atoms in the beta-lactam ring.

    OpenAIRE

    O'Sullivan, J; Gillum, A M; Aklonis, C A; Souser, M L; Sykes, R. B.

    1982-01-01

    The biosynthesis of monobactams by strains of Chromobacterium violaceum, Acetobacter sp., and Agrobacterium radiobacter was studied. Monobactams were produced during logarithmic growth by C. violaceum and Acetobacter sp. and during late log growth on glycerol and in stationary phase by A. radiobacter. The addition of various amino acids failed to significantly stimulate monobactam production in any of the producing organisms. Several 14C-amino acids and pyruvate were incorporated in vivo into...

  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. 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.

  12. 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

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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 ...

  18. 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...

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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...

  4. 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...

  5. 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 ...

  6. 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.

  7. 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...

  8. 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...

  9. 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

  10. Flocculating Properties and Production of the Compound Bioflocculant by Rhizobium Radiobacter F2 and Bacillus Sphaeicus F6

    Institute of Scientific and Technical Information of China (English)

    Lixin Li; Lingyan Feng; Fang Ma; Qianshen Zhao

    2015-01-01

    A compound bioflocculant CBF, produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its production and flocculating properties. The optimization of the culture medium constituents including carbon source, nitrogen source and C/N ratio, metal ions and ionic strength on CBF production were studied. Flocculating properties of CBF were examined by a series of experiments and CBF had good flocculating activities in kaolin suspension with divalent cations and stable over wide range of pH. Studies of the flocculating properties revealed that the flocculation could be stimulated by cations Ca2+, Mg2+, Fe2+, Al3+and Fe3+. In addition, it was stable at 4-30℃ in the presence of CaCl2 . It was found to be effective for flocculation of a kaolin suspension under neutral and weak alkaline conditions ( pH 7�0-9�0 ) , and flocculating activities of higher than 95% were obtained when the CBF concentrations among 6-14 mg/L at pH 8�0. The results of this study indicate that CBF is a potential replacement of conventional synthetic flocculants and is widely applied in water treatment and downstream processing of food and fermentation industries.

  11. 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...

  12. 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.

  13. 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.

  14. 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...

  15. 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 ...

  16. Evidence of transfer by conjugation of type IV secretion system genes between Bartonella species and Rhizobium radiobacter in amoeba.

    Directory of Open Access Journals (Sweden)

    Watcharee Saisongkorh

    Full Text Available BACKGROUND: Bartonella species cospeciate with mammals and live within erythrocytes. Even in these specific niches, it has been recently suggested by bioinformatic analysis of full genome sequences that Lateral Gene Transfer (LGT may occur but this has never been demonstrated biologically. Here we describe the sequence of the B. rattaustraliani (AUST/NH4(T circular plasmid (pNH4 that encodes the tra cluster of the Type IV secretion system (T4SS and we eventually provide evidence that Bartonella species may conjugate and exchange this plasmid inside amoeba. PRINCIPAL FINDINGS: The T4SS of pNH4 is critical for intracellular viability of bacterial pathogens, exhibits bioinformatic evidence of LGT among bacteria living in phagocytic protists. For instance, 3 out of 4 T4SS encoding genes from pNH4 appear to be closely related to Rhizobiales, suggesting that gene exchange occurs between intracellular bacteria from mammals (bartonellae and plants (Rhizobiales. We show that B. rattaustraliani and Rhizobium radiobacter both survived within the amoeba Acanthamoeba polyphaga and can conjugate together. Our findings further support the hypothesis that tra genes might also move into and out of bacterial communities by conjugation, which might be the primary means of genomic evolution for intracellular adaptation by cross-talk of interchangeable genes between Bartonella species and plant pathogens. CONCLUSIONS: Based on this, we speculate that amoeba favor the transfer of genes as phagocytic protists, which allows for intraphagocytic survival and, as a consequence, promotes the creation of potential pathogenic organisms.

  17. 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

  18. 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 ...

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

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

    Directory of Open Access Journals (Sweden)

    Mohammad M. Rana

    2016-07-01

    Full Text Available Tea (Camellia sinensis L. is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose. Additionally, the reporter genes β-glucuronidase (gusA and cyan fluorescent protein (cfp were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

  5. 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...

  6. 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

  7. 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

  8. 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...

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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...

  15. 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...

  16. 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.

  17. 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

  18. 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.

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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...

  5. 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.

  6. Disease: H01157 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available omised or chronically debilitated hosts and patients with indwelling catheters. C...Edwards MS Agrobacterium radiobacter bacteremia in pediatric patients: case report and review. Pediatr Infec...t L, Andre M, Lonchay C, Holemans X, Maton JP, Canon JL Agrobacterium radiobacter bacteremia in oncologic and geriatric patients

  7. 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.

  8. 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...

  9. 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...

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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...

  1. 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...

  2. 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.

  3. 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.

  4. 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 ...

  5. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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...

  14. 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...

  15. 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....

  16. 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...

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Effects of Biocontrol Strain E26 on Some Ecological Factors

    Institute of Scientific and Technical Information of China (English)

    WANG Hui-min; SUN Yan-li; WANG Jian-hui

    2002-01-01

    This study was to evaluate the ecological risk of strain E26 (Agrobacterium sp. ) by detecting its survival in waters and its effects on rhizosphere microorganisms. The data showed that E26 could not be detected in distilled water, tap water, river water, and rainwater after 36, 36, 8, and 9 days, respectively. E26 did not reveal significant effects on the population of bacteria, fungi, and actinomyces in rhizosphere soil and on the root surface of grapevines.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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...

  11. 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...

  12. 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

  13. 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...

  14. 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.

  15. 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.

  16. 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.

  17. Response of Kalanchoe daigremontiana to wounding and infection with Agrobacterium tumefaciens

    OpenAIRE

    Tkalec, Mirta; Car, Diana; GOSPOČIĆ, JANKO; KRIŽAIĆ, IVA; DUŽ, KAROLINA; VIDAKOVIĆ-CIFREK, ŽELJKA

    2012-01-01

    Background and Purpose: Transformation of plant tissue with Agrobacterium tumefaciens includes wounding of plant and subsequent infection by bacteria. Polyphenol oxidase activity and oxidative stress parameters – the content of H2O2, as well as activity and isoenzymes of antioxidative enzymes catalase, pyrogallol and guaiacol peroxidase were investigated as markers of plant response to wounding and infection. Materials and Methods: Five tissue types – healthy tissue, wounded tissue, tissue in...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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

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

    Science.gov (United States)

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

    2015-12-01

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

  6. 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

  7. [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

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

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  9. 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

  10. 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

  11. Development of a biocontrol agent for plant disease control with special emphasis on the near commercial fungal antagonist Clonostachys rosea strain "IK726"

    DEFF Research Database (Denmark)

    Jensen, Dan Funck; Knudsen, Inge M.B.; Lübeck, Mette;

    2007-01-01

    . Among the success stories for control of seed- and soilborne diseases are fungal biocontrol agents based on Trichoderma harzianum, Clonostachys rosea and Conithyrium minitans, and bacterial biocontrol agents based on strains of Agrobacterium, Pseudomonas and Streptomyces. We have developed C. rosea...

  12. 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...

  13. 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

  14. 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

  15. 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

  16. 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...

  17. 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.

  18. 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...

  19. 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.

  20. 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...

  1. 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.

  2. 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.

  3. 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...

  4. 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

  5. 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...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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%.

  11. 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

  12. 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

  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. 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

  15. 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...

  16. 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....

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Polyana Kelly Martins

    2015-06-01

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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. Complete Genome Sequence of the Sesbania Symbiont and Rice Growth-Promoting Endophyte Rhizobium sp. Strain IRBG74

    OpenAIRE

    Crook, Matthew B.; Mitra, Shubhajit; Ané, Jean-Michel; Sadowsky, Michael J.; Gyaneshwar, Prasad

    2013-01-01

    Rhizobium sp. strain IRBG74 is the first known nitrogen-fixing symbiont in the Agrobacterium/Rhizobium clade that nodulates the aquatic legume Sesbania sp. and is also a growth-promoting endophyte of wetland rice. Here, we present the sequence of the IRBG74 genome, which is composed of a circular chromosome, a linear chromosome, and a symbiotic plasmid, pIRBG74a.

  16. 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...

  17. 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...

  18. 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

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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

  4. 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...

  5. 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

  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. 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

  8. 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)

  9. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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

  12. 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

  13. [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

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

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

    Directory of Open Access Journals (Sweden)

    Mariena Ketudat-Cairns

    2011-02-01

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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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

  9. 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

  10. 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

  11. ВПЛИВ LACTOBACILLUS PLANTARUM НА УТВОРЕННЯ ПУХЛИН , СПРИЧИНЕНИХ RHIZOBIUM RADIOBACTER

    OpenAIRE

    Ліманська, Н. В.; Коротаєва, Н. В.; Ямборко, Г. В.; Іваниця, В. О.

    2015-01-01

    Целью исследования было изучение влияния бактерий Lactobacillus plantarum и их метаболитов на образование опухолей у растений Kalanchoe daigremontiana Mill. при инокуляции возбудителями бактериального рака Rhizobium radiobacter. Методы. Листья тест-растений инокулировали культурами R. radiobacter С58 и трех штаммов Lactobacillus plantarum или их надосадочными жидкостями (НОЖ) как с первичными низкими значениями рН (4,1–4,3), так и с нейтрализованными рН (6,5) в соотношении 1:1. Через 45 дней ...

  12. 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...

  13. 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

  14. 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.

  15. [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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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...

  4. 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...

  5. 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.

  6. 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.

  7. 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

  8. 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。

  9. 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

    compounds is concentrated. Because of this,the production of hairy roots becomes an alternative technique for scale production of the metabolites of interest. In this research, a protocol was implemented using agroinfection of U. tomentosa shoot tips with wild strains of Agrobacterium rizhogenes (AR1500 and A4RS and maintenance of hairy roots obtained. In collaboration with the Laboratory of Molecular Biology of the PIPRA program (UC Davis, we determined the efficacy of this agroinfection protocol and the use of other molecular tools which showed satisfactory results in testing agroinfection under the methodologies established.

  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. 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...

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

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

    Directory of Open Access Journals (Sweden)

    Lashermes Philippe

    2011-05-01

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

  19. 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

  20. 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....

  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. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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

  7. Dicty_cDB: Contig-U12999-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CP001614 |pid:none) Teredinibacter turnerae T7901, c... 54 8e-06 M65066_1( M65066 |pid:none) Human cAMP-dep...0628_3079( CP000628 |pid:none) Agrobacterium radiobacter K84 c... 37 0.98 CP001614_2742( CP001614 |pid:none) Teredinibacter turner

  8. 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.

  9. Genetic analysis of agrocin 84 production and immunity in Agrobacterium spp.

    OpenAIRE

    Ryder, M H; Slota, J.E.; Scarim, A; Farrand, S K

    1987-01-01

    Mutations affecting agrocin production on the 48-kilobase (kb) plasmid, pAgK84, can be complemented in trans with cloned portions of the plasmid. Five complementation groups ranging in minimum size from 1.2 to 5.6 kb were identified within a 14-kb segment. Plasmid pAgK84-encoded immunity to agrocin 84 was located to two separate regions of the plasmid. Either region alone was sufficient to protect sensitive strains, and both loci mapped to the agrocin 84 biosynthesis region. One region is loc...

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. Geobacteraceae strains and methods

    Science.gov (United States)

    Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

    2015-07-07

    Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

  18. Muscle strain (image)

    Science.gov (United States)

    A muscle strain is the stretching or tearing of muscle fibers. A muscle strain can be caused by sports, exercise, a ... something that is too heavy. Symptoms of a muscle strain include pain, tightness, swelling, tenderness, and the ...

  19. Muscle strain treatment

    Science.gov (United States)

    Treatment - muscle strain ... Question: How do you treat a muscle strain ? Answer: Rest the strained muscle and apply ice for the first few days after the injury. Anti-inflammatory medicines or acetaminophen ( ...

  20. 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.

  1. 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 ...

  2. 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...

  3. 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...

  4. 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...

  5. Program Calibrates Strain Gauges

    Science.gov (United States)

    Okazaki, Gary D.

    1991-01-01

    Program dramatically reduces personnel and time requirements for acceptance tests of hardware. Data-acquisition system reads output from Wheatstone full-bridge strain-gauge circuit and calculates strain by use of shunt calibration technique. Program nearly instantaneously tabulates and plots strain data against load-cell outputs. Modified to acquire strain data for other specimens wherever full-bridge strain-gauge circuits used. Written in HP BASIC.

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

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  8. Strains and Sprains

    Science.gov (United States)

    ... ON THIS TOPIC Sports Medicine Center Preventing Children's Sports Injuries Computer-Related Repetitive Stress Injuries Knee Injuries Broken ... Knee Injuries Sports and Exercise Safety Dealing With Sports Injuries Groin Strain Ankle Sprains Strains and Sprains Stretching ...

  9. Strained Silicon Photonics

    OpenAIRE

    Wehrspohn, Ralf B; Jörg Schilling; Christian Bohley; Clemens Schriever

    2012-01-01

    A review of recent progress in the field of strained silicon photonics is presented. The application of strain to waveguide and photonic crystal structures can be used to alter the linear and nonlinear optical properties of these devices. Here, methods for the fabrication of strained devices are summarized and recent examples of linear and nonlinear optical devices are discussed. Furthermore, the relation between strain and the enhancement of the second order nonlinear susceptibility is inves...

  10. 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...

  11. 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.

  12. 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

  13. 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.

  14. "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

  15. 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

  16. 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

  17. Biological control of grape crown gall by strain f2/5 is not associated with agrocin production or competition for attachment sites on grape cells.

    Science.gov (United States)

    Burr, T J; Reid, C L; Tagliati, E; Bazzi, C; Süle, S

    1997-07-01

    ABSTRACT Agrocin-minus mutants of nontumorigenic Agrobacterium vitis strain F2/5 controlled grape crown gall as well as the wild-type strain, indicating that agrocin is not a major factor in the mechanism of biological control. Relative levels of attachment to grape cells by tumorigenic and biocontrol strains were also measured. Attachment of tumorigenic strains (CG49 and K306) and biological control strains (F2/5 and agrocin-minus mutant 1077) was often reduced when mixtures of the strains were applied. However, high populations (10(3) to 10(5) CFU/ml) of all strains attached following mixed inoculations, suggesting that competition for attachment sites is also not a factor in the mechanism of biological control. Transfer of T-DNA to grape by CG49 was prevented or greatly inhibited in the presence of F2/5 or 1077 as measured by expression of the GUS reporter gene. The Ti plasmid virulence genes, however, were induced by exudates from grape shoots that had been inoculated with F2/5. Sonicated and autoclaved preparations of F2/5 and 1077 did not control crown gall or inhibit T-DNA transfer. Control by F2/5 is specific to grape, since gall formation on tomato, sunflower, and Kalanchoe daigremontiana were not inhibited. PMID:18945092

  18. Diffraction tomography of strain

    OpenAIRE

    Lionheart, W R B; Withers, P. J.

    2015-01-01

    We consider whether it is possible to recover the three dimensional strain field tomographically from neutron and x-ray diffraction data for polycrystalline materials. We show that the distribution of strain transverse to a ray cannot be deduced from one diffraction pattern accumulated along that path, but that a certain moment of that data corresponds to the transverse ray transform of the strain tensor and so may be recovered by inverting that transform given sufficient data. We show that t...

