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1
Ophel, Kathleen Margaret. ""Agrobacterium" : plasmids and biovars /." Title page, contents and summary only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09pho61.pdf.
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Abarca, Grau Ana María. "Biopelículas en Agrobacterium spp." Doctoral thesis, Universitat Politècnica de València, 2011. http://hdl.handle.net/10251/11670.
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Diversas especies del género Agrobacterium producen tumores en gran cantidad de plantas cultivadas, causando la enfermedad conocida en inglés como "crown gall". En esta tesis se ha demostrado que cepas patógenas y no patógenas de las especies A. tumefaciens, A. rhizogenes y A. vitis son capaces de formar biopelículas tanto sobre superficies inertes como sobre raíces de tomate. Las cepas de A. tumefaciens y A. vitis se unieron a poliestireno y polipropileno, mientras que las de A. rhizogenes sólo se unieron a polipropileno. Se ha constatado que la formación de biopelículas in vitro sobre superficies abióticas en Agrobacterium spp. depende de la especie (biovar), la superficie y las condiciones de cultivo. Mediante microscopía electrónica de barrido y microscopía láser confocal, utilizando cepas marcadas con GFP, se demostró que las tres especies formaban estructuras complejas compuestas por numerosas células bacterianas dispuestas según alguno de los siguientes modelos: 1) tapices densos y continuos, 2) grandes agregados irregulares embebidos en material extracelular, o 3) en forma de setas globulares atravesadas internamente por canales. Los resultados sugieren que la formación de biopelículas puede estar asociada a la colonización y supervivencia de estas especies bacterianas en la rizosfera. Además, la capacidad del agente de biocontrol A. rhizogenes K84 de formar biopelículas durante su interacción con la planta podría ser una característica relevante para el control de la enfermedad. Con el propósito de ahondar y conocer genes implicados en la formación de biopelículas en este agente de biocontrol se han utilizado dos estrategias. Mediante genética clásica, se analizó una librería de mutantes al azar de la cepa K84 y se identificaron dos mutantes afectados en la formación de biopelículas. Uno fue incapaz de unirse y formar biopelículas sobre polipropileno, pero por el contrario se unió y formó biopelículas sobre ápices radiculares. El otro mutante produjo más biopelícula que la cepa K84, tanto en superficie abiótica como en ápices radiculares.Abarca Grau, AM. (2011). Biopelículas en Agrobacterium spp [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11670
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3
Faria, Maria José Sparça Salles de. "Red raspberry transformation using agrobacterium." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69522.
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Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry.The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.
4
Spencer, Paul Anthony. "Signal compound specificity in agrobacterium tumefaciens." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28401.
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Agrobacterium tumefaciens , a soil-borne gram negative bacterium, is the causative agent of crown gall disease and one of the most promising vectors for genetic engineering in plants. It is known to respond to the presence of certain plant-derived phenolic compounds by expressing an essential set of genes for virulence (vir) (Bolton et al., 1986; Stachel et al., 1985-a). However, only one report has described the isolation and identification of virulence inducing phytochemicals producecd by a host plant (Stachel etal., 1985-a). These compounds are acetosyringone (AS) and a-hydroxyacetosyringone (OH-AS), or 3,5-dimethoxy-4-hydroxy- and α-hydroxy-3,5-dimethoxy-4-hydroxy-acetophenone, respectively. Since these compounds have never previously been reported from plant tissues and are not likely to be of widespread occurrence, it seemed unlikely that these were the only signal compounds for this wide host range pathogen. In addition, the results of Bolton et al. (1986), who found that a mixture of lower molecular weight phenolics could also induce vir gene expression, raised the question as to exactly which chemical structures could act as vir -inducers.
This thesis reports a quantitative re-examination of the results of Bolton et al. (1986), describes more fully the structure-activity specificity of vir -induction in a wide host range (WHR) strain of A. tumefaciens than did Stachel et al. (1985-a), and presents results which indicate that WHR Agrobacterium is capable of detecting phytochemicals which are ubiquitous or at least widespread amongst susceptible hosts. The relative vir - inducing activities of the lignin precursors coniferyl and sinapyl alcohols, a variety of cinnamic acid derivatives and two chalcones are presented and discussed in terms of the early events in crown gall tumorogenesis and the sophisticated use of Agrobacterium in Ti plasmid-mediated transformation of plants.Science, Faculty of
Botany, Department of
Graduate
5
Janssen, Bart-Jan. "Agrobacterium-mediated gene transfer into kiwifruit." Thesis, University of Auckland, 1991. http://hdl.handle.net/2292/2313.
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A system has been developed to aid in the establishment of Agrobacterium-mediated transformation for new plant species. A series of binary vectors have been constructed that express a chimaeric β-D-glucuronidase (GUS) gene in plants cells but not in bacterial cells. This feature allows GUS activity from transformed plant cells to be assayed in the presence of Agrobacterium. Preliminary experiments examined the expression of these chimaeric GUS genes in transformed petunia leaf discs. GUS expression was detectable 2 days after inoculation, peaked at 3 – 4 days and then declined; if selection was imposed expression increased again after 10 - 14 days. The amount of expression observed 4 days after inoculation correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3 - 4 days after inoculation. Surprisingly, GUS expression was concentrated in localized zones on the circumference of the disc; within these zones essentially all the cells appeared to be expressing GUS. These results suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of the petunia leaf explants, but that the frequency of stable integration is several orders of magnitude lower. A reliable Agrobacterium-mediated transformation system has been established for kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward) by using transient expression of GUS to monitor gene transfer frequencies. In vitro culture of kiwifruit plants and conditions for regeneration of plants from leaf discs have been established. Several factors were found to improve gene transfer frequencies in kiwifruit: (i) healthy actively growing source tissue; (ii) the use of Agrobacterium strain A281; (iii) the presence of a layer of moistened filter paper between the leaf explants and the cocultivation media; and (iv) the presence of 20 μM acetosyringone in both the bacterial culture media and in the cocultivation media. Pre-culture of leaf explants significantly inhibited gene transfer, particularly at the cut edge of the explants. Using the optimized transformation system, at least one transgenic plant can be regenerated from each leaf inoculated. Stable transformation frequencies have been shown to vary significantly between different binary vectors. Phenotypic, PCR, and Southern analysis has confirmed the presence of stably integrated T-DNA in several transgenic kiwifruit plants.
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Ashby, Alison Mary. "Agrobacterium tumefaciens : chemotaxis and crop protection." Thesis, Durham University, 1988. http://etheses.dur.ac.uk/6723/.
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Chemotaxis in Agrobacierium tumefaciens was studied. Several plant derived monocyclic phenolic compounds were analysed for their ability to act as chemoattractants for A. tumefaciens C58C (^1) and as inducers of the Ti-plasmid virulence operons. The results divided the phenolics into 4 groups. A strong correlation between vir- inducing ability and Ti-plasmid requirement for chemo taxis was established and chemical structure rules for vir induction and chemo taxis are outlined. Furthermore, virA and virG were found to be the Ti-plasmid virulence genes required for chemo taxis towards the monocyclic phenolic compound acetosyringone. Chemotaxis towards both monocotyledonous and dicotyledonous plant extracts was analysed. Undiluted shoot and root extracts from both sources elicited a response from both Ti-plasmid harbouring and cured A. tumefaciens C58C(^1) However, when diluted extracts of Wheat and Kalanchoe shoot homogenate were analysed, a distinct enhancement of chemotaxis was conferred by the Ti-plasmid, suggesting that recognition of, and attraction towards, susceptible plants is not the step blocked in monocot transformation. Analysis of cell wall material revealed that native cell wall components are not required for chemotaxis of A. tumefaciens C58C (^1) towards plant extracts. Results obtained on chemotaxis along with current knowledge of vir- induction allowed the development of a novel idea involving Agrobacterium as a biocontrol agent. A chitinase gene from Serratia marcescens was manipulated such that its promotor was removed. The promotorless cassette was linked to the virB pro-motor from an octopine Ti-plasmid and the construct introduced into Agrobacterium harbouring virA and virG. The potential benefit of this biocontrol system with respect to other existing biocontrol systems is that expression of the pesticidal gene is regulated by components of wound exudate and therefore is a conservative process, pesticide being produced only when a plant is wounded, at a time when it is most susceptible to attack by plant pathogens, and then exclusively in the microrhizosphere around the wound site.