  19. Strained Silicon Photonics

    Directory of Open Access Journals (Sweden)

    Ralf B. Wehrspohn

    2012-05-01

    Full Text Available A review of recent progress in the field of strained silicon photonics is presented. The application of strain to waveguide and photonic crystal structures can be used to alter the linear and nonlinear optical properties of these devices. Here, methods for the fabrication of strained devices are summarized and recent examples of linear and nonlinear optical devices are discussed. Furthermore, the relation between strain and the enhancement of the second order nonlinear susceptibility is investigated, which may enable the construction of optically active photonic devices made of silicon.

  20. Strain-engineered MOSFETs

    CERN Document Server

    Maiti, CK

    2012-01-01

    Currently strain engineering is the main technique used to enhance the performance of advanced silicon-based metal-oxide-semiconductor field-effect transistors (MOSFETs). Written from an engineering application standpoint, Strain-Engineered MOSFETs introduces promising strain techniques to fabricate strain-engineered MOSFETs and to methods to assess the applications of these techniques. The book provides the background and physical insight needed to understand new and future developments in the modeling and design of n- and p-MOSFETs at nanoscale. This book focuses on recent developments in st

  1. Wpływ szczepów bakterii wyizolowanych z hydroponicznej uprawy sałaty (Lactuca sativa L. na wzrost siewek sałaty, rosnących w obecnosci rożnych form pożywienia azotowego [Influence of bacterial strains isolated from hydroponic cultures of lettuce (Lactuca sativa L. on the growth of lettuce seedlings growing in the presence of various forms of nitrogen nutrition

    Directory of Open Access Journals (Sweden)

    Z. Kobierzyńska-Gołąb

    2015-06-01

    Full Text Available 320 bacterial strains isolated from the surface of cultivated plants, as well as from other parts of hydroponic cultures showed stimulating (49 bacterial strains or inhibitory (9 bacterial strains properties in respect to the investigated plant. The following bacteria were isolated: Pseudomonas, Flavobacterium, Agrobacterium, Achromobacter and Chromobacterium. The effects of active bacterial strains on the growth of seedlings were investigated in dependence on the kind of inorganic form of nitrogen present in the nutrient solutions. The same bacterial strains exerted a stimulating effect on seedlings growing on nitrates, weaker stimulation was observed in cultures with ammonium nitrate; the growth of lettuce seedlings on nutrient solution with ammonium only, was, as a rule, inhibited by the bacteria.

  2. Highly Strained Organophosphorus Compounds

    NARCIS (Netherlands)

    Slootweg, J.C.

    2005-01-01

    In our research on small, strained organophosphorus ring systems we became interested in the synthesis and applications of species that are even more strained than the parent phosphirane, by introducing an exocyclic double bond (methylenephosphirane), and by cyclopropyl spirofusion to the edge (e.g

  3. Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

    Directory of Open Access Journals (Sweden)

    Das Pralay

    2011-05-01

    Full Text Available Abstract Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide, respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

  4. 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

  5. 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

  6. THE SCIENTIFIC BASIS FOR THE CREATION OF NEW FORMS OF MICROBIAL BIOCHEMICALS

    OpenAIRE

    A.P. KOZHEMYAKOV; Yu.V. LAKTIONOV; T.A. POPOVA; A.G. ORLOVA; A.L. KOKORINA; Vaishlya, O. B.; E.V. AGAFONOV; S.A. GUZHVIN; A.A. CHURAKOV; M.T. YAKOVLEVA

    2015-01-01

    Herein we summarize the results of a comprehensive study aimed on the creation of liquid form of biological products for symbiotic and associative rhizobacteria. The objects of study were nodule bacteria from the rhizosphere of galegae (Rhizobium galegae), soybean (Bradyrhizobium japonicum) and associative rhizobacteria (Arthrobacter mysorens, Azospirillum brasilense, Agrobacterium radiobacter), as well as plants of soybean (Glycine max L.), barley (Hordeum L.), alfalfa (Medicago L.), etc. As...

  7. Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate

    OpenAIRE

    van Loo, Bert; Permentier, Hjalmar P.; Kingma, Jaap; Baldascini, Helen; Janssen, Dick B.

    2008-01-01

    Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followe...

  8. Sprains and Strains

    Science.gov (United States)

    ... people at risk for strains. Gymnastics, tennis, rowing, golf, and other sports that require extensive gripping can ... Trials and You was designed to help people learn more about clinical trials, why they matter, and ...

  9. The strained state cosmology

    CERN Document Server

    Tartaglia, Angelo

    2015-01-01

    Starting from some relevant facts concerning the behaviour of the universe over large scale and time span, the analogy between the geometric approach of General Relativ- ity and the classical description of an elastic strained material continuum is discussed. Extending the elastic deformation approach to four dimensions it is shown that the accelerated expansion of the universe is recovered. The strain field of space-time repro- duces properties similar to the ones ascribed to the dark energy currently called in to explain the accelerated expansion. The strain field in the primordial universe behaves as radiation, but asymptotically it reproduces the cosmological constant. Subjecting the theory to a number of cosmological tests confirms the soundness of the approach and gives an optimal value for the one parameter of the model, i.e. the bulk modulus of the space-time continuum. Finally various aspects of the Strained State Cosmology (SSC) are discussed and contrasted with some non-linear massive gravity theor...

  10. 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.

  11. An Ultrasonic Strain Gauge

    OpenAIRE

    Kersemans, Mathias; Allaer, Klaas; Degrieck, Joris; Van Den Abeele, Koen; Pyl, Lincy; Zastavnik, Filip; Sol, Hugo; Van Paegem, Wim

    2014-01-01

    A method is introduced for the measurements of strain exploiting the interaction between ultrasound waves and characteristics of the insonified specimen. First, the response of obliquely incident harmonic waves to a deterministic surface roughness is utilized. Analysis of backscattered amplitudes in Bragg diffraction geometry then yields a measure for the in-plane strain field by mapping any shift in angular dependency. Secondly, the analysis of the reflection characteristics of normal incide...

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. MEMS Graphene Strain Sensor

    Science.gov (United States)

    Young, Clinton Wen-Chieh

    Graphene is a two dimensional honeycomb structure of sp2 hybridized carbon atoms that has possibilities in many applications due to its excellent mechanical and electrical properties. One application for Graphene is in the field of sensors. Graphene's electronic properties do not degrade when it undergoes mechanical strain which is advantageous for strain sensors. In this thesis, certain properties, such as the piezo-resistivity and flexibility, of graphene will be explored to show how they can be utilized to make a strain sensing device. Our original fabrication process of patterning graphene and the transfer process of graphene onto a flexible substrate will be discussed. The development of a stretchable and flexible graphene based rosette strain sensor will also be detailed. Developing a novel, reliable patterning process for the graphene is the first step to manufacture a stretchable graphene based sensor. The graphene was patterned using a photolithography and etching process that was developed by our research team, then it was transferred to a flexible polymer substrate with the use of a combination of soft lithography and wet etching of the Ni foil with ferric chloride solution. Graphene patterning is an essential step in fabricating reliable and sensitive sensors. With this process, graphene can be consistently patterned into different shapes and sizes. To utilize the graphene as the sensing material it also needs to be transferred onto a flexible substrate. The innovative transfer process developed by our research team consistently adheres graphene to a flexible PDMS substrate while removing the original nickel substrate. In the end, the graphene was transferred from the metal substrate to the desired flexible substrate. This process was repeated multiple times to create a stack and multilayer device. While many graphene-based strain sensors have been developed, they are uni-directional and can only measure the strain applied on the sensor in a principle

  2. Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Lixing Weng

    2014-04-01

    Full Text Available Quorum sensing (QS has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 µg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 µg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI and cognate receptor (lasR and rhlR genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s interferes with the las system and significantly inhibits biofilm formation.

  3. Longitudinal misalignment based strain sensor

    OpenAIRE

    Andrews, Jeffrey Pratt

    1989-01-01

    A practical fiber optic strain sensor has been developed to measure strains in the range of 0.0 to 2.0 percent strain with a resolution ranging between 10 and 100 microstrain depending on sensor design choices. This intensity based sensor measures strain by monitoring strain induced longitudinal misalignment in a novel fiber interconnection. This interconnection is created by aligning fibers within a segment of hollow core fiber. Related splice loss mechanisms are investigated ...

  4. ConStrains identifies microbial strains in metagenomic datasets.

    Science.gov (United States)

    Luo, Chengwei; Knight, Rob; Siljander, Heli; Knip, Mikael; Xavier, Ramnik J; Gevers, Dirk

    2015-10-01

    An important fraction of microbial diversity is harbored in strain individuality, so identification of conspecific bacterial strains is imperative for improved understanding of microbial community functions. Limitations in bioinformatics and sequencing technologies have to date precluded strain identification owing to difficulties in phasing short reads to faithfully recover the original strain-level genotypes, which have highly similar sequences. We present ConStrains, an open-source algorithm that identifies conspecific strains from metagenomic sequence data and reconstructs the phylogeny of these strains in microbial communities. The algorithm uses single-nucleotide polymorphism (SNP) patterns in a set of universal genes to infer within-species structures that represent strains. Applying ConStrains to simulated and host-derived datasets provides insights into microbial community dynamics. PMID:26344404

  5. The strained state cosmology

    Science.gov (United States)

    Tartaglia, Angelo

    2016-01-01

    Starting from some relevant facts concerning the behavior of the universe over large scale and time span, the analogy between the geometric approach of General Relativity and the classical description of an elastic strained material continuum is discussed. Extending the elastic deformation approach to four dimensions it is shown that the accelerated expansion of the universe is recovered. The strain field of space-time reproduces properties similar to the ones ascribed to the dark energy currently called in to explain the accelerated expansion. The strain field in the primordial universe behaves as radiation, but asymptotically it reproduces the cosmological constant. Subjecting the theory to a number of cosmological tests confirms the soundness of the approach and gives an optimal value for the one parameter of the model, i.e. the bulk modulus of the space-time continuum. Finally various aspects of the Strained State Cosmology (SSC) are discussed and contrasted with some non-linear massive gravity theories. The possible role of structure topological defects is also mentioned. The conclusion is that SSC is at least as good as the ΛCDM standard cosmology, giving a more intuitive interpretation of the physical nature of the phenomena.

  6. 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

  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. 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...

  9. 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...

  10. Novel strained superjunction VDMOS

    Science.gov (United States)

    Naugarhiya, Alok; Dubey, Shashank; Kondekar, Pravin N.

    2015-09-01

    In this paper, we have proposed novel strained superjunction (s-SJ) vertical double diffused MOS (VDMOS). Through channel engineering, we have introduced strain effects in s-SJ device using thin separate p-type silicon-germanium (p-SiGe) layer over silicon p-pillar. Further, we have designed process flow for the possible fabrication of s-SJ VDMOS. The proposed s-SJ devices fitted with less input capacitance (Cin) and 1.2∼3 times higher output current density than conventional SJ VDMOS. Therefore, 40% less gate charge (Qg) is required to turn-on the s-SJ VDMOS and Ron A is optimized in between 12% and 46%.

  11. Strains in general relativity

    International Nuclear Information System (INIS)

    The definition of relative accelerations and strains among a set of comoving particles is studied in connection with the geometric properties of the frame adapted to a 'fiducial observer'. We find that a relativistically complete and correct definition of strains must take into account the transport law of the chosen spatial triad along the observer's congruence. We use special congruences of (accelerated) test particles in some familiar spacetimes to elucidate such a point. The celebrated idea of Szekeres' compass of inertia, arising when studying geodesic deviation among a set of free-falling particles, is here generalized to the case of accelerated particles. In doing so we have naturally contributed to the theory of relativistic gravity gradiometer. Moreover, our analysis was made in an observer-dependent form, a fact that would be very useful when thinking about general relativistic tests on space stations orbiting compact objects like black holes and also in other interesting gravitational situations

  12. Strains in General Relativity

    CERN Document Server

    Bini, Donato; Geralico, Andrea

    2014-01-01

    The definition of relative accelerations and strains among a set of comoving particles is studied in connection with the geometric properties of the frame adapted to a "fiducial observer." We find that a relativistically complete and correct definition of strains must take into account the transport law of the chosen spatial triad along the observer's congruence. We use special congruences of (accelerated) test particles in some familiar spacetimes to elucidate such a point. The celebrated idea of Szekeres' compass of inertia, arising when studying geodesic deviation among a set of free-falling particles, is here generalized to the case of accelerated particles. In doing so we have naturally contributed to the theory of relativistic gravity gradiometer. Moreover, our analysis was made in an observer-dependent form, a fact that would be very useful when thinking about general relativistic tests on space stations orbiting compact objects like black holes and also in other interesting gravitational situations.

  13. Geodetic surveying of strains

    Energy Technology Data Exchange (ETDEWEB)

    Meixner, H.

    1980-11-01

    This article evaluates methods and apparatus used in the German Democratic Republic to assess vertical and horizontal deformations and strains caused by coal mining and mining of other deposits. Each of the methods is characterized; type of measuring apparatus and its accuracy is given. The following conventional methods of surveying are characterized: (I.) vertical strains: measurement with hydrostatic tube balance (apparatus produced by the Praezisionsmechanik Freiberg, with accuracy of 0.01 mm), trigonometric levelling (error up to 10 mm), geometric levelling (NI 007 and NI 008 apparatus produced by Carl Zeiss JENA), and direct height measuring; (II.) horizontal strains: direct surveying with accuracy higher than 5 mm/50 m, indirect surveying (theodolite THEO 010 A produced by the Carl Zeiss JENA), and alignment method (using apparatus produced by Praezisionsmechanik, Freiberg). Advanced surveying methods are also discussed: electro-optical method (using EOK 2000 range finder produced by Carl Zeiss JENA, with average error ranging from 5 mm to 10 mm), laser method (LF-1 and LFG-1 lasers produced by Carl Zeiss JENA), measuring gyroscope used to measure contorsion (MRK 2 gyroscope produced by the Praezisionsmechanik, Freiberg); photogrammetry: stereophotogrammetry and so-called parallel photogrammetry. (15 refs.) (In Polish)

  14. Temperature controlled strain gaged extensometer

    Science.gov (United States)

    Ramos, G. L.; Seplow, S.

    1968-01-01

    Temperature controlled strain-gaged extensometer measures longitudinal and girth deflections of pressure vessels in excess of one percent strain during pressurization and depressurization with cryogenic fluids at cryogenic temperatures. The device is of beryllium-copper strips.

  15. MEMS Resonant Strain Sensor Integration

    OpenAIRE

    Myers, David Richard

    2010-01-01

    Despite commercial availability since the 1950's, silicon strain sensors have not experienced the same success as other microdevices, such as accelerometers, pressure sensors, and inkjet heads. Strain sensors measure mechanical deformation and could be used in many structural components, improving safety, controls, and manufacturing tolerances. This thesis examines major strain sensing techniques and highlights both advantages and disadvantages of each. MEMS resonant strain gauges are iden...

  16. Strain calibration of optical FBG-based strain sensors

    Science.gov (United States)

    Roths, Johannes; Wilfert, Andre; Kratzer, Peter; Jülich, Florian; Kuttler, Rolf

    2010-09-01

    A facility for strain sensitivity calibration of optical FBG-based strain sensors according to the German VDI/VDE 2660 guideline was established and characterized. Statistical analysis of several calibration measurement series performed with one single type of FBG strain sensor and application technique showed a reproducibility of 0.15%. Strain sensitivities for FBGs inscribed in two different types of optical fibres (GF1B and PR2008) showed significantly different strain sensitivities of k = 0.7885+/-0.0026 and k = 0.7758+/-0.0024, respectively.