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Lilley, Catherine Jane. "Heterologous expression from Agrobacterium virulence promoters." Thesis, Durham University, 1991. http://etheses.dur.ac.uk/6202/.
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The aim of this work was twofold: to construct plasmids with a gene encoding a pesticidal protein expressed from an Agrobacterium tumefaciens virulence promoter and to determine, in planta, the sites of Agrobacterium vir-induction. A number of methods were employed to detect in situ vir-induction and, to this end, genes encoding β-glucuronidase (GUS) and bioluminescence (lux) were linked in plasmid constructs to Agrobacterium vir-promoters. In each case, expression of the gene was shown to be induced by the v/r-inducing phenolic compound acetosyringone. An existing plasmid, in which the lacZ gene was under control of the virB promoter was utilised to demonstrate v/r-induction occurring at sites of injury on the roots of mung bean seedlings. Pesticidal genes expressed from Agrobacterium vimlence promoters would form the basis of a biological control system. A microbial inoculant harbouring such a construct would produce the pesticidal protein only when in the presence of vi-inducing compounds in plant wound exudates. A chitinase gene, chiB, from Serratia marcescens was characterised and sequenced and, following removal of its promoter region, was linked to an Agrobacterium virB promoter. Plasmids were also constructed in which the chiA gene of S. marcescens was brought under the control of a virB or virE promoter. All the constructs specified acetosyringone- inducible production of chitinase. Chitinase is effective in the biological control of chitin containing organisms such as fungi.
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Khidr, Yehia. "Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-1963.
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Kempton, Julie B. "A mechanistic investigation of Agrobacterium [beta]-glucosidase." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/29141.
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The mechanism of glucoside hydrolysis by Agrobacterium β-glucosidase has been investigated through the study of linear free energy relationships and a-secondary deuterium kinetic isotope effects. A two-step mechanism has previously been proposed for this process, consisting of;
(1) cleavage of the glucosidic bond and formation of a covalent glucosyl-enzyme
intermediate ("glucosylation"), and
(2) hydrolysis of the intermediate to yield free enzyme and glucose
("deglucosylation").
Values of kcat and Km were determined for enzymic hydrolysis of fifteen substituted phenyl β-D-glucopyranosides with leaving group pKa's ranging from 3.96 to 10.34. A linear free energy correlation of log(kcat) vs. leaving group pKa resulted in a concave-downward plot with a break near pKa 8, indicating a change in rate-determining step of a multistep reaction. The rates of hydrolysis of substrates with leaving group pKa's < 8 are independent of phenol structure, indicating that deglucosylation is rate-limiting. Glucosides with leaving group pKa's > 8 exhibit a linear dependence of hydrolysis rate upon pKa, and thus it is proposed that glucosylation is the rate-determining step for these substrates. The value of the Hammett reaction constant, ρ, is 1.6, indicating that cleavage of the glucosidic bond is significantly advanced at the transition state.
The α-secondary deuterium kinetic isotope effects on hydrolysis of five substituted phenyl β-D-glucopyranosides were determined (deuterium substitution at the anomeric center), and the values were found to be segregated into two groups. The faster substrates (leaving group pKa < 8) exhibited kH/kD values of approximately 1.11, while values for the slower substrates averaged 1.06. These results support the hypothesis of a change in rate-determining step as leaving group pKa decreases. The magnitude of the isotope effect on glucosylation indicates a small amount of sp³ to sp² rehybridization at the transitionstate, which combined with the ρ value for this process suggests a substantial degree of nucleophilic participation of the enzymic carboxylate.
2-Deoxy-2-fluoro-D-glucosides with highly activated leaving groups are potent covalent inactivators of Agrobacterium β-glucosidase which operate by trapping the
enzyme as its glucosyl-enzyme intermediate. Values of ki and Ki were determined for six substituted phenyl 2-deoxy-2-fluoro-β-D-glucopyranosides whose leaving group pKa's
ranged from 3.96 to 7.18. A linear correlation was observed for both log(ki) and log(ki/Ki) vs. leaving group pKa, with ρ values of 2.0 and 2.7, respectively, which
indicates that cleavage of the glycosidic bond is virtually complete at the transition state. Such an observation of a linear free energy relationship between the rate of enzyme inactivation and the electronic structure of the inactivator is rarely accomplished in enzymology. Preliminary investigation of the α-secondary deuterium kinetic isotope effect on enzyme inactivation by 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside indicates that the effect is quite small, probably 0-5%. These results suggest that the inactivation proceeds via an essentially concerted mechanism and that the transition state has little oxocarbonium ion character.Science, Faculty of
Chemistry, Department of
Graduate
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Labarre, Marie. "Les hémoglobines tronquées de Agrobacterium tumefaciens C58." Québec : Université Laval, 2006. http://www.theses.ulaval.ca/2006/23598/23598.pdf.
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Labarre, Marie. "Les hémoglobines tronquées de Agrobacterium tumefaciens C58." Thesis, Université Laval, 2006. http://www.theses.ulaval.ca/2006/23598/23598.pdf.
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Résumé
Les hémoglobines tronquées (Hbtrs) sont retrouvées chez plusieurs organismes et leurs fonctions sont encore inconnues pour la plupart. Les Hbtrs du pathogène de plante Agrobacterium tumefaciens C58 ont été inactivées pour vérifier leurs implications dans la production de tumeur chez les plantes. Les expériences ont montré que les souches mutantes pour les Hbtrs AtuHb2 et AtuHb3 étaient capables d’induire la production de tumeur chez la plante Kalanchoe daigremontiana. La protéine recombinante AtuHb2 a été caractérisée par spectroscopie d’absorption et de résonance Raman. L’analyse, par spectroscopie d’absorption, montre que la protéine est hexacoordonnée dans la forme ferrique et ferreuse, qu’il est possible pour AtuHb2 de former des complexes stables avec les ligands CO, CN- et NO, mais pas avec l’O2 et que le complexe formé entre la forme ferreuse et le NO est pentacoordonné. L’analyse par spectroscopie de résonance Raman a montré que le ligand CO est peu stabilisé.
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Azhakanandam, K. "Agrobacterium-mediated rice (Oryza sativa L.) transformation." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285569.
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Lecomte, Solène. "Anaerobic respiration diversification in Agrobacterium fabrum C58." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1231.
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La respiration anaérobie peut être un trait essentiel dans le mode de vie, la colonisation de l'environnement et la survie. Jusqu'à présent, la seule respiration anaérobie confirmée chez Agrobacterium spp. est la dénitrification. De façon intéressante, cette voie est inégalement répandue chez les agrobactéries. Ces observations m'ont amené à mon hypothèse, à savoir la respiration anaérobie et notamment la dénitrification pourraient expliquer la coexistence d'agrobactéries et leur distribution dans des niches spécifiques de la rhizosphère. Ma thèse visait à explorer les stratégies de respiration anaérobie d’Agrobacterium spp. et de les relier à l'adaptation de niche écologiques différentes. Les objectifs de ma thèse étaient (1) de caractériser tous les gènes impliqués dans la dénitrification chez A. fabrum C58 in vitro, (2) d'explorer les gènes de la dénitrification nécessaires à la colonisation des racines du maïs et (3) de découvrir de nouvelles respirations anaérobies pendant la colonisation racinaire du maïs (Figure 16). Réaliser des mutants et les tester dans des conditions particulières est le moyen classique de déterminer l'implication d'un gène dans une voie spécifique. Cependant, cette méthode implique une vision à priori et des connaissances solides sur les gènes cibles et ne peut pas être appliquée à toutes les situations. Nous avons alors dû développer une méthode plus adaptée pour identifier les gènes essentiels impliqués dans la croissance dans des conditions anaérobies spécifiques. - Gènes de dénitrification chez A. fabrum C58 in vitro. Pour compléter la voie de dénitrification chez A. fabrum C58 et identifier tous les gènes et régulateurs impliqués dans la dénitrification, nous avons adopté deux stratégies : Premièrement, une vision à priori pour (1) identifier la nitrate réductase impliquée dans la première étape de la dénitrification et (2) valider le rôle d'un ARN non codant dans le contrôle de la dénitrification. Pour ce faire, nous avons construit un mutant napA de A. fabrum C58 et un mutant de l'ARN non codant NopR et nous avons évalué leur croissance et leur capacité à produire du N2O dans des conditions anoxiques. Deuxièmement, pour identifier tous les gènes impliqués dans la dénitrification, nous avons construit une banque de transposons mutants de C58 et testé sa croissance dans des conditions de dénitrification in vitro en présence de nitrate ou de nitrite. - Rôle des gènes de la denitrification de A. fabrum C58 dans la colonisation racinaire du maïs. Il est bien connu que le séquençage de transposons (Tn-Seq) est une méthode très puissante pour déterminer les gènes nécessaires à la croissance bactérienne en présence de leur hôte. Pour déterminer les gènes de dénitrification impliqués dans la colonisation des racines en anoxie, nous avons utilisé la banque construite chez C58 et l’avons inoculée sur les plants de maï cultivées sur un sol fertile et cultivées dans des conditions inondées mimant des conditions anaérobies. Le séquençage des cellules d ‘A. fabrum C58 récupérées mettra en évidence les gènes impliqués dans la colonisation anaérobie de cette niche spécifique. - Découverte de nouvelles voies de respiration anaérobie chez A. fabrum C58. Pour découvrir de nouvelles voies de respiration anaérobie, nous avons mis en place des tests de croissance de C58 dans des conditions anoxiques en présence de sources de C et de N en tant qu'accepteurs terminaux d'électrons. De façon interéssante, en cultivant des souches WT et mutée dans le gène napA au contact de la racine de maïs dans des conditions anoxiques (chapitre 1), nous avons montré une croissance des deux souches. Ce résultats suggère que les exsudats de racine servent d'accepteurs d'électrons terminaux pour la croissance anaérobie de C58. Pour déterminer quels composés exsudés du maïs peuvent servir de TEA, les principaux métabolites ont été identifiés par HPLC et certains ont été testés en tant que TEA dans des conditions anoxiquesAnaerobic respiration may be an essential trait in lifestyle, environment colonization and survival. Until now, the only confirmed anaerobic respiration in Agrobacterium spp. is denitrification. Interestingly, this pathway is unequally widespread among Agrobacteria. These observations led me to my hypothesis which is anaerobic respiration and notably denitrification could explain the coexistence of Agrobacteria and their distribution in specific niches in the rhizosphere. My thesis was undertaken to explore the anaerobic respiration strategies of Agrobacterium spp. and to relate them to niche adaptation. The objectives of my thesis were to (1) characterize all the genes involved in denitrification in A. fabrum C58 in vitro, (2) explore the genes of denitrification that are needed during maize root colonization and (3) discover new anaerobic respirations that occur during maize root colonization (Figure 16). Mutational analysis is the classic way to determine the involvement of a gene in specific pathway. However, this method implies an a priori view and solid knowledge on target genes and cannot be applied for every situation. We have to develop a more adapted method to identify essential genes involved in growth in specific anaerobic conditions. - Denitrification genes in A. fabrum C58 in vitro. To complete denitrification pathway in A. fabrum C58 and identify all the genes and regulators involved in the denitrification function, we adopted two strategies: Firstly, an a priori view to (1) identify the nitrate reductase involved in the first step of denitrification and (2) validate the role of a non-coding RNA in denitrification control. To do so, we constructed a mutant of napA of A. fabrum C58 and a mutant of the non-coding RNA NopR and we evaluated their growth and capacity to produce N2O under anoxic conditions. Secondly, to identify all the genes involved in denitrification, we constructed a mutant transposon library of C58 and tested its growth under denitrification conditions in vitro in the presence of either nitrate or nitrite. - Role of A. fabrum C58 denitrifying genes in the root colonization of maize. It is well known that Transposon-sequencing (Tn-Seq) is a very powerful method to determine genes required for bacterial growth in the presence of their host. To determine denitrifying genes involved in root colonization under anaerobic conditions, we used the library constructed in C58 and performed in planta assays. The mutant library was inoculated on maize plants grown on fertile-ground and cultured under flooded conditions miming anaerobic conditions. Sequencing the recovered A. fabrum C58 cells will evidence the genes involved in this anaerobically specific niche colonization. - Discovery of new anaerobic respiration pathways in A. fabrum C58. To discover new anaerobic respiration pathways, we set-up growth assays of C58 under anoxic conditions in the presence C and N sources as terminal electrons acceptors. Interestingly, by culturing WT and NapA-deficient strains in contact with maize root under anoxic conditions (Chapter 1), we showed growth of both strains, suggesting that root exudates serve as terminal electrons acceptors for anaerobic growth of C58. To determine which maize exuded compounds can serve as TEAs, primary metabolites were identified by HPLC and some were tested as TEAs under the set-up conditions
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Egger, Truong Tuan Van. "Optimisation de la formation directe de tiges sur section d'entre-noeuds de pomme de terre inoculée par Agrobacterium rhizogenes (souches à agropine et à cucumopine)." Paris 11, 1994. http://www.theses.fr/1994PA112199.
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Le but de cette étude était d'améliorer le taux de néoformation de tiges sur la section inoculée d'entre-nœuds pensant de cette façon accroître la fréquence de transformation de la pomme de terre (variété Fanette) par souches à agropine et à cucumopine d'Agrobacterium rhizogenes. En premier lieu sont présentés les effets de différents facteurs sur le taux de néoformation de tiges. L'action stimulante de certains traitements du milieu d'induction et notamment de la zéatine à la concentration de 5 mg. L-1 a été constatée. L'influence de l'état physiologique de la plante mère (dépendant du milieu de culture) et du niveau de prélèvement des entre-nœuds a été également étudiée. Un net effet caulogène des souches à cucumopine (2659 et 2659 GUS) a été observé. La deuxième partie a concerné la caractérisation de l'état transformé des tiges néoformées par la mise en évidence des opines et de l'activité du gène codant pour la β-glucuronidase. La transformation a été obtenue dans notre travail uniquement avec l'emploi de la souche 2659 GUS. Il est clairement apparu que la néoformation de tiges et leur transformation sont deux phénomènes totalement indépendants.