  17. Precision determination of strains by strain measurement method

    International Nuclear Information System (INIS)

    Method for precision strain measurement determination of strains measurement determination of strains by direct measurement of the resistance of strain gauge resistors put on the sample, by constant current bridge MOD-61 of 0.05 class using a zero method by single bridge double-clamp circuit is described. Strain measurement technique was used to investigate residual stresses in tubular samples. Sample external diameter made up 30 mm, the length - 180, the wall thickness-11 mm. To evaluate the method sensitivity, annealed samples were investigated (annealing temperature-850-880 deg C, hold-up-2 h, cooling in the furnace). It is ascertained, that the method provides for 0.3-0.5 μm strain measurement accuracy even if sample strains do not exceed 3.0-3.6 μm and correspond to residual stress level of no more than 20 MPa

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

    Institute of Scientific and Technical Information of China (English)

    程琴; 金刚; 李慧敏; 彭欣怡; 黎萍; 王丽萍

    2014-01-01

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

  19. Development of reversible strain gage

    International Nuclear Information System (INIS)

    A high-temperature strain gage which can be peeled after taking required apparent strain measurements in a furnace and can be attached reverse-side-up at the point of interest on a test structure was developed. Using the ''reversible'' strain gage with selected room-temperature curing type polyester adhesive, one can expect to measure thermal strain accurately, especially for on large structures, at the first test in temperature up to 250 deg C. The repeatability of apparent strains for about 100 reversible gages was within 50 microstrains of difference at 250 deg C (within 30 microstrains of difference for 80 % of the test gages). (author)

  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. [Echinococcus and strain concepts].

    Science.gov (United States)

    Utük, Armağan Erdem; Simsek, Sami

    2008-01-01

    Hydatid disease (echinococcosis) is one of the most important parasitic zoonoses and remains a public health and economic problem all over the world. Echinococcus granulosus includes a number of genetic variants and, up to date, analyses of mitochondrial DNA sequences have identified ten distinct genetic types (genotypes G1-10). This categorization follows closely the pattern of strain variation emerging based on biological characteristics. The extensive variation in E. granulosus may influence life-cycle patterns, host specificity, development rate, antigenicity, transmission dynamics, sensitivity to chemotherapeutic agents, and pathology. In this review, the recent genetic characterizations of Echinococcus genus have been summarized. PMID:18351549

  2. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast to...... uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...... against the UPEC strain CFT073 was also studied. The different ABU strains displayed a wide variety of the measured characteristics. Half of the ABU strains displayed functional type 1 fimbriae while only one expressed functional P fimbriae. A good correlation between the growth rate of a particular...

  3. Reconstruction of axisymmetric strain distributions via neutron strain tomography

    International Nuclear Information System (INIS)

    Predicting the behaviour of structural components under a particular set of loading conditions requires knowledge of the residual elastic strain distribution throughout the bulk of these components. Characterising the 3D strain state at any particular point involves the measurement of six independent components which make up the second order strain tensor. Mapping the complete strain distribution throughout large volumes thus presents significant practical challenges. One possible solution to this problem is to reconstruct the 3D variation of strain components using tomographic techniques. The basic principle underpinning this idea is that the multi-component strain tensor can be reconstructed from a redundant set of lower order projection data. Here we demonstrate this fundamental concept for two samples: a shrink fit ‘ring-and-plug’ sample, and a spray-quenched circular cylinder, both possessing axially symmetric internal strain distribution. We present and contrast different approaches to the strain tomography problem. The methods described here can also be readily applied to high-energy X-ray diffraction measurements and represent an important step toward developing the tomographic reconstruction framework for strain tensor distributions of arbitrary complexity. The major benefit of neutron strain tomography is that the incident beam flux is utilised more fully, greatly reducing the data collection times. Using micro-channel plate (MCP) neutron detectors, a spatial resolution of the order of 0.1 mm can be achieved .

  4. Improved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vitreoscilla hemoglobin.

    Science.gov (United States)

    Mora-Lugo, Rodrigo; Madrigal, Marvin; Yelemane, Vikas; Fernandez-Lahore, Marcelo

    2015-11-01

    The biotechnological value of Aspergillus sojae ATCC 20235 (A. sojae) for production of pectinases in solid-state fermentation (SSF) has been demonstrated recently. However, a common drawback of fungal solid-state cultures is the poor diffusion of oxygen into the fungi that limits its growth and biological productivity. The bacterial Vitreoscilla hemoglobin (VHb) has favored the metabolism and productivities of various bacterial and yeast strains besides alleviating hypoxic conditions of its native host, but the use of VHb in filamentous fungi still remains poor explored. Based on the known effects of VHb, this study assessed its applicability to improve A. sojae performance in SSF. The VHb gene (vgb) under control of the constitutive Aspergillus nidulants gpdA promoter was introduced into the genome of A. sojae by Agrobacterium-mediated transformation. Successful fungal transformants were identified by fluorescence microscopy and polymerase chain reaction (PCR) analyses. In solid-state cultures, the content of protease, exo-polygalacturonase (exo-PG), and exo-polymethylgalacturonase (exo-PMG) of the transformed fungus (A. sojae vgb+) improved were 26, 60, and 44 % higher, respectively, in comparison to its parental strain (A. sojae wt). Similarly, biomass content was also 1.3 times higher in the transformant strain. No significant difference was observed in endo-polygalacturonase (endo-PG) content between both fungal strains, suggesting dissimilar effects of VHb towards different enzymatic productions. Overall, our results show that biomass, protease, and exo-pectinase content of A. sojae in SSF can be improved by transformation with VHb. PMID:26224427

  5. 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

  6. Inhibitory and Toxic Effects of Volatiles Emitted by Strains of Pseudomonas and Serratia on Growth and Survival of Selected Microorganisms, Caenorhabditis elegans, and Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Alexandra A. Popova

    2014-01-01

    Full Text Available In previous research, volatile organic compounds (VOCs emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans. VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds—ketones (2-nonanone, 2-heptanone, 2-undecanone and dimethyl disulfide—were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

  7. Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster.

    Science.gov (United States)

    Popova, Alexandra A; Koksharova, Olga A; Lipasova, Valentina A; Zaitseva, Julia V; Katkova-Zhukotskaya, Olga A; Eremina, Svetlana Iu; Mironov, Alexander S; Chernin, Leonid S; Khmel, Inessa A

    2014-01-01

    In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions. PMID:25006575

  8. 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

  9. 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

  10. Strain in silicon nanowire beams

    Science.gov (United States)

    Ureña, Ferran; Olsen, Sarah H.; Šiller, Lidija; Bhaskar, Umesh; Pardoen, Thomas; Raskin, Jean-Pierre

    2012-12-01

    In this work, strain in silicon free standing beams loaded in uniaxial tension is experimentally and theoretically investigated for strain values ranging from 0 to 3.6%. The fabrication method allows multiple geometries (and thus strain values) to be processed simultaneously on the same wafer while being studied independently. An excellent agreement of strain determined by two non-destructive characterization techniques, Raman spectroscopy and mechanical displacement using scanning electron microscopy (SEM) markers, is found for all the sample lengths and widths. The measured data also show good agreement with theoretical predictions of strain based upon continuum mechanical considerations, giving validity to both measurement techniques for the entire range of strain values. The dependence of Young's modulus and fracture strain on size has also been analyzed. The Young's modulus is determined using SEM and compared with that obtained by resonance-based methods. Both methods produced a Young's modulus value close to that of bulk silicon with values obtained by resonance-based methods being slightly lower. Fracture strain is analyzed in 40 sets of samples with different beam geometries, yielding values up to 3.6%. The increase in fracture strain with decreasing beam width is compared with previous reports. Finally, the role of the surface on the mechanical properties is analyzed using UV and visible lasers having different penetration depths in silicon. The observed dependence of Raman shift on laser wavelength is used to assess the thermal conductivity of deformed silicon.

  11. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast to...... uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...

  12. Hydrogen production from microbial strains

    Science.gov (United States)

    Harwood, Caroline S; Rey, Federico E

    2012-09-18

    The present invention is directed to a method of screening microbe strains capable of generating hydrogen. This method involves inoculating one or more microbes in a sample containing cell culture medium to form an inoculated culture medium. The inoculated culture medium is then incubated under hydrogen producing conditions. Once incubating causes the inoculated culture medium to produce hydrogen, microbes in the culture medium are identified as candidate microbe strains capable of generating hydrogen. Methods of producing hydrogen using one or more of the microbial strains identified as well as the hydrogen producing strains themselves are also disclosed.

  13. 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 preliminary experiment of this type and have successfully complemented an A. tumefaciens CelC mutant with the homologous gene (yhjM) from E. coli.

  14. 产生物胺菌株群体感应AHLs检测及分析%Detection and analysis of quorum sensing about N-acylhomoserine lactones of biogenic amine strains

    Institute of Scientific and Technical Information of China (English)

    陆静; 卢士玲; 刘玉晗; 尹罗罗; 李想鹏; 潘竞勇; 高雄颖

    2014-01-01

    为探讨发酵肉制品中生物胺的产生、累积机理,采用高效液相法分析了新疆熏马肠样品产生物胺优势菌株克雷伯氏菌(Klebsiella sp.)、阴沟肠杆菌(Enterobacter cloacae strain)和成团泛菌(Pantoea agglomerans strain)产生物胺能力.并选取克雷伯氏菌、阴沟肠杆菌、成团泛菌作为群体感应现象研究对象,利用微生物传感菌Agrobacterium tumefaciens A136及Agrobacterium tumefaciens KYC55平行线法检测AHLs.检测表明,3株产生物胺菌能够发生群体感应现象.通过β-半乳糖苷酶活性检测法测定AHLs活性试验、HPLC法对产生物胺菌株AHLs定性分析,结果表明,β-半乳糖苷酶活性越高,产生物胺菌株分泌的AHLs越多;Klebsiella sp.、Enterobacter cloacae strain均能分泌:N-butanoyl-L-homoserine lactone (C4-HSL)、N-hexanoyl-DL-homoserine homoserine lactone(C6-HSL)、N-octanoyl-DL-homoserine lactone(C8-HSL)类型的信号分子.

  15. Will the swine strain crowd out the seasonal influenza strain?

    CERN Document Server

    Schinazi, Rinaldo B

    2010-01-01

    We use spatial and non spatial models to argue that competition alone may explain why two influenza strains do not usually coexist. The more virulent strain is likely to crowd out the less virulent one. This can be seen as a consequence of the Exclusion Principle of Ecology. We exhibit, however, a spatial model for which coexistence is possible.

  16. Tissue Velocity, Strain und Strain Rate bei Hunden mit Mitralklappenendokardiose

    OpenAIRE

    Javornik, Ana

    2007-01-01

    In der vorliegenden Arbeit „Tissue Velocity, Strain und Strain Rate bei Hunden mit Mitralklappenendokardiose“ wurden erstmals die Geschwindigkeits- und Verformungsparameter zur Evaluierung von Hunden mit Mitralklappenendokardiose angewendet. Diese erweisen sich als neue und viel versprechende Möglichkeiten zur Quantifizierung der regionalen Myokardfunktion. Die myokardialen Funktionsparameter finden in der Humanmedizin weit verbreitete Anwendung und auch in der Tiermedizin erlangen sie zunehm...

  17. TL transgenic mouse strains

    International Nuclear Information System (INIS)

    As a result of abnormal development of the thymus of these mice, TCR αβ lineage of the T cell differentiation is disturbed and cells belonging to the TCR γδ CD4- CD8- double negative (DN) lineage become preponderant. The γδ DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, indicating that the γδ cells are incapable of participating in these reactions. Molecular and serological analyses of T-cell lymphomas reveal that they belong to the γδ lineage. Tg.Tlaa-3-1 mice should be useful in defining the role of TL in normal and abnormal T cell differentiation as well as in the development of T-cell lymphomas, and further they should facilitate studies on the differentiation and function of γδ T cells. We isolated T3b-TL gene from B6 mice and constructed a chimeric gene in which T3b-TL is driven by the promoter of H-2Kb. With the chimeric gene, two transgenic mouse strains, Tg. Con.3-1 and -2 have been derived in C3H background. Both strains express TL antigen in various tissues including skin. The skin graft of transgenic mice on C3H and (B6 X C3H)F1 mice were rejected. In the mice which rejected the grafts, CD8+TCRαβ cytotoxic T cells (CTL) against TL antigens were recognized. The recognition of TL by CTL did not require the antigen presentation by H-2 molecules. The results indicated that TL antigen in the skin becomes a transplantation antigen and behaves like a typical allogeneic MHC class I antigen. The facts that (B6 X C3H)F1 mice rejected the skin expressing T3b-TL antigen and induced CTL that killed TL+ lymphomas of B6 origin revealed that TL antigen encoded by T3b-TL is recognized as non-self in B6 mice. Experiments are now extended to analyze immune responses to TL antigen expressed on autochthonous T cell lymphomas. (J.P.N.)

  18. 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.

  19. What Are Sprains and Strains?

    Science.gov (United States)

    ... and Strains Find a Clinical Trial Journal Articles Sprains and Strains PDF Version Size: 119 KB Audio Version Time: 07: ... Twist a knee. Where Do Sprains Usually Occur? Sprains happen most often in the ankle. Sometimes when people fall and land on their ...

  20. Strain measurements at railway wheels

    International Nuclear Information System (INIS)

    The development of the radial strain component in railway wheels was investigated by neutron diffraction. In the as manufactured state four railway wheels were investigated. In all four wheels no significant strains were found. After 18,400 km usage first strain gradients close to the outer surface of the wheels were detected. In axial middle ranges the changes in strain are weak. After an usage of 61,000 km the gradients becomes strong at the axial outer position between about +700 με close below the tread and -500 με in a depth of 12.5 mm below the tread. At axial middle positions also strain gradients are formed. The end of live state (510,000 km) differs only slightly from the state measured after a usage of 61,000 km

  1. Pin clad strains in Phenix

    International Nuclear Information System (INIS)

    The Phenix reactor has operated for 4 years in a satisfactory manner. The first 2 sub-assembly loadings contained pins clad in solution treated 316. The principal pin strains are: diametral strain (swelling and irradiation creep), ovality and spiral bending of the pin (interaction of wire and pin cluster and wrapper). A pin cluster irradiated to a dose of 80 dpa F reached a pin diameter strain of 5%. This strain is principally due to swelling (low fission gas pressure). The principal parameters governing the swelling are instantaneous dose, time and temperature for a given type of pin cladding. Other types of steel are or will be irradiated in Phenix. In particular, cold-worked titanium stabilised 316 steel should contribute towards a reduction in the pin clad strains and increase the target burn-up in this reactor. (author)

  2. 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

  3. Recent advances in echocardiography: strain and strain rate imaging

    Science.gov (United States)

    Mirea, Oana; Duchenne, Jurgen; Voigt, Jens-Uwe

    2016-01-01

    Deformation imaging by echocardiography is a well-established research tool which has been gaining interest from clinical cardiologists since the introduction of speckle tracking. Post-processing of echo images to analyze deformation has become readily available at the fingertips of the user. New parameters such as global longitudinal strain have been shown to provide added diagnostic value, and ongoing efforts of the imaging societies and industry aimed at harmonizing methods will improve the technique further. This review focuses on recent advances in the field of echocardiographic strain and strain rate imaging, and provides an overview on its current and potential future clinical applications.

  4. Magnetostriction strain measurement: heterodyne laser interferometry versus strain gauge technique

    OpenAIRE

    Gorji Ghalamestani, Setareh; Vandevelde, Lieven; J.J. Dirckx, Joris; Melkebeek, Jan

    2013-01-01

    Deformation of the ferromagnetic material, known as magnetostriction, causes vibrations and noise of electrical machines and transformer cores. A setup by using heterodyne laser interferometers has been built to measure the magnetostriction strains as a function of the applied magnetic field. The measurement results on a sample of nonoriented electrical steel are presented in this work. These results are compared with those obtained by using a strain gauge setup. The laser measurements are l...