15
Brightwell, Gale. "Molecular genetics of exopolysaccharide synthesis in Agrobacterium radiobacter." Thesis, University of East Anglia, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.482034.
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Deakin, William James. "Molecular characterisation of flagellar genes from agrobacterium tumefaciens." Thesis, Durham University, 1994. http://etheses.dur.ac.uk/5858/.
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Three behavioural mutants of A. tumefaciens C58C1 (mot-l, mot-12 and fla-15) generated by transposon (Tn5) mutagenesis were studied. Analysis was initially at the molecular level, as a cosmid, pDUB1900, from a representative genomic library of C58C1 had been isolated that complemented the mutants. A region of 8624 nucleotides to which the Tn5 insertion sites of the three mutants had been mapped was sequenced completely in both directions. The comparison of this sequence with sequence databases and other computer analyses revealed six flagellar gene homologues (flgI,flgH,fliP,flaA,flaB,flaC), three open reading frames (ORFA, B and C) with no significant sequence identity to any open reading frames in the databases and the partial sequence of the flagellar gene homologue flgG. Computer analysis also showed that theflgH,flgI andfliP homologues, and ORFs A, B and C, could form the downstream region of a larger operon involved in chemotactic and motility functions. However putative transcription signals were also found within the operon. A new mutant (MANl) was created in the last gene (fliP) of the putative operon to investigate the function of possible transcription signals in the open reading frame immediately upstream of it (ORFC). The mot-12 mutant phenotype of fully synthesised but paralysed flagella is brought about by the insertion of Tn5 in ORFC. ORFC contains a possible promoter for fliP. The Tn5 insertion in ORFC should have polar effects upon the expression of fliP, unless the putative promoter can cause expression of fliP. The MANl mutant had a flagella-less phenotype. FliP in other bacteria is required early in the synthesis of flagella and the null phenotype is/7a-. Thus for flagella to be present in mot-12 suggests fliP must have a promoter. The ORFC sequence is highly conserved in R. meliloti and the overall regulation of these flagellar gene homologues may be as an operon with other regulatory signals. Evidence from other operons (including motility operons) with multiple transcription signals is discussed. The flaABC homologues were multiple copies of the gene encoding the flagellin protein of the flagellum. The mot-l phenotype of severely truncated filaments was caused by a Tn5 insertion in flaA. Analysis of the sequence showed flaABC to each have transcription signals that could lead to separate transcription. Transcription analysis by Northern blotting showed flaA to be transcribed monocistronically. Flagella were isolated from A. twnefaciens and the flagellins separated by SDS-PAGE. The migrated distances (relative to those of markers) was not as predicted from the nucleotide sequence. This anomaly could be caused by unequivalent binding of SDS or post-translational modification of FlaA. The A. tumefaciens flagellar genes were most similar to those of R. meliloti. However A. tumefaciens flagella do not exhibit the characteristic cross-hatching of the complex flagella of R. meliloti. This study also showed A. tumefaciens flagella not to be dependent on divalent cations for subunit associations unlike R. meliloti. These properties of A. tumefaciens flagella were similar to those of R. leguminosarum.The open reading frames found were isolated, radiolabelled and used as probesagainst Southern blots containing chromosomal DNA from a variety of soil bacteria, and cosmids known to contain motility genes in R. meliloti. Hybridisation revealed homologous DNA sequences in a number of these bacteria. All the A. tumefaciens open reading frames hybridised to homologous DNA in R. meliloti and are found in the same order in both species. This suggests that there are similarities at the molecular level in motility and chemotaxis functions between R. meliloti and A. tumefaciens as well as in the patterns of chemotaxis and motility observed previously.
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Brown, Adrian. "Molecular characterisation of behavioural functions in Agrobacterium tumefaciens." Thesis, Durham University, 1992. http://etheses.dur.ac.uk/6019/.
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Tn5 insertion behavioural mutants of A. tumefaciens C58C(^1) were available. Cloning of the kanamycin resistance gene allowed isolation of Tn5 flanking sequences from a number of the mutants. Flanking sequences from five mutants were used to isolate cosmids, overlapping the Tn5 insertion sites of the mutantsfrom a C58C(^1) library. Two cosmids, pDUB1900 and 1905 have been characterised. pDUB1900 contains the insertion sites of eight motility mutants, with another immediately adjacent. The pDUB1905 insert overlaps sequences interrupted in another three mutants. Behavioural genes in Agrobacterium are clustered together on the chromosome, as in other motile bacteria. Restriction maps of the isolated cosmids show that none of motility mutants analysed was the result of mactivation of pscA or chvB which would lead to an altered behavioural phenotype. Flanking sequences from three of the mutants hybridised to R. meliloti chromosomal DNA, but not to DNA from other motile genera. DNA adjacent to the insertion site of fla-11 hybridised to the insert of pRZ-2, a cosmid containing behavioural genes from R. meliloti. Experiments were undertaken to investigate the occurrence of proteins homologous to the MCP's of enteric bacteria in C58C(^1) DNA hybridisation to oligonucleotide probes showed DNA that could potentially code for MCP-like proteins exists m both Agrobacterium and Rhizobium spp. In addition, an antibody raised against the E. coli MCP Tar cross-reacted with a protein of approximately 60kDa. in C58C(^1). In vivo protein methylation experiments using C58C(^1) resulted in the labelling of a 45kDa protein, whose methylation pattern did not change upon addition of chemostimuli. Possible reasons for the difference in size between the labelled protein and that revealed by the antibodv are discussed.
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Loake, Gary John. "The genetic dissection of chemotaxis in Agrobacterium tumefaciens." Thesis, Durham University, 1989. http://etheses.dur.ac.uk/6735/.
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A range of sugars, many of them characteristic of plant extracts were tested as potential chemoattractants for Agrobacierium .The results divided the sugars into 4 groups of attrcictants and indicated the presence of a highly sensitive chemotaxis system in A. tumefaciens. Motility in Agrobactewrium consisted of long straight runs, with relatively few tumbles or stops. The propulsive mechanism seemed to resemble that of Rhizobium. Methionine-starved methionine auxotrophs of A. tumefaciens , although fully motile, were non-chemotactic to sucrose or acetosyringone, unless supplemented with exogenous methionine. Neither ethionine nor a-methyl-DL-methionine could correct the non-chemotactic phenotype, while seleno- DL-methionine partially restored taxis. Pulse-labelling of A.tumefaciens with L- [methyl-(^3) H] - methionine in the presence of chloramphenicol, and an attractant resulted in the appearance of 2 radio-labelled proteins of approximately 55KDa. Thus, in A. tumefaciens, chemotactic responses may be associated with the transfer of methyl groups from methionine via S-eidenosyl methionine to MCPs. Using transposon mutagenesis a battery of A.tumefaciens chemotaxis mutants were generated and characterized. A number of mutated behavioural genes were isolated using the kanamycin resistant determinant of Tn5 as a positive selectable marker. Tn5 flanking sequences were used as probes to recover wild-type behavioural genes from a gene library constructed in the cosmid pLAFR3. Behavioural genes were found to be clustered on the A. tumefaciens chromosome and to possess similarity with behavioural genes from R.meliloti.
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McAdam-O'Connell, Darren John Patrick. "Agrobacterium-mediated genetic modification of Bramley's seedling apple." Thesis, Queen's University Belfast, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396078.
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Kanvinde, Laxmi Anant. "Studies on the diazotrophic nature of Agrobacterium tumefaciens." Thesis, University of Leeds, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303441.
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Donaldson, Pauline A. (Pauline Alison) Carleton University Dissertation Biology. "Nodulation and tumorgenesis by Agrobacterium carrying Rhizobium plasmids." Ottawa, 1985.
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Rodrigues-Otubo, Benedita Maria. "Regeneração organogênica de soja sob transformação via agrobacterium." Universidade Estadual de Londrina. Centro de Ciências Agrárias. Programa de Pós-Graduação em Agronomia, 2011. http://www.bibliotecadigital.uel.br/document/?code=vtls000164924.
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A soja [Glycine max (L.) Merrill] é uma das culturas mundialmente mais importantes pelas altas concentrações de óleo e proteína presentes nos grãos. Com o avanço das novas tecnologias, o melhoramento dessa leguminosa, por meio da transformação, viabilizou a introdução de características, até então, impossíveis por outros métodos. A transformação genética tem sido uma ferramenta utilizada para transpor barreiras entre as espécies possibilitado a obtenção de plantas transgênicas, sendo o seu sucesso altamente dependente de um bom protocolo de regeneração in vitro. No entanto, a inexistência de protocolos altamente reproduzíveis e com altas taxas de transformação tem dificultado o emprego desta tecnologia. Este trabalho teve por objetivo selecionar cultivares de soja com maior potencial organogênico, e avaliar o efeito do tempo no meio de indução de brotações, tempo no meio de recuperação, tempo de sonicação e das concentrações de acetosiringona em plantas regeneradas de eixos embrionários de sementes maduras da cultivar Conquista, sob transformação via Agrobacterium. Foram apresentados dois artigos. No primeiro artigo foram delineados dois experimentos, onde no primeiro foi avaliado a regeneração organogênica das cultivares Conquista, Valiosa RR, Perdiz, V.Max, Pintado, Tucunaré, Potência, BRS-232 e Tabarana, no delineamento em blocos ao acaso, com quatro repetições e parcelas com dez eixos embrionários. Aos 70 dias do início da cultura foram avaliados a frequência de regeneração, o número médio de brotos por eixo e a frequência de multibrotação. No segundo experimento, foram avaliadas a regeneração organogênica e a eficiência de expressão do gene GUS em eixos embrionários da cultivar Conquista, cultivados no meio de indução de brotações durante um e dois dias e a manutenção por 10, 15 e 20 dias na fase de recuperação. Utilizou-se o delineamento de blocos ao acaso, no esquema fatorial, de 2 x 3, quatro repetições e parcelas com cinco eixos embrionários. Aos 70 dias do início da cultura, foram avaliadas a regeneração de plântulas e a eficiência da expressão estável do gene GUS. No segundo artigo (primeiro experimento), foram avaliados o efeito da manutenção de eixos embrionários da cultivar Conquista no meio de indução de brotações por um, dois e três dias com sonicação de 0,0; 2,5; 7,5; 15,0 e 30,0 segundos na fase de agroinfecção, na regeneração organogênica e na eficiência de expressão do gene GUS, sob transformação via Agrobacterium na OD600 0,5. Utilizou-se o delineamento em blocos casualizados, no esquema fatorial 3 x 5, quatro repetições e parcelas de 12 eixos. No segundo experimento, foram avaliados os efeitos das concentrações de 100 e 200 µM de acetosiringona na agroinfecção de eixos embrionários da cultivar Conquista com sonicação de 0,0; 2,5; 7,5; 15,0 e 30,0 segundos, na regeneração organogênica e na eficiência de expressão do gene GUS, sob transformação via Agrobacterium na OD600 0,2. Utilizou-se o delineamento de blocos casualizados, no esquema fatorial 2 x 5, com 100 e 200 µM de acetosiringona e tempos de sonicação de 0,0; 2,5; 7,5; 15,0 e 30,0 segundos, quatro repetições e parcelas com 12 eixos; sendo as avaliações dos experimentos realizadas aos 70 dias do início dos cultivos. No primeiro artigo (primeiro experimento), as cultivares apresentam potencial para regeneração organogênica. O maior potencial para número de brotos é observado nas cultivares Valiosa RR e Conquista e, para frequência de multibrotação na cultivar Valiosa RR. No segundo experimento, o cultivo de eixos embrionários da cultivar Conquista por dois dias no meio de indução e 20 dias no meio de recuperação estimulam maior regeneração de plântulas e maior expressão do gene GUS. No segundo artigo (primeiro experimento). A maior elongação das plântulas e maior expressão do gene GUS ocorrem em eixos embrionários da cultivar Conquista mantidos por dois e três dias no meio de indução e com 2,5 e 15,0 segundos de sonicação na suspensão de Agrobacterium OD600 0,5. No segundo experimento, a maior elongação das plântulas e maior expressão do gene GUS ocorrem em 100 µM de acetosiringona, com 7,5 e 30,0 segundos de sonicação de eixos embrionários da cultivar Conquista na suspensão de Agrobacterium OD600 0,2.Soybean [Glycine max (L.) Merrill] is one of the world's most important crops by high concentrations of oil and protein present in grains. With the advancement of new technologies, improvement of legume, by means of transformation, made possible the introduction of features hithert impossible by other methods. The genetic transformation has been a tool used to overcome barriers between species made it possible to obtain transgenic plants, and its success is highly dependent on a good protocol for regeneration in vitro. However, the lack of protocols and highly reproducible with high rates of transformation has hindered the use of this technology. This study aimed to select soybean cultivars with higher organogenic potential, and evaluate the effect of time in the shoot induction medium and recovery time, sonication time and concentration acetosiringona in plants regenerated embryonic axes from mature seeds Conquista the cultivar under transformation via Agrobacterium. In the first article two experiments were designed, where the first was evaluated organogenic regeneration of Valiosa RR, Perdiz, V.Max, Pintado, Tucunaré, Potência, BRS-232 Tabarana cultivars, in a randomized block design with four replications with ten embryonic axes. At 70 days of onset of culture were assessed the frequency of regeneration, the average number of shoots per axis and the frequency of multiple shoots. In the second experiment, we evaluated the organogenic regeneration and the efficiency of GUS gene expression in embryonic axes Conquista cultivar, grown in the shoot induction during one and two days and maintained for 10, 15 and 20 days in the recovery phase. We used the design of randomized blocks in factorial scheme 2 x 3, four replications and plots with five embryonic axes. At 70 days of the onset of culture, we assessed the regeneration of seedlings and the efficiency of stable GUS gene expression. In the second article (first experiment), we evaluated the effect of the maintenance of embryonic axis Conquista cultivar in the shoots induction medium per one, two and three days with sonication of 0.0, 2.5, 7.5, 15.0 and 30.0 seconds in agroinfection phase in organogenic regeneration and efficiency of GUS gene expression under transformation via Agrobacterium in 0.5 OD600. We used a randomized block design in 3 x 5 factorial design, four replications and plots of 12 axes. In the second experiment, we assessed the effects of concentrations of 100 and 200 µM acetosiringone in agroinfection of embryonic axis Conquista cultivar with sonication of 0.0, 2.5, 7.5, 15.0 and 30.0 seconds, organogenic regeneration and efficiency GUS gene expression under transformation via Agrobacterium 0.2 OD600. We used a randomized block design, in factorial scheme 2 x 5 with 100 and 200 µM acetosiringone and sonication times of 0.0, 2.5, 7.5, 15.0 and 30.0 seconds, four replications and plots with 12 axes, and the evaluations of the experiments performed at 70 days of start of cultivation. In the first article (first experiment), the cultivars have potential for organogenic regeneration. The greatest potential for number of shoots is observed on the Valiosa RR and Conquista cultivars, and to frequency multiple shoots in Valiosa RR. In the second experiment, the cultivation of embrionic axes of Conquista cultivar for two days in induction medium and 20 days in recovery medium stimulate regeneration greater of seedlings and expression greater of the GUS gene in Conquista cultivar. In the second article (first experiment), the greatest elongation of seedlings and increased expression of the GUS gene occur in embryonic axes of Conquista cultivar maintained by two and three days in induction medium and with 2.5 and 15.0 seconds of sonication in Agrobacterium suspension of 0.5 OD600. In the second experiment, the greatest elongation of seedlings and increased expression of the GUS gene occur in 100 µM acetosiringone with 7.5 and 30.0 seconds of sonication in embrionic axes Conquista cultivar in Agrobacterium suspension of 0.2 OD600.