  5. Mercury retention in several strains and strain crosses of chickens

    Energy Technology Data Exchange (ETDEWEB)

    Miller, V.L.; Bearse, G.E.; Csonka, E.

    1970-01-01

    The retention of mercury from injections of mercuric chloride was determined in 32 samples of chicks from various strains and strain crosses of egg type stock. The chicks retaining the most mercury had some four times as much mercury in the liver and kidneys as the chicks retaining the lowest amount. The chicks selected for resistance to leukosis retained more mercury than the susceptible chicks within several leukosis breeding programs.

  6. Low TCR nanocomposite strain gages

    Science.gov (United States)

    Gregory, Otto J. (Inventor); Chen, Ximing (Inventor)

    2012-01-01

    A high temperature thin film strain gage sensor capable of functioning at temperatures above 1400.degree. C. The sensor contains a substrate, a nanocomposite film comprised of an indium tin oxide alloy, zinc oxide doped with alumina or other oxide semiconductor and a refractory metal selected from the group consisting of Pt, Pd, Rh, Ni, W, Ir, NiCrAlY and NiCoCrAlY deposited onto the substrate to form an active strain element. The strain element being responsive to an applied force.

  7. Roll bonding of strained aluminium

    DEFF Research Database (Denmark)

    Staun, Jakob M.

    2003-01-01

    This report investigates roll bonding of pre-strained (å ~ 4) aluminium sheets to produce high strain material from high purity aluminium (99.996%) and commercial pure aluminium (99.6%). The degree of bonding is investigated by optical microscopy and ultrasonic scanning. Under the right...... circumstances both materials show good bonding, but the high purity material is excluded because of recrystallisation and the resulting loss of mechanical properties. The effect of cross stacking and roll bonding pre-strained sheets of the commercial purity material is investigated and some dependence of the...... cross rolled volume fraction is found. To further asses this effect, and the anisotropy, it is necessary to acquire knowledge about both texture and microstructure, e.g. by TEM. Roll bonding of pre-strained aluminium is found to be a possible alternative to ARB in the quest for ultra-fine grained...

  8. Covalent functionalization of strained graphene

    OpenAIRE

    Boukhvalov, Danil W.; Son, Young-Woo

    2012-01-01

    Enhancement of the chemical activity of graphene is evidenced by first-principles modelling of chemisorption of the hydrogen, fluorine, oxygen and hydroxyl groups on strained graphene. For the case of negative strain or compression, chemisorption of the single hydrogen, fluorine or hydroxyl group is energetically more favourable than those of their pairs on different sublattices. This behaviour stabilizes the magnetism caused by the chemisorption being against its destruction by the pair form...

  9. Hypervirulent strains of Clostridium difficile

    OpenAIRE

    Cookson, Barry

    2007-01-01

    North America has seen increasing numbers of hospitalised patients and others in nursing homes and the community, with more severe Clostridium difficile associated diarrhoea. This is also described in Northern Europe and surveillance systems are being developed or improved to monitor the situation. One strain (ribotype O27) is described in detail and, like other emerging strains, is demonstrating increasing antimicrobial resistance, notably to quinolone antibiotics. However, its association w...

  10. Biocontrol mechanisms of Thrichoderma strains

    OpenAIRE

    Benítez Fernández, Concepción Tahía; Rincón Romero, Ana María; Limón Mirón, María del Carmen; Carballo Codón, Antonio

    2004-01-01

    The genus Trichoderma comprises a great number of fungal strains that act as biological control agents, the antagonistic properties of which are based on the activation of multiple mechanisms. Trichoderma strains exert biocontrol against fungal phytopathogens either indirectly, by competing for nutrients and space, modifying the environmental conditions, or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by mechanisms such as mycoparasitism. These indirect a...

  11. Strain evaluation in fatigue analysis

    International Nuclear Information System (INIS)

    Thermoplastic analysis and more precisely fatigue analysis of nuclear components working at high temperature is usually very difficult and very expensive. Simplified methods have been proposed and are generally used to perform analysis of these structures. These simplified methods require elastic analysis of the component. For a fatigue analysis it is necessary to obtain a very precise value of the strains so the elastic strains must be corrected in order to take plasticity into account. This later appears in two different ways. Non linearity of material which induces strain amplification when working in the plastic field especially near by geometric discontinuities. That can be taken into account with a coefficient Ke. Triaxiality and isovolumic character of strains in the plastic field. That also induces strain amplification with regard to elastic computation. It is proposed in this work to introduce a coefficient Kv to estimate this strain amplification. Several elastic and plastic calculations for typical geometries, have been done in order to test the importance of these parameters and to set up methods for evaluating the coefficients Ke and Kv

  12. 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...

  13. 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

  14. Genetic diversity and distribution of a distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman.

    Science.gov (United States)

    Khan, Akhtar J; Akhtar, Sohail; Al-Zaidi, Amal M; Singh, Achuit K; Briddon, Rob W

    2013-10-01

    Tomato and pepper are widely grown in Oman for local consumption. A countrywide survey was conducted during 2010-2011 to collect samples and assess the diversity of begomoviruses associated with leaf curl disease of tomato and pepper. A virus previously only identified on the Indian subcontinent, chili leaf curl virus (ChLCV), was found associated with tomato and pepper diseases in all vegetable grown areas of Oman. Some of the infected plant samples were also found to contain a betasatellite. A total of 19 potentially full-length begomovirus and eight betasatellite clones were sequenced. The begomovirus clones showed >96% nucleotide sequence identity, showing them to represent a single species. Comparisons to sequences available in the databases showed the highest levels of nucleotide sequence identity (88.0-91.1%) to isolates of the "Pakistan" strain of ChLCV (ChLCV-PK), indicating the virus from Oman to be a distinct strain, for which the name Oman strain (ChLCV-OM) is proposed. An analysis for recombination showed ChLCV-OM likely to have originated by recombination between ChLCV-PK (the major parent), pepper leaf curl Lahore virus and a third strain of ChLCV. The betasatellite sequences obtained were shown to have high levels of identity to isolates of tomato leaf curl betasatellite (ToLCB) previous shown to be present in Oman. For the disease in tomato Koch's postulates were satisfied by Agrobacterium-mediated inoculation of virus and betasatellites clones. This showed the symptoms induced by the virus in the presence of the betasatellite to be enhanced, although viral DNA levels were not affected. ChLCV-OM is the fourth begomovirus identified in tomato in Oman and the first in Capsicum. The significance of these findings is discussed. PMID:23911631

  15. 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.

  16. Genome Sequence of Pseudomonas chlororaphis Strain 189

    Science.gov (United States)

    Town, Jennifer; Audy, Patrice; Boyetchko, Susan M.

    2016-01-01

    Pseudomonas chlororaphis strain 189 is a potent inhibitor of the growth of the potato pathogen Phytophthora infestans. We determined the complete, finished sequence of the 6.8-Mbp genome of this strain, consisting of a single contiguous molecule. Strain 189 is closely related to previously sequenced strains of P. chlororaphis. PMID:27340063

  17. Strain Growth in Containment Vessels

    Institute of Scientific and Technical Information of China (English)

    DONG Q; LI Q M; ZHENG J Y

    2006-01-01

    Strain growth is a phenomenon observed in containment vessels subjected to internal blast loading.The elastic response of the vessel may become larger in a later stage compared to its response during the initial stage.The dynamic responses of infinitely long cylindrical containment vessels subjected to uniformly-distributed internal blast loading are studied using LS-DYNA.The development of bending modes and the interaction between the breathing mode and bending modes are observed.The methodology developed for dynamic elastic buckling analysis is employed to study the strain growth phenomenon in explosion containment vessels.It is shown that the dynamic instable vibration of a containment vessel is the basic mechanism of strain growth.

  18. Thermoelectric properties of strained silicon

    Energy Technology Data Exchange (ETDEWEB)

    Hinsche, Nicki F.; Zahn, Peter [Martin-Luther-Universitaet, Institut fuer Physik, Von-Seckendorff-Platz 1, 06120 Halle/S. (Germany); Mertig, Ingrid [Martin-Luther-Universitaet, Institut fuer Physik, Von-Seckendorff-Platz 1, 06120 Halle/S. (Germany); Max-Planck-Institut fuer Mikrostrukturphysik, Weinberg 2, D-06120 Halle (Germany)

    2011-07-01

    Starting from bulk silicon, we study the change in thermoelectric properties due to symmetry breaking in rolled-up and layered Si which might lead to nanostructured thermoelectrics. Valley splitting in strained Si caused by tetragonal distortion was studied recently with respect to the enhancement of electron mobility. Our results show that the tetragonal distortion has a strong influence on the electronic transport properties. The electronic structure is calculated self consistently within the framework of density functional theory. The transport properties are studied in the diffusive limit applying the Boltzmann theory in relaxation time approximation. In detail, the anisotropy of the electrical conductivity, the thermopower and the resulting powerfactor in the in-plane and off-plane directions are studied in dependence on strain, doping level and temperature. It is shown, that the powerfactor at a given temperature can be enhanced slightly by strain for p-doping, while no enhancement is obtained for n-doping.

  19. Spin transport in graphene superlattice under strain

    Science.gov (United States)

    Sattari, Farhad

    2016-09-01

    In this paper, the spin-dependent transport and the spin polarization properties for graphene superlattice with Rashba spin-orbit interaction (RSOI) in the presence of zigzag and armchair direction strain are studied. It is found that for the zigzag direction strain the angular range of the spin-inversion can be efficiently controlled by the strain strength. In addition, the efficiency of spin-inversion and spin-dependent conductivity decreases by increasing the strain strength. When the armchair direction strain is applied to a monolayer graphene superlattice the spin polarization can be observed and increases by increasing the strain strength, whereas for the zigzag direction strain it is zero.

  20. Myocardial Strain and Strain Rate Imaging: Comparison between Doppler Derived Strain Imaging and Speckle Tracking Echocardiography

    Directory of Open Access Journals (Sweden)

    Anita Sadeghpour

    2013-05-01

    Full Text Available Regional myocardial function has been traditionally assessed by visual estimation (1. Echocardiographic strain imaging which is known as deformation imaging, has been emerged as a quantitative technique to accurately estimate regional myocardial function and contractility. Currently, strain imaging has been regarded as a research tool in the most echocardiography laboratories. However, in recent years, strain imaging has gain momentum in daily clinical practice (2. The following two techniques have dominated the research arena of echocardiography: (1 Doppler based tissue velocity measurements, frequently referred to tissue Doppler or myocardial Doppler, and (2 speckle tracking on the basis of displacement measurements (3. Over the past two decades, Tissue Doppler Imaging (TDI and Doppler –derived strain (S and strain rate (SR imaging were introduced to quantify regional myocardial function. However, Doppler–derived strain variables faced criticisms, with regard to the angle dependency, noise interference, and substantial intraobserver and interobserver variability. The angle dependency is the major weakness of Doppler based methodology; however, it has the advantage of online measurements of velocities and time intervals with excellent temporal resolution, which is essential for the assessment of ischemia (4. Speckle-tracking echocardiography (STE or Non Doppler 2D strain echocardiography is a relatively new, largely angle-independent technique that analyzes motion by tracking natural acoustic reflections and interference patterns within an ultrasonic window. The image-processing algorithm tracks elements with approximately 20 to 40 pixels containing stable patterns and are described as ‘‘speckles’’ or ‘‘fingerprints’’. The speckles seen in grayscale B-mode (2D images are tracked consecutively frame to frame (5, 6. Assessment of 2D strain by STE is a semiautomatic method that requires definition of the myocardium

  1. Photoacoustic spectroscopy of Entamoeba histolytica strains

    Science.gov (United States)

    Acosta-Avalos, D.; Alvarado-Gil, J. J.; Silva, E. F.; Orozco, E.; de Menezes, L. F.; Vargas, H.

    2005-06-01

    Pathogenic and non-pathogenic strains of E. histolytica are studied using photoacoustic spectroscopy. It is shown that the pathogenic strain presents a spectrum similar to that of iron sulfur proteins. The non-pathogenic strain does not show any relevant absorption at the studied wavelength range. The differences observed between the optical absorption spectra of both strains opens the possibility of using photoacoustic spectroscopy as a reliable and simple technique to identify different types of E. histolytica strains.

  2. Computational Strain Gradient Crystal Plasticity

    DEFF Research Database (Denmark)

    Niordson, Christian Frithiof; Kysar, Jeffrey W.

    2011-01-01

    A model for strain gradient crystal visco-plasticity is formulated along the lines proposed by Fleck andWillis (2009) for isotropic plasticity. Size-effects are included in the model due to the addition of gradient terms in both the free energy as well as through a dissipation potential. A finite...

  3. Job strain and alcohol intake

    DEFF Research Database (Denmark)

    Heikkilä, Katriina; Nyberg, Solja T; Fransson, Eleonor I;

    2012-01-01

    The relationship between work-related stress and alcohol intake is uncertain. In order to add to the thus far inconsistent evidence from relatively small studies, we conducted individual-participant meta-analyses of the association between work-related stress (operationalised as self-reported job...... strain) and alcohol intake....

  4. Trials with a Strain Gauge.

    Science.gov (United States)

    Auty, Geoff

    1996-01-01

    Describes an attempt to match the goals of the practical demonstration of the use of a strain gauge and the technical applications of science and responding to student questions in early trials, while keeping within the level of electronics in advanced physics. (Author/JRH)

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Dicty_cDB: Contig-U02712-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 015 1 ( FE523757 ) mat2920444 Radiata pine transition earlywood cDNA... 54 0.015 ...e) Agrobacterium radiobacter K84 c... 45 0.007 FM177140_2194( FM177140 |pid:non...clear 8.0 %: endoplasmic reticulum 8.0 %: vacuolar 8.0 %: mitochondrial 4.0 %: cytoskeletal 4.0 %: Golgi >> prediction for Con...e) Kineococcus radiotolerans SRS30... 54 1e-05 BT065333_1( BT065333 |pid:non...CP000885_1913( CP000885 |pid:none) Clostridium phytofermentans ISD... 46 0.004 AE017198_264( AE017198 |pid:none) Lactobaci

  10. Material mechanical characterization method for multiple strains and strain rates

    Energy Technology Data Exchange (ETDEWEB)

    Erdmand, III, Donald L.; Kunc, Vlastimil; Simunovic, Srdjan; Wang, Yanli

    2016-01-19

    A specimen for measuring a material under multiple strains and strain rates. The specimen including a body having first and second ends and a gage region disposed between the first and second ends, wherein the body has a central, longitudinal axis passing through the first and second ends. The gage region includes a first gage section and a second gage section, wherein the first gage section defines a first cross-sectional area that is defined by a first plane that extends through the first gage section and is perpendicular to the central, longitudinal axis. The second gage section defines a second cross-sectional area that is defined by a second plane that extends through the second gage section and is perpendicular to the central, longitudinal axis and wherein the first cross-sectional area is different in size than the second cross-sectional area.

  11. Engineering piezoresistivity using biaxially strained silicon

    DEFF Research Database (Denmark)

    Pedersen, Jesper Goor; Richter, Jacob; Brandbyge, Mads;

    2008-01-01

    piezocoefficient on temperature and dopant density is altered qualitatively for strained silicon. In particular, we find that a vanishing temperature coefficient may result for silicon with grown-in biaxial tensile strain. These results suggest that strained silicon may be used to engineer the iezoresistivity to......We calculate the shear piezocoefficient of p-type silicon with grown-in biaxial strain using a 66 k·p method. We find a significant increase in the value of the shear piezocoefficient for compressive grown-in biaxial strain, while tensile strain decreases the piezocoefficient. The dependence of the...