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Oliveira, Marcelo Tempesta. "Transformação genética de Candida spp via Agrobacterium tumefaciens." Universidade Estadual de Londrina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Microbiologia, 2007. http://www.bibliotecadigital.uel.br/document/?code=vtls000126403.
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Este trabalho descreve o estabelecimento de um sistema de transformação eficiente para as espécies de leveduras oportunistas Candida albicans, C. tropicalis e C. glabrata, importantes patógenos humanos, pelo emprego da metodologia de transformação mediada por Agrobacterium tumefaciens. As células fúngicas foram transformadas em diferentes condições de co-cultivo utilizando o vetor binário pBTS-4, que contém o gene de resistência ao antibiótico Higromicina B (hph) como marcador de seleção. Nossos resultados indicam uma freqüência de transformação para C. albicans, C. tropicalis e C. glabrata de até 1471, 1612 e 83 transformantes obtidos por co-cultivo, respectivamente. Após quatro repiques em meio não seletivo, todos os transformantes analisados cresceram quando expostos novamente ao meio de seleção, sendo desta forma 100% estáveis mitóticamente. A PCR e o dot-blot confirmaram molecularmente a transformação das espécies de Candida, gerando o produto de amplificação esperado de 600pb, e hibridado positivamente com os transformantes quando o mesmo foi utilizado como sonda. A habilidade da linhagem AGL-1 em aderir às células fúngicas nas condições de co-cultivo foi observada através do emprego de microscopia eletrônica de varredura e transmissão. A reprodutibilidade do sistema de transformação descrito neste trabalho pode fornecer um método para a manipulação genética destes patógenos que irá facilitar a análise molecular detalhada destas espécies fúngicas.This paper describes the establishment of an efficient transformation system for the opportunistic yeasts species C. albicans, C. tropicalis and C. glabrata, by the employment the Agrobacterium-mediated transformation method (AMT). Yeast cells were transformed to hygromycin B resistance using the binary vector pBTS4 carrying a hygromycin B resistance gene (hph) as a selection marker under different co-cultivation conditions. Overall, our data indicate a transformation frequency of up to 1472, 1612 and 83 transformants/co-cultivation for C. albicans, C. tropicalis and C. glabrata, respectively. Following 4 serial passages of transformants on non-selective medium, 100% of the transformants were found to be mitotically stable for the selection mark. PCR and dot-blot targeted at a 600 bp fragment from hph gene confirmed the transformation of Candida species. The ability of Agrobacterium strain AGL-1 to attach to Candida cells under co-cultivation was analysed under scanning and transmission electron microscopy. The reproducible transformation system described in this work may provide a method for the genetic manipulation of these pathogens, which will facilitate detailed molecular analysis of these fungal species.
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Greenberg, Norman Michael. "Cellulase gene transcription in Cellulomonas fimi and an Agrobacterium." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28836.
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Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping.
Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Korban, Martine. "Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.)." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41644.
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Regeneration and shoot multiplication of common bean (Phaseolus vulgaris L. 'ICA Pijao') from half-cotyledonary nodes was achieved on modified Murashige and Skoog (1962) basal medium amended with 5 $ mu$M 6-benzylaminopurine. Histological studies confirmed the adventitious origin of the regenerated buds. Shoots were rooted ex vitro and developed into morphologically normal plants compared with seed-grown controls. The relative susceptibility of bean tissues to infection by a collection of wild-type Agrobacterium strains was tested. Positive transformation events were evaluated based on morphological and biochemical changes observed following Agrobacterium infection. The A. tumefaciens strain C58 was particularly virulent on greenhouse-grown plants, in vitro-derived stem sections, half-cotyledonary nodes and seedlings. A sensitive and rapid method was developed to detect opines using thin layer chromatography. Transient $ beta$-glucuronidase (GUS) gene expression was detected in 'ICA Pijao' bean buds regenerated from half-cotyledonary nodes following Agrobacterium-mediated gene transfer with the binary vector pGV1040 or p35SGUSINT. Four out of eight putative transformants contained the chimeric GUSINT gene following polymerase chain reaction (PCR) analysis. This was confirmed by Southern analysis of blotted PCR gels. However, there was no stable integration of the GUSINT gene as none of the R1 progeny showed an amplified GUSINT fragment with PCR.
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Knight, Claire Jane. "Investigation into agrobacterium-mediated transformation of fungi in nauture." Thesis, University of Bristol, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500434.
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Parsons, Stephen H. "Comparing orchid transformation using agrobacterium tumefaciens and particle bombardment." Virtual Press, 1995. http://liblink.bsu.edu/uhtbin/catkey/941350.
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The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities.Department of Biology
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Marashi, Sayyed Hassan. "Identification and characterisation of behavioural genes of Agrobacterium tumefaciens." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4222/.
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Three behavioural mutants (fla-8, mot-6 and cheL) generated by transposon Tn5 mutagenesis and localised on cosmid pDUB1905 were studied. The cosmid pDUB1905, from a representative genomic library of the Agrobacterium tumefaciens C58C1 chromosome has previously been partly mapped and found to contain genes concerned with flagella. In this study a region of 5860 nucleotides from a 12 kb BamHl fragment of cosmid pDUB1905 was sequenced completely in both directions. Homology searches of this sequence with sequence databases and other computer analysis revealed two flagellar-related genes (flhA and fliR), a chemotactic gene (cheL) and four open reading frames (orfX, orfW, orfY and orfZ) with no significant sequence identity to any open reading frame in databases. A putative promoter-like sequence was also found upstream of orfZ. The FlhA and FliR are the inner members of type III flagellum-specific export apparatus which are responsible for delivering the flagellar subunits lacking a signal peptide leader to the surface of the cell. These have counterparts in the type III secretion proteins system responsible for transporting pathogen proteins to host cells. The function of CheL has not yet been identified. Three ORFs have chaperone characteristics. A mutant was created by insertion of a neomycin resistance cassette in the fliR homologue to determine the effects of the gene on motility. Phenotype analysis of the mutant showed no flagella and motility with small swarming pattern comparing to wild type, indicating that fliR is indeed a flagellar gene. In this study two more members of flagellum-specific export, a chemotactic gene, three open reading frames which could have specific chaperon activity, and an unknown open reading frame were identified in A. tumefaciens C58C'.
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Hodson, de Jaramillo Elizabeth. "Agrobacterium-mediated transformation of Passiflora edulis for Potyvirus resistance." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301691.
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Cancino, Escalante Giovanni Orlando. "Tissue culture and Agrobacterium-mediated transformation studies in Passiflora." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342022.
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Al-Forkan, Mohammad. "Agrobacterium-mediated transformation of Indica rice (Oryza sativa L.)." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342480.
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Joubert, Dirk Albert 1973. "Development of an Agrobacterium vitis transformation system for grapevine." Thesis, Stellenbosch : Stellenbosch University, 2000. http://hdl.handle.net/10019.1/51687.
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Thesis (MSc)--University of Stellenbosch, 2000.ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis.
AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme, soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat deur gelokaliseerde selafsterwing en nekrose gekenmerk word. Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf deur die betreklike homogenisiteit met A. tumefaciens. In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word), sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon word. Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras. In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem. Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51 - en 31 - areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul potensiële rol in die evolusionêre ontwikkeling van die ras. Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke kenmerke van een van sy patogene, A. vitis, uit te buit.
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Renou, Jean-Pierre. "Transformation génétique du chrysanthème (dendranthema grandiflora tzvelev) par agrobacterium." Angers, 1992. http://www.theses.fr/1992ANGE0009.
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La possibilité de transformation génétique du chrysanthème par agrobacterium a été étudiée. Des racines transformées ont pu être obtenues après inoculation avec a. Rhizogenes. Cependant la régénération de plantes transformées ne fut possible que dans de rares cas, après des temps de culture in vitro très longs. Des plantes transformées furent ensuite obtenues grâce à la souche d'agrobacterium. Eha 101 contenant un plasmide vecteur binaire qui comporte les gènes : npt ii, hpt et gus. Ces plantes ont été obtenues par sélection sur la résistance à la kanamycine. Différents protocoles ont conduit à l'obtention de plantes transformées, mais les conditions optimales furent l'inoculation de nuds immédiatement transférés sur un milieu sélectif contenant du cefotaxime. La transformation de plantes gus positives a été confirmée par hybridation moléculaire de l'ADN et amplification génique. Des plasmides vecteurs binaires contenant des gènes d'intérêt agronomiques ont été construits : les gènes rol a, b et c du ri tl-dna afin d'étudier leur effet sur le port de la plante, et le gène de la dihydroflavonol réductase de l'antirrhinum majus afin de permettre une modification de la couleur des fleurs de chrysanthème.
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Tjokrokusumo, Donowati. "Plant transformation using pollen vacuum infiltrated with Agrobacterium tumefaciens." Thesis, Tjokrokusumo, Donowati (1998) Plant transformation using pollen vacuum infiltrated with Agrobacterium tumefaciens. Masters by Research thesis, Murdoch University, 1998. https://researchrepository.murdoch.edu.au/id/eprint/52417/.
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Bechtold et al. (1993) obtained transformed seedlings from Arabidopsis thaliana inflorescences vacuum infiltrated with Agrobacterium tumefaciens. In this thesis vacuum infiltration of pollen with Agrobacterium tumefaciens and other pollen treatments were studied to evaluate whether these methods could achieve transformation of the dicotyledon Petunia hybrida, var. Peach Ice and the monocotyledon Zea mays, var. Super Sweet.
Pollen germination in vitro from both petunia and com was optimised. The highest germination of petunia pollen (62 %) was achieved in medium containing 20 % sucrose, 100 mg!'1 boric acid and 300 mgl’1 CaCl2. 2H20. In com, the maximum germination observed was 73 % in medium containing 10 % sucrose, 100 rngf1 boric acid and 300 mgl'1 CaCl2. 2H20.
The Agrobacterium tumefaciens used in this research carried the plasmid pCGP1258 containing the GUS gene and the bar gene both controlled by the 35S promoter.
The effect of vacuum on petunia pollen suspended in germination medium showed that germination was not significantly depressed by exposure of 20 minutes vacuum (80 kPa) and that this treatment induced an increase in pollen tube length. In com, vacuum treatment of pollen reduced germination when applied for more than 1 minute. Pollen treated for 1 minute developed longer pollen tubes than control pollen. Agrobacterium tumefaciens cells placed under vacuum for up to 30 minutes were unaffected.
Pollen of petunia which was vacuum-infiltrated with Agrobacterium for 20 minutes was used to pollinate the stigmas. Similarly com pollen vacuum treated with Agrobacterium for 1 minute was used to pollinate the silks. At the same time pollination was also carried out using untreated pollen and applying a drop of Agrobacteria solution to the stigma at the time of pollination. The resultant T, seedlings were screened in vitro on medium containing 3 mg Y of the herbicide Basta for petunia, while seedlings of com were screened by applying 20 mg 1-1 of Basta R to the leaves of 15 day old plants.
In petunia 9 % of 1806 seedlings tested were resistant to BastaR, while in com 32 % of 372 plants tested were resistant to BastaR. BastaR resistance was seen in seedlings resulting from both the vacuum and the drop treatments.
Putatively transformed T] plants of petunia were grown further in the glasshouse and 10 % of plants showed GUS expression in tissues of the leaf, pistil and young anthers. Amongst putatively transformed T, com plants 20 % showed GUS expression in the silks.
PCR analyses showed that for the T1 putatively transformed petunia plants, 66 % were positive for the presence of the GUS gene although only 10 % expressed GUS. For com plants 44 % of putatively transformed T1 plants showed the presence of the GUS gene through PCR, compared with 20 % from GUS expression. No carryover bacterial contamination of the transformed plants could be detected.
Southern hybridisation analyses showed that iscoRI-digested DNA of putatively transformed petunia plants tested with the bar gene probe hybridised twice to genomic DNA fragments of approximately 6.5 kb and 4.3 kb.
Some T2 plants were produced by crossing two putatively transformed T1 plants. Amongst the T2 progeny; 5 % were resistant to Basta , 5 % of these expressed GUS, and PCR analysis showed 61 % of those resistant to Basta were positive for the presence of the GUS gene. Two different T2 plants and the putatively transformed ^ plants showed almost identical banding patterns when a bar gene probe was used in a Southern hybridisation, indicating inheritance of the transgene.
In summary it was shown that vacuum infiltration of pollen with Agrobacterium is unnecessary to achieve transformation of petunia and com. Transformation can be achieved if there are bacteria present while the germinating pollen is growing on the stigma and style. It seems likely that this was also the mechanism through which vacuum infiltrated inflorescences of Arabidopsis produced the transformed progeny reported by Bechtold et al. (1993).
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Kováčová, Viera. "IZOLACE TRANSGENNÍCH ROSTLIN NICOTIANA TABACUM A SILENE VULGARIS." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2010. http://www.nusl.cz/ntk/nusl-216657.
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This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.
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Esteban, Fernández Berta. "Biochemische Untersuchungen mit den prokaryotischen Phytochromen Cph1 aus Synechocystis PCC6803 und Agp1 aus Agrobacterium tumefaciens." [S.l.] : [s.n.], 2005. http://www.diss.fu-berlin.de/2005/13/index.html.
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Marzouk, Amani M. "Transformed root cultures for production of secondary metabolites." Thesis, University of Strathclyde, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248740.
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Benzle, Kyle Arthur. "Isolation of Novel Agrobacterium and Transient Expression Assays in Soybean (Glycine max) and Sunflower (Helianthus annuus)." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405555898.
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Almerei, Ayman. "Agrobacterium-mediated transformation of Syrian maize with anti-stress genes." Thesis, University of Plymouth, 2016. http://hdl.handle.net/10026.1/5336.