  12. Plane strain test for metal sheet characterization

    OpenAIRE

    Flores, Paulo; Bonnet, Félix; Habraken, Anne

    2007-01-01

    This article shows the influence of a plane strain test specimen geometry on the measurable strain field and the influence of free edge effects over the stress computation. The experimental strain field distribution is measured over the whole deformable zone of a plane strain test specimen by an optical strain gauge. The chosen material is the DC06 IF steel of 0.8 mm thickness. The stress field is computed for several geometries at different strain levels by a Finite Element (FE) commer...

  13. Computational strain gradient crystal plasticity

    DEFF Research Database (Denmark)

    Niordson, Christian Frithiof; Kysar, Jeffrey W.

    2014-01-01

    A numerical method for viscous strain gradient crystal plasticity theory is presented, which incorporates both energetic and dissipative gradient effects. The underlying minimum principles are discussed as well as convergence properties of the proposed finite element procedure. Three problems...... of plane crystal plasticity are studied: pure shear of a single crystal between rigid platens as well as plastic deformation around cylindrical voids in hexagonal close packed and face centered cubic crystals. Effective in-plane constitutive slip parameters for plane strain deformation of specifically...... oriented face centered cubic crystals are developed in terms of the crystallographic slip parameters. The effect on geometrically necessary dislocation structures introduced by plastic deformation is investigated as a function of the ratio of void radius to plasticity length scale....

  14. Electronic Transport in Strained Graphene

    OpenAIRE

    Aguilera, Juan Luis

    2015-01-01

    Graphene is a single atomic layer material with exceptional electronic and mechanical properties. Graphene has formed the basis of many nanoelectromechanical and strain sensing devices. However, the ultimate limit of miniaturization of such sensors has not yet been ascertained. In this work we present the fabrication and electrical characterization of nanoscale pressure sensors realized from suspended graphene membrane devices.We start in chapter 1 by describing the elemental electronic prope...

  15. Using Strain Analysis For Landslides

    Czech Academy of Sciences Publication Activity Database

    Talich, Milan

    Vol. 4. Kyoto : International Consortium on Landslides (ICL), 2014, s. 178-184. [World Landslide Forum 3. Beijing (CN), 02.06.2014-06.06.2014] R&D Projects: GA MPO FR-TI4/436 Institutional support: RVO:67985556 Keywords : strain analysis * deformation analysis * displacement vectors * mechanics of continuum * landslides Subject RIV: DE - Earth Magnetism, Geodesy, Geography http://library.utia.cas.cz/separaty/2015/ZOI/talich-0443019.pdf

  16. Temperature-Compensating Inactive Strain Gauge

    Science.gov (United States)

    Moore, Thomas C., Sr.

    1993-01-01

    Thermal contribution to output of active gauge canceled. High-temperature strain gauges include both active gauge wires sensing strains and inactive gauge wires providing compensation for thermal contributions to gauge readings. Inactive-gauge approach to temperature compensation applicable to commercially available resistance-type strain gauges operating at temperatures up to 700 degrees F and to developmental strain gauges operating at temperatures up to 2,000 degrees F.

  17. Strain Monitoring of Flexible Structures

    Science.gov (United States)

    Litteken, Douglas A.

    2017-01-01

    , such as tensile testing, fatigue testing, and shear testing, but common measurement techniques cannot be used on fabric. Measuring strain in a material and during a test is a critical parameter for an engineer to monitor the structure during the test and correlate to an analytical model. The ability to measure strain in fabric structures is a challenge for NASA. Foil strain gauges, for example, are commonplace on metallic structures testing, but are extremely difficult to interface with a fabric substrate. New strain measuring techniques need to be developed for use with fabric structures. This paper investigates options for measuring strain in fabric structures for both ground testing and in-space structural health monitoring. It evaluates current commercially available options and outlines development work underway to build custom measurement solutions for NASA's fabric structures.

  18. Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains.

    Directory of Open Access Journals (Sweden)

    Dharanesh Gangaiah

    Full Text Available Although cutaneous ulcers (CU in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin?To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin.These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

  19. Lewis Strain Gauge Laboratory: Status and plans

    International Nuclear Information System (INIS)

    An in-house lab was established for developing, testing, and evaluating high-temperature strain gauges and to aid in in-house applications of high-temperature strain instrumentation. The lab is automated to provide computer control of oven temperatures, imposed strain, and data sampling

  20. Biotransformation of codeine to 14-OH-codeine derivatives by Rhizobium radiobacter R89-1

    Czech Academy of Sciences Publication Activity Database

    Kyslíková, Eva; Babiak, Petr; Štěpánek, Václav; Zahradník, Jiří; Palyzová, Andrea; Marešová, Helena; Valešová, Renata; Hájíček, J.; Kyslík, Pavel

    2013-01-01

    Roč. 87, MAR 2013 (2013), s. 1-5. ISSN 1381-1177 R&D Projects: GA MŠk 2B08064 Institutional support: RVO:61388971 Keywords : Biotransformation * codeine * morphine Subject RIV: CE - Biochemistry Impact factor: 2.745, year: 2013

  1. Influence of particles on work hardening strain and recovery strain

    Czech Academy of Sciences Publication Activity Database

    Blum, W.; Dvořák, Jiří; Král, Petr; Sklenička, Václav

    Brno: Ústav fyziky materiálů AV ČR, v. v. i., 2015 - (Dlouhý, A.; Kunz, L.). s. 137-137 ISBN 978-80-87434-07-9. [ICSMA-17 International Conference on the Strength of Materials /17./. 09.08.2015-14.08.2015, Brno] R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081723 Keywords : recovery strain * CuZr particles * crystallite boundaries * work hardening Subject RIV: JG - Metallurgy

  2. Sensibility of different wheat varieties (strains) to Ar+ implantation

    International Nuclear Information System (INIS)

    The sensibility of different wheat varieties (strains) to Ar+ implantation was studied. The results showed that the survival rate of 21 wheat varieties (strains) at the dose of 6 x 1016 Ar+/cm2 could be divided into five groups: surplus sensitive varieties (strains), sensitive varieties (strains), transitional varieties (strains), obtuse varieties (strains) and surplus obtuse varieties (strains). The sensibility of wheat varieties (strains) to Ar+ injection is high-moisture-fertility wheat varieties (strains) > medium-moisture-fertility wheat varieties (strains) > dry land wheat varieties (strains). The study has provided theoretical basis in induced mutation medial lethal dose of different wheat varieties (strains) to Ar+ implantation. (authors)

  3. Monoclonal antibodies to Toxoplasma gondii strain 119 identify recently isolated Danish strains as one group

    DEFF Research Database (Denmark)

    Jensen, L.; Petersen, E.; Henriksen, S.A.; Dietz, Hans-Henrik; Lind, Peter

    1998-01-01

    Four mAb raised against the Danish Toxoplasma gondii strain 119, were selected by screening hybridoma supernatants by indirect immunofluorescence against tachyzoites of the RH strain in order to obtain strain restricted markers. Strain restriction extended beyond discrimination of the 119 and RH...

  4. 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.

  5. Local strains in waste tank deflagration analysis

    International Nuclear Information System (INIS)

    In recent years extensive effort has been expended to qualify buried nuclear waste storage tanks under accident conditions. One of these conditions is deflagration of the combustible gases which may build up over time. While much work has been done to calculate the general strain state, less effort has been made to address the local strains at structural discontinuities. An analytical method is presented for calculating these local strains and combining them with the general strain state. A closed form solution of the local strains is compared to a finite element solution

  6. Spontaneous abortion and physical strain around implantation

    DEFF Research Database (Denmark)

    Hjollund, N H; Jensen, Tina Kold; Bonde, Jens Peter;

    2000-01-01

    Existing studies of physical strain and spontaneous abortion are mainly retrospective or based only on pregnancies that have survived the first trimester. Furthermore, almost all studies have relied on averaged measures of physical strain, which tend to blur an effect if peak values during short...... pregnancy the women recorded physical strain prospectively in a structured diary. Physical strain around the time of implantation was associated with later spontaneous abortion. The adjusted risk ratio for women who reported physical strain higher than average at day 6 to 9 after the estimated date of...

  7. Transient temperature response of strain gages

    International Nuclear Information System (INIS)

    When an electrical resistance strain gage bonded to a material is subjected to a temperature change, the resistance of the strain gage changes even though the material is free of stress. This temperature-induced resistance change is generally expressed in strain units and is commonly called apparent strain or thermal output. The thermal expansion coefficients and the temperature coefficient of resistance are temperature-dependent quantities and not necessarily linearly related to temperature. For transient temperature conditions, the two temperatures are different. The magnitude and effect of this temperature difference on apparent strain is studied

  8. Strain Engineering of Transition Metal Dichalcogenides

    Science.gov (United States)

    Dadgar, Ali; Pasupathy, Abhay; Herman, Irving; Wang, Dennis; Kang, Kyungnam; Yang, Eui-Hyeok

    The application of strain to materials can cause changes to bandwidth, effective masses, degeneracies and even structural phases. In the case of the transition metal dichalcogenide (TMD) semiconductors, small strain (around 1 percent) is expected to change band gaps and mobilities, while larger strains are expected to cause phase changes from the triangular 2H phase to orthorhombic 1T' phases. We will describe experimental techniques to apply small and large (around 10 percent) strains to one or few layer samples of the TMD semiconductors, and describe the effect of the strain using optical (Raman, photoluminescence) and cryogenic transport techniques.

  9. Stress-strain properties of railway steel at strain rates of upto 105 per second

    International Nuclear Information System (INIS)

    This paper presents the stress-strain characteristics of railway steel at strain rates of up to 105/s at room temperature determined by a new technique. In determining the results, account has been taken of the strain-rate variation, the total strain and the strain rate history. The effect of friction, material inertia and temperature rise is also assessed and an empirical constitutive equation describing the strain-rate and strain sensitive flow stress for this type of steel is proposed. (orig.)

  10. Comparison between strain values of plates measured by ESPI technique and strain gauge method

    International Nuclear Information System (INIS)

    Comparison between strain values of plates measured by Electronic Speckle Pattern Interferometry(ESPI) method and strain gauge method is discussed. Unlike traditional method, such ax strain gauge and moire method, ESPI method requires no special surface preparations or attachments and can be measured in-plane displacement with non-contacting in real-time. In this study, specimens are loaded in parallel using tensile testing machine. In front of a specimen three strain gauges are bonded with 5 mm gap from edge of a specimen strain values are measured at each strain gauge position and compared. The results by ESPI method agree well with the result by strain gauge method.

  11. Strain flexibility identification of bridges from long-gauge strain measurements

    Science.gov (United States)

    Zhang, Jian; Xia, Qi; Cheng, YuYao; Wu, ZhiShen

    2015-10-01

    Strain flexibility, defined as the strain response of a structure's element to a unit input force, is import for structural safety evaluation, but its identification is seldom investigated. A novel long-gauge fiber optic sensor has been developed to measure the averaged strain within a long gauge length. Its advantage of measuring both local and global information of the structure offers an excellent opportunity of developing the strain flexibility identification theory. In this article, the method to identify structural strain flexibility from long-gauge dynamic strain measurements is proposed. It includes the following main steps: (a) macro strain frequency response function (FRF) estimation from macro strain measurements and its feature characterization; (b) general strain modal parameter identification; (c) scaling factor calculation, and (d) strain flexibility identification. Numerical and experimental examples successfully verify the effectiveness of the proposed method.

  12. Numerical demonstration of MEMS strain sensor

    Science.gov (United States)

    Saboonchi, Hossain; Ozevin, Didem

    2012-04-01

    Silicon has piezoresistive property that allows designing strain sensor with higher gauge factor compared to conventional metal foil gauges. The sensing element can be micro-scale using MEMS, which minimizes the effect of strain gradient on measurement at stress concentration regions such as crack tips. The challenge of MEMS based strain sensor design is to decouple the sensing element from substrate for true strain measurement and to compensate the temperature effect on the piezoresistive coefficients of silicon. In this paper, a family of MEMS strain sensors with different geometric designs is introduced. Each strain sensor is made of single crystal silicon and manufactured using deposition/ etching/oxidation steps on a n- doped silicon wafer in (100) plane. The geometries include sensing element connected to the free heads of U shape substrate, a set of two or more sensing elements in an array in order to capture strain gradients and two directional sensors. The response function and the gauge factor of the strain sensors are identified using multi-physics models that combine structural and electrical behaviors of sensors mounted on a strained structure. The relationship between surface strain and strain at microstructure is identified numerically in order to include the relationship in the response function calculation.

  13. Strain engineering of graphene: a review

    Science.gov (United States)

    Si, Chen; Sun, Zhimei; Liu, Feng

    2016-02-01

    Graphene has intrigued the science community by many unique properties not found in conventional materials. In particular, it is the strongest two-dimensional material ever measured, being able to sustain reversible tensile elastic strain larger than 20%, which yields an interesting possibility to tune the properties of graphene by strain and thus opens a new field called ``straintronics''. In this article, the current progress in the strain engineering of graphene is reviewed. We first summarize the strain effects on the electronic structure and Raman spectra of graphene. We then highlight the electron-phonon coupling greatly enhanced by the biaxial strain and the strong pseudomagnetic field induced by the non-uniform strain with specific distribution. Finally, the potential application of strain-engineering in the self-assembly of foreign atoms on the graphene surface is also discussed. Given the short history of graphene straintronics research, the current progress has been notable, and many further advances in this field are expected.

  14. Strain gauges as intrusion detection sensors

    International Nuclear Information System (INIS)

    Strain gauges have been studied for use as intrusion detection sensors in several applications being investigated at Sandia National Laboratories. Strain gauges are attached to a metal structure to monitor the quiescent strain in the structure. The change in the quiescent strain, when an intruder adds weight to the structure provides the alarm mechanism. The basic theory of force sensors is covered to lay the foundation for this application. In this paper, how this basic theory is applied to security sensors is discussed, and how this class of sensors is applicable to security at Department of Energy facilities is covered. Several applications are described for strain gauges as security sensors. Test results are presented from a six-month test conducted at a Department of Energy facility using the strain gauge to monitor overhead lines crossing a security perimeter. Monitoring these overhead lines with strain gauges is valuable because the cost is much less than the cost to bury the lines

  15. Generalizing the Fermi velocity of strained graphene from uniform to nonuniform strain

    OpenAIRE

    Oliva-Leyva, M.; Naumis, Gerardo G.

    2014-01-01

    The relevance of the strain-induced Dirac point shift to obtain the appropriate anisotropic Fermi velocity of strained graphene is demonstrated. Then a critical revision of the available effective Dirac Hamiltonians is made by studying in detail the limiting case of a uniform strain. An effective Dirac Hamiltonian for nonuniform strain is thus reported, which takes into account all strain-induced effects: changes in the nearest-neighbor hopping parameters, the reciprocal lattice deformation a...

  16. A Method to Measure Reference Strain in FBG Strain Sensor Interrogation System Involving Actuators

    OpenAIRE

    Rajan, Ginu; Semenova, Yuliya; Wu, Qiang; Farrell, Gerald; Wang, Pengfei

    2007-01-01

    A method for reference strain measurement for FBG strain sensor in the testing stage while applying strain using actuators like piezo translators or micro screw is introduced. Unlike conventional methods of surface mounting, in our method the strain gauge is affixed directly to the optical fibre, which allows it to use with systems where the strain is applied directly using actuators while testing the FBG sensing system. Different bonding techniques were tested and a comparison with the resul...

  17. Application of capsule type strain gage and fiber optic grating strain sensor for measurement of strain under irradiation environment

    International Nuclear Information System (INIS)

    In Japan Atomic Energy Research Institute, in-pile strain measurement techniques have been developing by using JMTR. In order to evaluate the performance of capsule type strain gage and fiber optic grating sensor under irradiation environment, the heat-up tests in electric furnace before irradiation and in-pile tests were performed. As for capsule type strain gage, it is found that gage factor almost did not change and both its electric resistance and strain output decreased by irradiation effect. A correlation was found between the change ratio of the electric resistance of the capsule type strain gage and fast neutron fluence. As for fiber optic grating sensor, it is possible to measure strain under irradiation environment below 1x1023 n/m2 (E>1 MeV) by this sensor, because in-pile temperature characteristic was in agreement with out-of-pile test results. (author)

  18. Modeling competition between yeast strains

    Science.gov (United States)

    de Gee, Maarten; van Mourik, Hilda; de Visser, Arjan; Molenaar, Jaap

    2016-04-01

    We investigate toxin interference competition between S. cerevisiae colonies grown on a solid medium. In vivo experiments show that the outcome of this competition depends strongly on nutrient availability and cell densities. Here we present a new model for S. cerevisiae colonies, calculating the local height and composition of the colonies. The model simulates yeast colonies that show a good fit to experimental data. Simulations of colonies that start out with a homogeneous mixture of toxin producing and toxin sensitive cells can display remarkable pattern formation, depending on the initial ratio of the strains. Simulations in which the toxin producing and toxin sensitive species start at nearby positions clearly show that toxin production is advantageous.

  19. Investigation of Electronic Devices for Strain Measurement

    Directory of Open Access Journals (Sweden)

    Ričardas Masiulionis

    2011-08-01

    Full Text Available Importance of strain measuring for safety of buildings is shown. The strain monitoring should be one of the buildings security systems. Often used balanced and non-balanced Wheatstone bridge strain measurement methods are analyzed. The Wheatstone bridge method with feedback is improved. A new method based on small resistance changes by the digital balancing currents is presented. Computer and experimental models of measurement are investigated. The received results confirm theoretical assumptions.Article in Lithuanian

  20. Mechanical strain and degradation of laser heterostructures

    Science.gov (United States)

    Ptashchenko, Alexander A.; Ptashchenko, Fedor A.; Maslejeva, Natalia V.; Sadova, Galina V.

    2001-02-01

    The effect of mechanical strain on degradation processes in GaAs-AlGaAs laser heterostructures (LHS) with stripe geometry and in light emitting diodes (LED) was experimentally studied. The strain was produced either by axial pressure or by indentation with a Wickers pyramid. We show that degradation affects the degree of polarization and the far-field distribution of laser emission. The effect of strain on the degradation intensity is estimated.

  1. Aggregation and distribution of strains in microparasites.

    OpenAIRE

    Lord, C. C.; Barnard, B.; Day, K; Hargrove, J. W.; McNamara, J J; Paul, R E; Trenholme, K; Woolhouse, M. E.

    1999-01-01

    Recent research has shown that many parasite populations are made up of a number of epidemiologically distinct strains or genotypes. The implications of strain structure or genetic diversity for parasite population dynamics are still uncertain, partly because there is no coherent framework for the interpretation of field data. Here, we present an analysis of four published data sets for vector-borne microparasite infections where strains or genotypes have been distinguished: serotypes of Afri...

  2. Flat band superconductivity in strained Dirac materials

    OpenAIRE

    Kauppila, V. J.; Aikebaier, F.; Heikkilä, T. T.

    2016-01-01

    We consider superconducting properties of a two-dimensional Dirac material such as graphene under strain that produces a flat band spectrum in the normal state. We show that in the superconducting state, such a model results in a highly increased critical temperature compared to the case without the strain, inhomogenous order parameter with two-peak shaped local density of states and yet a large and almost uniform and isotropic supercurrent. This model could be realized in strained graphene o...

  3. Strain rate sensitivity of automotive sheet steels : influence of plastic strain, strain rate, temperature, microstructure, bake hardening and pre-strain

    OpenAIRE

    Larour, Patrick

    2010-01-01

    This experimental work shows the different parameters influencing the strain rate sensitivity behaviour of automotive sheet steel grades in crash conditions. Most investigations have been performed in the strain rate range [0,001-200/s] and temperature range [233-373K] with servohydraulic tensile testing machines. Additional Split-Hopkinson bar testing results up to 1000/s have also been included at room temperature. The focus has been laid on the “apparent” strain rate sensitivity, determine...

  4. Nonlinear strain gradient elastic thin shallow shells

    OpenAIRE

    Lazopoulos, K.A.

    2011-01-01

    Abstract The governing equilibrium equations for strain gradient elastic thin shallow shells are derived, considering non-linear strains and linear constitutive strain gradient elastic relations. Adopting Kirchhoff's theory of thin shallow structures, the equilibrium equations, along with the boundary conditions, are formulated through a variational procedure. It turns out that new terms are introduced, indicating the importance of the cross-section area in bending of thin plates. ...

  5. High strain rate loading of zircaloy

    International Nuclear Information System (INIS)

    High strain rate tensile loading behaviours of Zircaloys subjected to various treatments were investigated at room temperature and at 3000C, and compared with those of static cases. It was observed that the dynamic stress-strain profiles were different depending upon the specimen treatment and that the higher peak stress and the lower sectional area reduction were always recognized in the high strain rate cases

  6. Film germanium strain gauges for cryogenic temperatures

    International Nuclear Information System (INIS)

    Strain-measuring characteristics of strain gauges (SG) based on germanium films on gallium arsenide designed for operation in 4-100 K temperature interval and strain range ε∼(±0.3%) are presented. SG are characterized by weak temperature dependences of resistance and strain sensitivity in the temperature range measured. It is shown that in the low-temperature region SG based on heteroepitaxial germanium films on gallium arsenide are no worse than the best domestic and foreign semiconducting and metal SG and are perspective for cryogenic object diagnostics under magnetic field effect

  7. High strain rate damage of Carrara marble

    Science.gov (United States)

    Doan, Mai-Linh; Billi, Andrea

    2011-10-01

    Several cases of rock pulverization have been observed along major active faults in granite and other crystalline rocks. They have been interpreted as due to coseismic pervasive microfracturing. In contrast, little is known about pulverization in carbonates. With the aim of understanding carbonate pulverization, we investigate the high strain rate (c. 100 s-1) behavior of unconfined Carrara marble through a set of experiments with a Split Hopkinson Pressure Bar. Three final states were observed: (1) at low strain, the sample is kept intact, without apparent macrofractures; (2) failure is localized along a few fractures once stress is larger than 100 MPa, corresponding to a strain of 0.65%; (3) above 1.3% strain, the sample is pulverized. Contrary to granite, the transition to pulverization is controlled by strain rather than strain rate. Yet, at low strain rate, a sample from the same marble displayed only a few fractures. This suggests that the experiments were done above the strain rate transition to pulverization. Marble seems easier to pulverize than granite. This creates a paradox: finely pulverized rocks should be prevalent along any high strain zone near faults through carbonates, but this is not what is observed. A few alternatives are proposed to solve this paradox.

  8. Comparison of Thermal Creep Strain Calculation Results Using Time Hardening and Strain Hardening Rules

    International Nuclear Information System (INIS)

    One of the design criteria for the fuel rod in PGSFR is the thermal creep strain of the cladding, because the cladding is exposed to a high temperature for a long time during reactor operation period. In general, there are two kind of calculation scheme for thermal creep strain: time hardening and strain hardening rules. In this work, thermal creep strain calculation results for HT9 cladding by using time hardening and strain hardening rules are compared by employing KAERI's current metallic fuel performance analysis code, MACSIS. Also, thermal creep strain calculation results by using ANL's metallic fuel performance analysis code, LIFE-METAL which adopts strain hardening rule are compared with those by using MACSIS. Thermal creep strain calculation results for HT9 cladding by using time hardening and strain hardening rules were compared by employing KAERI's current metallic fuel performance analysis code, MACSIS. Also, thermal creep strain calculation results by using ANL's metallic fuel performance analysis code, LIFE-METAL which adopts strain hardening rule were compared with those by using MACSIS. Tertiary creep started earlier in time hardening rule than in strain hardening rule. Also, calculation results by MACSIS with strain hardening and those obtained by using LIFE-METAL were almost identical to each other

  9. The treatment of secondary strains within a strain-based failure assessment diagram

    International Nuclear Information System (INIS)

    In a companion paper, proposals for the shape of a strain-based failure assessment diagram (FAD) have been made. The developments in that paper and in related works on strain-based fracture assessment have largely been for remotely applied strains, such as those produced by welding. This paper re-writes existing methods for treating secondary stresses within stress-based FADs in the framework of the proposed strain-based FAD. It is shown that residual strains must be included fully in the elastic regime but at large applied strains their contribution to fracture can be relaxed considerably. Practical methods for inclusion of secondary strains within the strain-based FAD are then developed and shown to be conservative relative to some detailed finite element calculations. -- Highlights: ► Methods for treating secondary stresses are written in the framework of a strain-based FAD. ► Residual strains have a full effect in the elastic regime but their influence is relaxed considerably at large applied strains. ► Practical methods for inclusion of secondary strains within a strain-based FAD are developed. ► The methods are shown to be conservative relative to some detailed finite element calculations

  10. True stress–strain curves of cold worked stainless steel over a large range of strains

    International Nuclear Information System (INIS)

    True stress–strain curves for cold worked stainless steel were obtained over a range of strains that included a large strain exceeding the strain for the tensile strength (post-necking strain). A specified testing method was used to obtain the stress–strain curves in air at room temperature. The testing method employed the digital image correlation (DIC) technique and iterative finite element analyses (FEA) and was referred to as IFD (Iteration FEA procedure based on DIC measurement) method. Although hourglass type specimens have been previously used for the IFD method, in this study, plate specimens with a parallel gage section were used to obtain accurate yield and tensile strengths together with the stress–strain curves. The stress–strain curves including the post-necking strain were successfully obtained by the IFD method, and it was shown that the stress–strain curves for different degrees of cold work collapsed onto a single curve when the offset strain was considered. It was also shown that the Swift type constitutive equation gave good regression for the true stress–strain curves including the post-necking strain regardless of the degree of cold work, although the Ramberg–Osgood type constitutive equation showed poor fit. In the regression for the Swift type constitutive equation, the constant for power law could be assumed to be nS = 0.5

  11. 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

  12. A comparison of eastern North American seismic strain-rates to glacial rebound strain-rates

    Science.gov (United States)

    James, Thomas S.; Bent, Allison L.

    1994-01-01

    Glacial rebound strain-rates computed using a simple Laurentide glacial loading model are of the order of 10(exp -9) per year within the region of glaciation and extending several hundred kilometers beyond. The horizontal strain-rates receive approximately equal contributions from horizontal and vertical velocities, a consequence of the spherical geometry adopted for the Earth model. In the eastern United States and southeastern Canada the computed strain-rates are 1-3 orders of magnitude greater than an estimate of the average seismic strain-rate (Anderson, 1986) and approximately 1 order of magnitude greater than predicted erosional strain-rates. The predicted glacial rebound strain-rates are not, in general, oriented in such a way as to augment the observed state of deviatoric stress, possibly explaining why the seismic strain-rates are much smaller than the glacial rebound strain-rates. An exception to this may be seismically active regions in the St. Lawrence valley.

  13. Predicting creep rupture from early strain data

    International Nuclear Information System (INIS)

    To extend creep life modelling from classical rupture modelling, a robust and effective parametric strain model has been developed. The model can reproduce with good accuracy all parts of the creep curve, economically utilising the available rupture models. The resulting combined model can also be used to predict rupture from the available strain data, and to further improve the rupture models. The methodology can utilise unfailed specimen data for life assessment at lower stress levels than what is possible from rupture data alone. Master curves for creep strain and rupture have been produced for oxygen-free phosphorus-doped (OFP) copper with a maximum testing time of 51,000 h. Values of time to specific strain at given stress (40-165 MPa) and temperature (125-350 deg. C) were fitted to the models in the strain range of 0.1-38%. With typical inhomogeneous multi-batch creep data, the combined strain and rupture modelling involves the steps of investigation of the data quality, extraction of elastic and creep strain response, rupture modelling, data set balancing and creep strain modelling. Finally, the master curves for strain and rupture are tested and validated for overall fitting efficiency. With the Wilshire equation as the basis for the rupture model, the strain model applies classical parametric principles with an Arrhenius type of thermal activation and a power law type of stress dependence for the strain rate. The strain model also assumes that the processes of primary and secondary creep can be reasonably correlated. The rupture model represents a clear improvement over previous models in the range of the test data. The creep strain information from interrupted and running tests were assessed together with the rupture data investigating the possibility of rupture model improvement towards lower stress levels by inverse utilisation of the combined rupture based strain model. The developed creep strain model together with the improved rupture model is

  14. The Stress-Strain Condition Estimation of Detail in Crack Tip by Integral Strain Gauges

    Science.gov (United States)

    Syzrantsev, V.; Syzrantseva, K.

    2016-04-01

    The paper considers the task of stress-strain condition calculation of experimental sample in fatigue crack tip on weld boundary at its cyclic deforming. For this task decision authors use the information obtained by original means of cyclic strains measurement: Integral Strain Gauges. The results of carried experimental researches are compared with data of stress-strain condition estimation of detail in crack tip calculated by Finish Element Method.

  15. Effect of strain rate and temperature at high strains on fatigue behavior of SAP alloys

    DEFF Research Database (Denmark)

    Blucher, J.T.; Knudsen, Per; Grant, N.J.

    1968-01-01

    Fatigue behavior of three SAP alloys of two nominal compositions (7 and 13% Al2O3) was studied in terms of strain rate and temperature at high strains; strain rate had no effect on life at 80 F, but had increasingly greater effect with increasing temperature above 500 F; life decreased with...... decreasing strain rate, above 500 F, and with increasing temperature; ductility at fracture in tension test is shown to be important factor in determining life under conditions of test. (20318)...

  16. Multiplying decomposition of stress/strain, constitutive/compliance relations, and strain energy

    OpenAIRE

    Lee, Hyunsuk; Kim, Jinkyu

    2012-01-01

    To account for phenomenological theories and a set of invariants, stress and strain are usually decomposed into a pair of pressure and deviatoric stress and a pair of volumetric strain and deviatoric strain. However, the conventional decomposition method only focuses on individual stress and strain, so that cannot be directly applied to either formulation in Finite Element Method (FEM) or Boundary Element Method (BEM). In this paper, a simpler, more general, and widely applicable decompositio...

  17. Influence of Azospirillum Strains on the Nodulation of Clovers by Rhizobium Strains

    OpenAIRE

    Plazinski, Jacek; Rolfe, Barry G.

    1985-01-01

    Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cel...

  18. Crack growth monitoring by strain measurements

    International Nuclear Information System (INIS)

    Cracks detected during in-service inspections are not always removed when they are judged as hazardous. It is important to monitor the crack growth in order to secure the integrity of the cracked components. The author and a co-worker proposed a crack growth monitoring method, in which the elastic strain caused by internal pressure is continuously measured. The elastic strain acting at the outside surface of a pressurized pipe changes due to growth of a crack in the inside surface, and the magnitude of its change depends on the growth size. In this study, the author uses multiple strain gages to monitor the elastic strain acting on the cracked part of a pipe. An axial crack was introduced at the butt welding portion inside a carbon steel pipe. The strains were then measured under static internal pressure. The crack size was estimated based on the change in strains measured by strain gages attached onto the outside surface of the pipe. This study reveals that such a monitoring procedure could successfully identify not only the crack depth but also the surface length. The maximum estimation errors were 2.2 mm and 0.97 mm for the surface length and depth, respectively. The accuracy of the estimation improved as the number of strain gages increased. It was also apparent that the residual stress had subtle effect on the size estimation, albeit it may have significant influence when the crack propagates. (author)

  19. On lower order strain gradient plasticity theories

    DEFF Research Database (Denmark)

    Niordson, Christian Frithiof; Hutchinson, J. W.

    2002-01-01

    By way of numerical examples, this paper explores the nature of solutions to a class of strain gradient plasticity theories that employ conventional stresses, equilibrium equations and boundary conditions. Strain gradients come into play in these modified conventional theories only to alter the...

  20. On lower order strain gradient plasticity theories

    DEFF Research Database (Denmark)

    Niordson, Christian Frithiof; Hutchinson, J. W.

    2003-01-01

    By way of numerical examples, this paper explores the nature of solutions to a class of strain gradient plasticity theories that employ conventional stresses, equilibrium equations and boundary conditions. Strain gradients come into play in these modified conventional theories only to alter the...

  1. Nanocomposite Strain Gauges Having Small TCRs

    Science.gov (United States)

    Gregory, Otto; Chen, Ximing

    2009-01-01

    Ceramic strain gauges in which the strain-sensitive electrically conductive strips made from nanocomposites of noble metal and indium tin oxide (ITO) are being developed for use in gas turbine engines and other power-generation systems in which gas temperatures can exceed 1,500 F (about 816 C). In general, strain gauges exhibit spurious thermally induced components of response denoted apparent strain. When temperature varies, a strain-gauge material that has a nonzero temperature coefficient of resistance (TCR) exhibits an undesired change in electrical resistance that can be mistaken for the change in resistance caused by a change in strain. It would be desirable to formulate straingauge materials having TCRs as small as possible so as to minimize apparent strain. Most metals exhibit positive TCRs, while most semiconductors, including ITO, exhibit negative TCRs. The present development is based on the idea of using the negative TCR of ITO to counter the positive TCRs of noble metals and of obtaining the benefit of the ability of both ITO and noble metals to endure high temperatures. The noble metal used in this development thus far has been platinum. Combinatorial libraries of many ceramic strain gauges containing nanocomposites of various proportions of ITO and platinum were fabricated by reactive co-sputtering from ITO and platinum targets onto alumina- and zirconia-based substrates mounted at various positions between the targets.

  2. High-Temperature Resistance Strain Gauges

    Science.gov (United States)

    Lei, Jih-Fen

    1994-01-01

    Resistance strain gauges developed for use at high temperatures in demanding applications like testing aircraft engines and structures. Measures static strains at temperatures up to 800 degrees C. Small and highly reproducible. Readings corrected for temperature within small tolerances, provided temperatures measured simultaneously by thermocouples or other suitable devices. Connected in wheatstone bridge.

  3. Whole genome sequences of four Brucella strains.

    Science.gov (United States)

    Ding, Jiabo; Pan, Yuanlong; Jiang, Hai; Cheng, Junsheng; Liu, Taotao; Qin, Nan; Yang, Yi; Cui, Buyun; Chen, Chen; Liu, Cuihua; Mao, Kairong; Zhu, Baoli

    2011-07-01

    Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine. PMID:21602346

  4. Job strain and time to pregnancy

    DEFF Research Database (Denmark)

    Hjollund, N H; Jensen, Tina Kold; Bonde, J P; Henriksen, T B; Kolstad, H A; Andersson, A M; Ernst, E; Giwercman, A; Skakkebaek, N E; Olsen, J

    1998-01-01

    The association between fertility and job strain defined as high job demands and low job control has not previously been studied. A follow-up study was conducted with prospective collection of information on job strain among women, achievement of pregnancy, and potential confounding variables....

  5. Marital Role Strain and Sexual Satisfaction.

    Science.gov (United States)

    Frank, Ellen; And Others

    1979-01-01

    Responses to a questionnaire pertaining to discrepancies between an individual's ideal and actual marital role behaviors and level of sexual satisfaction indicated lower levels of role strain in nonpatient couples. A higher level of role strain correlated with increased sexual dissatisfaction. (Author)

  6. A Natural Vaccine Candidate Strain Against Cholera

    Institute of Scientific and Technical Information of China (English)

    LIUYAN-QING; QIGUO-MING; 等

    1995-01-01

    El Tor Vibrio cholerae(EVC)strains may be classified into two kinds-epidemigenic(EEVC)strains and non-epidemigenic(NEEVC)strains-based on a phage-biotyping system.A large number of EEVC strains have been screened for toxigenic and putative colonization attributes.One such naturally occurring strain(designated IEM101)has been found which is devoid of genes encoding cholera toxin(CT),accessory cholera enterotoxin(ACE),zonula occludens toxin(ZOT),but possesses RS1 sequences and toixn-coregulated pilus A gene(tcpA)although tcpA is poorly expressed.It expresses type B pili but does not posses type C pili.It is an El Tor Ogawa strain and does not cause fluid accumulation in rabbit ileal loop tests.Active immunization of rabbits with strain IEM101 elicited good protection against challenge with virulent strains of V.cholerae Ol.Oral administration cased no side effects in 15 human volunteers.colonized the gut for four to ten days and elicited good immune responses.

  7. On fracture in finite strain gradient plasticity

    DEFF Research Database (Denmark)

    Martínez Pañeda, Emilio; Niordson, Christian Frithiof

    2016-01-01

    In this work a general framework for damage and fracture assessment including the effect of strain gradients is provided. Both mechanism-based and phenomenological strain gradient plasticity (SGP) theories are implemented numerically using finite deformation theory and crack tip fields are...... investigated. Differences and similarities between the two approaches within continuum SGP modeling are highlighted and discussed. Local strain hardening promoted by geometrically necessary dislocations (GNDs) in the vicinity of the crack leads to much higher stresses, relative to classical plasticity...... predictions. These differences increase significantly when large strains are taken into account, as a consequence of the contribution of strain gradients to the work hardening of the material. The magnitude of stress elevation at the crack tip and the distance ahead of the crack where GNDs significantly alter...

  8. Mechanical strain effects on black phosphorus nanoresonators.

    Science.gov (United States)

    Wang, Cui-Xia; Zhang, Chao; Jiang, Jin-Wu; Park, Harold S; Rabczuk, Timon

    2016-01-14

    We perform classical molecular dynamics simulations to investigate the effects of mechanical strain on single-layer black phosphorus nanoresonators at different temperatures. We find that the resonant frequency is highly anisotropic in black phosphorus due to its intrinsic puckered configuration, and that the quality factor in the armchair direction is higher than in the zigzag direction at room temperature. The quality factors are also found to be intrinsically larger than those in graphene and MoS2 nanoresonators. The quality factors can be increased by more than a factor of two by applying tensile strain, with uniaxial strain in the armchair direction being the most effective. However, there is an upper bound for the quality factor increase due to nonlinear effects at large strains, after which the quality factor decreases. The tension induced nonlinear effect is stronger along the zigzag direction, resulting in a smaller maximum strain for quality factor enhancement. PMID:26649476

  9. Dark field electron holography for strain measurement

    International Nuclear Information System (INIS)

    Dark field electron holography is a new TEM-based technique for measuring strain with nanometer scale resolution. Here we present the procedure to align a transmission electron microscope and obtain dark field holograms as well as the theoretical background necessary to reconstruct strain maps from holograms. A series of experimental parameters such as biprism voltage, sample thickness, exposure time, tilt angle and choice of diffracted beam are then investigated on a silicon-germanium layer epitaxially embedded in a silicon matrix in order to obtain optimal dark field holograms over a large field of view with good spatial resolution and strain sensitivity. -- Research Highlights: → Step by step explanation of the dark field electron holography technique. → Presentation of the theoretical equations to obtain quantitative strain map. → Description of experimental parameters influencing dark field holography results. → Quantitative strain measurement on a SiGe layer embedded in a silicon matrix.

  10. Pseudomagnetic fields and triaxial strain in graphene

    DEFF Research Database (Denmark)

    Settnes, Mikkel; Power, Stephen; Jauho, Antti-Pekka

    2016-01-01

    Pseudomagnetic fields, which can result from nonuniform strain distributions, have received much attention in graphene systems due to the possibility of mimicking real magnetic fields with magnitudes of greater than 100 T. We examine systems with such strains confined to finite regions ("pseudoma......Pseudomagnetic fields, which can result from nonuniform strain distributions, have received much attention in graphene systems due to the possibility of mimicking real magnetic fields with magnitudes of greater than 100 T. We examine systems with such strains confined to finite regions......-binding calculations of single pseudomagnetic dots in extended graphene sheets confirm these predictions, and are also used to study the effect of rotating the strain direction with respect to the underlying graphene lattice, and varying the size of the pseudomagnetic dot....

  11. Review of graphene-based strain sensors

    Institute of Scientific and Technical Information of China (English)

    Zhao Jing; Zhang Guang-Yu; Shi Dong-Xia

    2013-01-01

    In this paper,we review various types of graphene-based strain sensors.Graphene is a monolayer of carbon atoms,which exhibits prominent electrical and mechanical properties and can be a good candidate in compact strain sensor applications.However,a perfect graphene is robust and has a low piezoresistive sensitivity.So scientists have been driven to increase the sensitivity using different kinds of methods since the first graphene-based strain sensor was reported.We give a comprehensive review of graphene-based strain sensors with different structures and mechanisms.It is obvious that graphene offers some advantages and has potential for the strain sensor application in the near future.

  12. Dark field electron holography for strain measurement

    Energy Technology Data Exchange (ETDEWEB)

    Beche, A., E-mail: armand.beche@fei.com [CEA-Grenoble, INAC/SP2M/LEMMA, F-38054 Grenoble (France); Rouviere, J.L. [CEA-Grenoble, INAC/SP2M/LEMMA, F-38054 Grenoble (France); Barnes, J.P.; Cooper, D. [CEA-LETI, Minatec Campus, F-38054 Grenoble (France)

    2011-02-15

    Dark field electron holography is a new TEM-based technique for measuring strain with nanometer scale resolution. Here we present the procedure to align a transmission electron microscope and obtain dark field holograms as well as the theoretical background necessary to reconstruct strain maps from holograms. A series of experimental parameters such as biprism voltage, sample thickness, exposure time, tilt angle and choice of diffracted beam are then investigated on a silicon-germanium layer epitaxially embedded in a silicon matrix in order to obtain optimal dark field holograms over a large field of view with good spatial resolution and strain sensitivity. -- Research Highlights: {yields} Step by step explanation of the dark field electron holography technique. {yields} Presentation of the theoretical equations to obtain quantitative strain map. {yields} Description of experimental parameters influencing dark field holography results. {yields} Quantitative strain measurement on a SiGe layer embedded in a silicon matrix.

  13. Near tip strain evolution under cyclic loading

    Directory of Open Access Journals (Sweden)

    J. Tong

    2013-07-01

    Full Text Available The concept of ratchetting strain as a crack driving force in controlling crack growth has previously been explored at Portsmouth using numerical approaches for nickel-based superalloys. In this paper, we report the first experimental observations of the near-tip strain evolution as captured by the Digital Image Correlation (DIC technique on a compact tension specimen of stainless steel 316L. The evolution of the near-tip strains with loading cycles was studied whilst the crack tip was maintained stationary. The strains were monitored over the selected distances from the crack tip for a given number of cycles under an incremental loading regime. The results show that strain ratchetting does occur with load cycling, and is particularly evident close to the crack tip and under higher loads. A finite element model has been developed to simulate the experiments and the simulation results are compared with the DIC measurements.

  14. Microstructure and strain hardening of steels

    International Nuclear Information System (INIS)

    In the present work, the relation between metallurgical microstructures and strain hardening behaviour of both fcc and bcc steels was investigated. In order to obtain large strains, a new experimental method was developed and the data mathematically prepared for further analysis. It was found that the strain hardening rate increases with decreasing particle size or increasing yield stress of ferritic steels. The strain hardening rate is found to be correlated to the specific microstructure. A new model was formulated to describe large deformations. This has been done on the basis of already existing models. According to the new model, the strain hardening in stage IV is caused by the dislocations in cell interiors instead of those in the cell walls. (orig.)

  15. Nanoscale strain mapping in battery nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Ulvestad, A., E-mail: aulvesta@ucsd.edu; Kim, J. W.; Dietze, S. H.; Shpyrko, O. G. [Department of Physics, University of California-San Diego, La Jolla, California 92093-0319 (United States); Cho, H. M.; Meng, Y. S. [Department of NanoEngineering, University of California-San Diego, La Jolla, California 92093-0448 (United States); Harder, R. [Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439 (United States); Fohtung, E. [Manuel Lujan Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Department of Physics, New Mexico State University, Las Cruces, New Mexico 88003 (United States)

    2014-02-17

    Coherent x-ray diffraction imaging is used to map the local three dimensional strain inhomogeneity and electron density distribution of two individual LiNi{sub 0.5}Mn{sub 1.5}O{sub 4−δ} cathode nanoparticles in both ex-situ and in-situ environments. Our reconstructed images revealed a maximum strain of 0.4%. We observed different variations in strain inhomogeneity due to multiple competing effects. The compressive/tensile component of the strain is connected to the local lithium content and, on the surface, interpreted in terms of a local Jahn-Teller distortion of Mn{sup 3+}. Finally, the measured strain distributions are discussed in terms of their impact on competing theoretical models of the lithiation process.

  16. 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

  17. Extreme resistance to two Brazilian strains of Potato virus Y (PVY in transgenic potato, cv. Achat, expressing the PVYº coat protein Resistência extrema a duas estirpes do Potato virus Y (PVY de batata transgênica, cv. Achat, expressando o gene da capa protéica do PVY O

    Directory of Open Access Journals (Sweden)

    Eduardo Romano

    2001-07-01

    Full Text Available The coat protein (CP gene of the potato virus Y strain "o" (PVY O was introduced into potato, cultivar Achat, via Agrobacterium tumefaciens-mediated transformation. Sixty three putative transgenic lines were challenged against the Brazilian strains PVY-OBR and PVY-NBR. An extremely resistant phenotype, against the two strains, was observed in one line, denominated 1P. No symptoms or positive ELISA results were observed in 16 challenged plants from this line. Another clone, named as 63P, showed a lower level of resistance. Southern blot analysis showed five copies of the CP gene in the extremely resistant line and at least three copies in the other resistant line. The stability of the integrated transgenes in the extreme resistant line was examined during several in vitro multiplications over a period of three years, with no modification in the Southern pattern was observed. The stability of the transgenes, the absence of primary infections and the relatively broad spectrum of resistance suggest that the extremely resistant line obtained in this work can be useful for agricultural purposes.O gene da capa protéica (CP do Potato virus Y estirpe "o", foi introduzido em batata cultivar Achat, via Agrobacterium tumefaciens. Sessenta e três linhas possivelmente transgênicas foram desafiadas com as estirpes brasileiras PVY-OBR e PVY-NBR. Uma linha apresentou extrema resistência às duas estirpes inoculadas, e foi denominado clone 1P. Não foram observados sintomas sistêmicos de infecção e as plantas foram negativas em Elisa. Outra linha, denominada clone 63P, mostrou algum nível de resistência. Análises por Southern blot indicaram a presença de pelo menos cinco cópias do gen CP no clone 1P e pelo menos três cópias no clone 63P. A estabilidade do gene introduzido no clone 1P foi avaliada durante três anos, após várias multiplicações in vitro. Não foram observadas mudanças no padrão do Southern blot. A estabilidade do transgene, na

  18. Irreducible decomposition of strain gradient tensor in isotropic strain gradient elasticity

    CERN Document Server

    Lazar, Markus

    2016-01-01

    In isotropic strain gradient elasticity, we decompose the strain gradient tensor into its irreducible pieces under the n-dimensional orthogonal group O(n). Using the Young tableau method for traceless tensors, four irreducible pieces (n>2), which are canonical, are obtained. In three dimensions, the strain gradient tensor can be decomposed into four irreducible pieces with 7+5+3+3 independent components whereas in two dimensions, the strain gradient tensor can be decomposed into three irreducible pieces with 2+2+2 independent components. The knowledge of these irreducible pieces is extremely useful when setting up constitutive relations and strain energy.

  19. Strain relaxation in nano-patterned strained-Si/SiGe heterostructure on insulator

    International Nuclear Information System (INIS)

    In order to evaluate the strain stability, arrays of strained Si/SiGe nano-stripes and nano-pillars were fabricated by Electron-Beam Lithography (EBL) and Reactive-Ion Etching (RIE). The strain relaxation in the patterned strained Si on SiGe-on-insulator (SGOI) was investigated by high-resolution UV micro-Raman spectroscopy. The Raman measurements before and after patterning indicate that most of the strain in the top strained Si is maintained until scaling down to 300 nm, and relaxation of <15% is observed in pillars with a dimension of 150 nm x 150 nm. In the nano-patterned heterostructure strained Si/SiGe, the observed relaxation is small, which is mainly attributed to the fully relaxed and dislocation-free SiGe virtual substrate fabricated by modified Ge condensation.

  20. Strain mapping analysis of textile composites

    Science.gov (United States)

    Ivanov, Dmitry; Ivanov, Sergey; Lomov, Stepan; Verpoest, Ignaas

    2009-03-01

    The focus of the work is meso-scale analysis (scale level of the fabric unit cell) of textile composite deformation and failure. The surface strain measurement is used for: (1) experimental investigation, which includes study of strain distribution at various stages of deformation, plasticity detection, damage initiation; (2) numerical validation of the correspondent finite element (FE) models. Two examples are considered: carbon-epoxy triaxial-braided and glass polypropylene-woven composite. The surface strain measurement (by digital image correlation technique) accompanies the tensile tests, aiming at: (1) elastic anisotropic constants characterisation, (2) study of non-linear material behaviour (for the thermoplastic composite), (3) control of homogeneity of the macro-strain distribution, and (4) analysis of damage initiation in brittle composites. Validation of meso-FE models by strain measurements encounters difficulties arising from (1) resolution of the strain measurements, (2) irregularities of the initial structure such as random layer nesting, ply interaction, and deviation of yarns from their theoretical position, which affects the measured strain fields. The paper discusses these difficulties and demonstrates a qualitative agreement with the FE analysis of idealised composite configurations.

  1. Thermoresistance in radioresistant strains of 'Drosophila nebulosa'

    International Nuclear Information System (INIS)

    The detection of thermoresistance in radioresistant strains of 'D. nebulosa' is described, as well as some conclusions on the genetic nature of these differences are presented. The strains used in this experiment were MF 204, from 'Morro de Ferro', in Pocos de Caldas (MG) (one of the biggest radioactive anomalies in the world) whose radioresistance is due to its additive genetic components (Kratz, 1973 and 1975); 85(87) R, an induced radioresistant strain; and MF K a control 'pooled' strain obtained near 'Morro do Ferro'. Survival tests, 72 hours after temperature shocks, performed in the interval of 360C to 390C showed a decreasing gradient of thermoresistance with the following regression coefficients: MF 204 b=-5,4; 85(87)R b=-7,2 and MF K b=-7,9. Bifactorial analysis (strains and sexes) performed at 380C and 390C confirmed differences among strains (P<01 and P<0,5, respectively) suggesting a poligenic control of the thermoresistance. Consitent and verified relations among strains, being simultaneously resistant to different kinds of mutagenic factors, are considered evidence of the existence of general mechanisms in mutagenic control. Therefore, the results, together with results of Tsukamoto, Ogaki and Kikkawa 1957, Ogaki 1962 and Nakashima-Tanaka 1966, Parsons 1969, add to the hypotheses of the existence of general mechanisms in mutagenic control

  2. Piezoelectric strain sensor/actuator rosettes

    International Nuclear Information System (INIS)

    In-plane anisotropy in the linear piezoelectric constitutive law for [011]c cut and poled PMN–0.29PT is demonstrated to enable its use as a sensor/actuator rosette. The equations for a 0°/45°/90° rosette are developed using the conditions of coupling between the in-plane strain of the crystal and a substrate, and zero out-of-plane stress on the crystal (plane stress conditions in the crystals). The crystals are bonded to a substrate aluminum plate that is instrumented with strain gages next to the crystals. The plate is subjected to bending about different axes and the resulting electric displacement change of the crystals is monitored. The strain components calculated using the change of electric displacement are compared with the strain components measured using strain gages. This sensor/actuator rosette approach is demonstrated to enable both sensing principal strain components and actuating principal strains in an electronically controllable direction. (fast track communication)

  3. PHYLOGENETIC STUDY OF SOME STRAINS OF DUNALIELLA

    Directory of Open Access Journals (Sweden)

    Duc Tran

    2013-01-01

    Full Text Available Dunaliella strains were isolated from a key site for salt production in Vietnam (Vinh Hao, Binh Thuan province. The strains were identified based on Internal Transcribed Spacer (ITS markers. The phylogenetic tree revealed these strains belong to the clades of Dunaliella salina and Dunaliella viridis. Results of this study confirm the ubiquitous nature of Dunaliella and suggest that strains of Dunaliella salina might be acquired locally worldwide for the production of beta-carotene. The identification of these species infers the presence of other Dunaliella species (Dunaliella tertiolecta, Dunaliella primolecta, Dunaliella parva, but further investigation would be required to confirm their presence in Vietnam. We anticipate the physiological and biochemical characteristics of these local species will be compared with imported strains in a future effort. This will facilitate selection of strains with the best potential for exploitation in the food, aquaculture and biofuel industries. The Dunaliella strains isolated and identified in this study are maintained at the Laboratory of Algal Biotechnology, International University and will be made available for research and educational institutions.

  4. Strain gradient plasticity: energetic or dissipative?

    Science.gov (United States)

    Fleck, N. A.; Willis, J. R.

    2015-08-01

    For an infinite slab of strain gradient sensitive material subjected to plane-strain tensile loading, computation established and analysis confirmed that passivation of the lateral boundaries at some stage of loading inhibits plastic deformation upon further loading. This result is not surprising in itself except that, remarkably, if the gradient terms contribute to the dissipation, the plastic deformation is switched off completely and only resumes at a clearly defined higher load, corresponding to a total strain , say. The analysis presented in this paper confirms the delay of plastic deformation following passivation and determines the exact manner in which the plastic flow resumes. The plastic strain rate is continuous at the exact point of resumption of plastic flow and, for the first small increment in the imposed total strain, the corresponding increment in plastic strain, , is proportional to . The constant A in the relation , where denotes the plastic strain increment at the centre of the slab, has been determined explicitly; it depends on the hardening modulus of the material. The presence of energetic gradient terms has no effect on the value of unless the dissipative terms are absent, in which case passivation reduces the rate of plastic deformation but introduces no delay. This qualitative effect of dissipative gradient terms opens the possibility of experimental discrimination of their presence or absence. The analysis employs an incremental variational formulation that is likely to find use in other problems.

  5. Revisiting the gauge fields of strained graphene

    OpenAIRE

    Iorio, Alfredo; Pais, Pablo(Centro de Estudios Científicos (CECS), Casilla, 1469 , Valdivia, Chile)

    2015-01-01

    We show that, when graphene is only subject to strain, the spin connection gauge field that arises plays no measurable role, but when intrinsic curvature is present and strain is small, spin connection dictates most the physics. We do so by showing that the Weyl field associated with strain is a pure gauge field and no constraint on the $(2+1)$-dimensional spacetime appears. On the other hand, for constant intrinsic curvature that also gives a pure-gauge Weyl field, we find a classical manife...

  6. STRAIN ANALYSIS OF LATERAL EXTRUSION PROCESS

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The strain distribution of equal-cross section lateral extrusion(ECSLE) has been simulated by finite element method. Considering the effect of friction and the width of sample, the simulation results are very close to the reality. The simulated results showed that, around the corner of die, the strain is distributed by sharp layers, and the gradient of the layers is very large, which means that the deformation is just plane shear deformation; the larger the width of sample or the smaller the friction, the more uniform the strain distribution is.

  7. Semiconductor strain metrology principles and applications

    CERN Document Server

    Wong, Terence KS

    2012-01-01

    This book surveys the major and newly developed techniques for semiconductor strain metrology. Semiconductor strain metrology has emerged in recent years as a topic of great interest to researchers involved in thin film and nanoscale device characterization. This e-book employs a tutorial approach to explain the principles and applications of each technique specifically tailored for graduate students and postdoctoral researchers. Selected topics include optical, electron beam, ion beam and synchrotron x-ray techniques. Unlike earlier references, this e-book specifically discusses strain metrol

  8. Self-diffusion in compressively strained Ge

    International Nuclear Information System (INIS)

    Under a compressive biaxial strain of ∼ 0.71%, Ge self-diffusion has been measured using an isotopically controlled Ge single-crystal layer grown on a relaxed Si0.2Ge0.8 virtual substrate. The self-diffusivity is enhanced by the compressive strain and its behavior is fully consistent with a theoretical prediction of a generalized activation volume model of a simple vacancy mediated diffusion, reported by Aziz et al.[Phys. Rev. B 73, 054101 (2006)]. The activation volume of (-0.65±0.21) times the Ge atomic volume quantitatively describes the observed enhancement due to the compressive biaxial strain very well.

  9. Strain gage balances and buffet gages

    Science.gov (United States)

    Ferris, A. T.

    1983-01-01

    One-piece strain gage force balances were developed for use in the National Transonic Facility (NTF). This was accomplished by studying the effects of the cryogenic environment on materials, strain gages, cements, solders, and moisture proofing agents, and selecting those that minimized strain gage output changes due to temperature. In addition, because of the higher loads that may be imposed by the NTF, these balances are designed to carry a larger load for a given diameter than conventional balances. Full cryogenic calibrations were accomplished, and wind tunnel results that were obtained from the Langley 0-3-Meter Transonic Cryogenic Tunnel were used to verify laboratory test results.

  10. Strains in pyrolytic graphite under irradiation

    International Nuclear Information System (INIS)

    The studies of strains in pyrolytic graphite caused by development of amorphization in the irradiation zone showed that the local strains attain tens of percent; their distribution is inhomogeneous both in magnitude and in sign. The tendency toward formation of a biaxial field of compressive strains becomes marked with an increase in the fluence, although the formation of small regions in the form of narrow sectors within whose boundaries compression is observed in the radial direction and extension is observed in the tangential direction is also possible. 14 refs., 6 figs

  11. Stability of germanene under tensile strain

    KAUST Repository

    Kaloni, Thaneshwor P.

    2013-09-01

    The stability of germanene under biaxial tensile strain and the accompanying modifications of the electronic properties are studied by density functional theory. The phonon spectrum shows that up to 16% strain the germanene lattice is stable, where the Dirac cone shifts towards higher energy and hole-doped Dirac states are achieved. The latter is due to weakening of the Ge-Ge bonds and reduction of the s-p hybridization. Our calculated Grüneisen parameter shows a similar dependence on the strain as reported for silicene (which is different from that of graphene). © 2013 Elsevier B.V. All rights reserved.

  12. Unit cell of strained GeSi

    International Nuclear Information System (INIS)

    The local structure within the unit cell of strained-GeSi layers grown on Si(001) has been examined by polarization-dependent extended x-ray-absorption fine structure. First-neighbor bond lengths are found to deviate only slightly from their unstrained values; however, the distortion of the cubic-unit cell by strain leads to measurable polarization-dependent changes in first-shell coordination and second-shell distances. A unifying picture of bond lengths and elasticity in strained-layer semiconductors is presented. copyright 1997 The American Physical Society

  13. Construction of Killer Wine Yeast Strain

    OpenAIRE

    Seki, Tetsuji; Choi, Eon-Ho; Ryu, Dewey

    1985-01-01

    A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the k...

  14. 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...

  15. 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

  16. A new radial strain and strain rate estimation method using autocorrelation for carotid artery

    Science.gov (United States)

    Ye, Jihui; Kim, Hoonmin; Park, Jongho; Yeo, Sunmi; Shim, Hwan; Lim, Hyungjoon; Yoo, Yangmo

    2014-03-01

    Atherosclerosis is a leading cause of cardiovascular disease. The early diagnosis of atherosclerosis is of clinical interest since it can prevent any adverse effects of atherosclerotic vascular diseases. In this paper, a new carotid artery radial strain estimation method based on autocorrelation is presented. In the proposed method, the strain is first estimated by the autocorrelation of two complex signals from the consecutive frames. Then, the angular phase from autocorrelation is converted to strain and strain rate and they are analyzed over time. In addition, a 2D strain image over region of interest in a carotid artery can be displayed. To evaluate the feasibility of the proposed radial strain estimation method, radiofrequency (RF) data of 408 frames in the carotid artery of a volunteer were acquired by a commercial ultrasound system equipped with a research package (V10, Samsung Medison, Korea) by using a L5-13IS linear array transducer. From in vivo carotid artery data, the mean strain estimate was -0.1372 while its minimum and maximum values were -2.961 and 0.909, respectively. Moreover, the overall strain estimates are highly correlated with the reconstructed M-mode trace. Similar results were obtained from the estimation of the strain rate change over time. These results indicate that the proposed carotid artery radial strain estimation method is useful for assessing the arterial wall's stiffness noninvasively without increasing the computational complexity.

  17. Information about the Current Strain of Clostridium difficile

    Science.gov (United States)

    ... treatment of this epidemic strain different? How does fluoroquinolone resistance affect management of this strain? What should ... guidelines on internet) Top of page How does fluoroquinolone resistance affect management of this strain? Increased fluoroquinolone ...

  18. Screening of Trichoderma strains tolerant to benzimidazole

    Institute of Scientific and Technical Information of China (English)

    LIU Kai-qi; XIANG Mei-mei; LIU Ren; ZENG Yong-san; ZHOU Hong-zi; YU Jin-feng; JIANG Xin-yin; ZHANG Yue-li

    2004-01-01

    @@ The screening of isolates and the assay of biocontrol mechanisms of Trichoderma were studied systematically in laboratory and greenhouse in vivo. The proteins tolerant to benzimidazole in Trichoderma strains were purified, and their physical and chemical properties were detected. Compared their biological activities in vitro and vivo in greenhouse, nine biocontrol strains (including Ty- 10-2, LTR-2, Tj-5-1, Tj-5-4, Ty- 11-1, Tj-11-3, Ty- 11-3, Tj-3-3-2, Tj-3-3-4) were screened. These biocontrol strains had faster rates of growth and higher inhibition to gray mould (Bortrytis cinerea),and the inhibition was stable. The effects of controlling gray mould in greenhouse with the screened Trichoderma strains were 70 % and 50 % in vivo.

  19. Axillary vein thrombosis mimicking muscular strain.

    OpenAIRE

    Louis, J.

    1999-01-01

    Axillary vein thrombosis may occur on strenuous activity with a clinical picture similar to a simple strain. It carries significant morbidity but a good outcome is possible with early treatment. The aetiology, investigation, and treatment are discussed.

  20. Energy Harvesting Wireless Strain Networks Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Prime Research LC (PPLC) and Virginia Tech (VT) propose to develop an energy harvesting wireless strain node technology that utilizes single-crystal piezoelectric...