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Agrobacterium is widely considered, when suitably modified, to be the most effective vector for gene transfer into plant cells. For a long time, many cereals crops (monocotyledonous plants) were recalcitrant species to genetic modification, mainly as a result of their recalcitrance to in-vitro regeneration and their resistance to Agrobacterium infection. However, recently Agrobacterium-mediated transformation has been used to transform monocot crops such as maize (Zea mays) but with severe restrictions on genotype suitability. This study was carried out to evaluate the transformation amenability of 2 Syrian maize varieties and 2 hybrids in comparison with the hybrid line Hi II by the Agrobacterium tumefaciens-mediated transformation technique using a callus induction based system from immature zygotic embryos IZEs. A. tumefaciens strains EHA101, harbouring the standard binary vector pTF102, and the EHA105 containing the pBINPLUS/ARS:PpCBF1 vector were used. The effects of genotypes and the size of IZEs explants on callus induction and development were investigated. Results showed that callus induction and subsequent callus growth were significantly affected by the initial explant size. Calli induction from IZEs explants sized 1.5-2.00mm was 76%. Callus weight however decreased to 8.2g, compared with 11.7g of callus derived from IZEs >2.00mm. Callus induction ranged between 73.6-78.9% for varieties and hybrids respectively. Calli derived from varieties weighed significantly more than those initiated from the hybrids. Results demonstrated that Syrian maize genotypes were efficiently transformed via the A. tumefaciens strains but there was variation in transformation frequency. A transformation frequency of 3.7-4.2% was achieved for hybrids and varieties respectively confirming that the transformation frequency was genotype-dependent. The transformation frequency averaged between 3.2-5.6% for the EHA105 and EHA101 respectively. Fertile transgenic plants were regenerated from mature somatic embryos with an average regeneration frequency of 59.2 and 17% respectively for varieties and hybrids. Transgenic seeds of R0 and R1 progenies were produced from 74% of the outcrosses attempted and more than 98% of transgenic plants were normal in morphology. Fertile transgenic maize plants carrying the transferred gene CBF were produced using the Agrobacterium EHA105/PpCBF1 and these plants were shown to be more salt tolerant. Transient expression of the GUS gene was confirmed in transgenic calli, shoots, leaves, roots and floral parts of transgenic R0 and R1 progenies using histochemical GUS assays. The presence of the introduced bar and CBF genes in the genomic DNA of the transformants was confirmed by the PCR amplification. Further, the stable expression of the CBF and bar transgenes in the maize genome of transgenic R1 progeny was confirmed by qRT-PCR. The transformation protocol developed using an A. tumefaciens standard binary vector system was an effective and reproducible method to transform Syrian maize with an anti-stress gene in which fertile salt-resistant transgenic plants were routinely produced. This approach has great potential for development of Syrian maize breeding programmes for abiotic stress resistance for application in many areas in Syrian maize production.
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Edwards, Glyn Alyn. "Plant transformation using an Agrobacterium tumefaciens Ti-plasmid vector system." Thesis, Durham University, 1988. http://etheses.dur.ac.uk/6600/.
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A plasmid, pDUB116 Was constructed, containing the Nos-NPT dominant selectable marker for expression in plants which was capable of being mobilised to Agrobacterium tumefaciens and forming cointegrates with pGV3850 and pGV3851. The frequencies of cointegrate formation of these plasmids were determined and the cointegrate structure established by Southern blotting. Inoculation of Kalanchoe diaigremontiana leaves and Nicotiana tabacum stems in vivo showed that pGV3851 is only weakly oncogenic. A fully oncogenic Ti plasmid (pTiGEl) was constructed which was capable of forming cointegrates with pDUB116 and suitable for use in in vivo plant transformation systems. A. tumefaciens GV3101 [pGV3850] was found to be highly resistant to cefotaxime and carbenicillin in plant tissue culture media, preventing its use in in vitro plant transformation proceedures. A. tumefaciens strains were therefore screened for sensitivity to a number of antibiotics, two of which, augmentin and timentin, were found to be inhibitory to the growth of A. tumefaciens but non-inhibitory to callusing, shooting or rooting of N. tabacum in tissue culture.The effect of the SV40 enhancer on the expression of the Nos promoter was investigated by constructing integrating plasmids (pDUBllG derivatives) with the SV40 enhancer 5' and 3'. and in both orientations with respect to the Nos-NPT gene. These plasmids were mobilised to A. tumefaciens and cointegrates with pTiGEl selected, characterised by Southern blotting, and inoculated in vivo onto leaves of K. diaigromentiana. Extracts from the resultant callus tissues were found to contain no detectable NPT activity in all cases. The same constructs were used to transform N. tabacum in vitro by a leaf disc transformation method and callus was selected on hormone free media and kanamycin. The callus induced by the constructs containing the SV40 enhancer showed no significant increase over the control construct, indicating that the SV40 enhancer does not function in these plants. Further improvements to the pBR322-homology mediated Ti vector system were made by constructing a new oncogenic Ti vector. pTiGE2. which has a smaller T-DNA containing a single copy of pBR322. giving a more defined T-DNA which is easier to analyse after cointegrate formation. New T-DNA integrating vectors containing the LacZ insertional inactivation region from pUC18 were constructed giving more unique restriction enzyme sites and making the selection of recombinants easier. A chimaeric CAMV-pea lectin gene was constructed and subcloned into a T-DNA integrating vector. Resultant cointegrates with pGV3850 were characterised and transgenic A'', tabacum plants regenerated. The T-DNA structure and copy number in the transgenic plants were investigated. Expression of the CAMV-pea lectin gene was characterised by northern blotting, western blotting, haemagglutination and ELISA and showed the gene to be expressed constitutively at high levels and the protein to be processed correctly. The subcellular site of lectin deposition in transgenic tobacco root was found to be the vacuole. Plants expressing lectin at high level were screened for resistance to a root-knot nematode and found not to be resistant to infection.
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Harighi, Behrouz. "Investigating the role of chemotaxis operon genes in Agrobacterium tumefaciens." Thesis, Durham University, 2003. http://etheses.dur.ac.uk/4083/.
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A 1.7 Kb chromosomal DNA of A. tumefaciens C58C1 downstream of chemotaxis operon was sequenced completely in both directions. The comparison of this sequence with sequence databases revealed one open reading frame with strong sequence identity to MCP gene in other bacteria. The sequencing of chromosomal DNA of A. tumefaciens C58 confirmed that this open reading frame has similarity with cytoplasmic domain of McpA. Four mutants of A. tumefaciens C58C1 (Cl/delYl, Cl/delY2, Cl/delB and Cl/delR) were created by in-frame deletion mutagenesis in cheYl, cheY2, cheB and cheR using pKlSmobsacB. Some phenotypic properties of mutants were studied. The cheY2, cheR and cheB mutants showed impaired chemotactic capabilities in both swarming and chemotaxis assays. Deletion of cheY1 appeared to have no significant effect on chemotaxis, under the conditions studied.
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Wright, Emma Louise. "Identification and molecular characterisation of chemotaxis genes in Agrobacterium tumefaciens." Thesis, Durham University, 1999. http://etheses.dur.ac.uk/4307/.
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Using heterologous probing, with fragments from the S. meliloti che operon, putative chemotaxis genes were identified in A. tumefaciens. The cosmid pDUB1911, from a representative genomic library of C58C1, was identified and found to contain a cluster of chemotaxis-related genes. A 9.6kb region of pDUB1911 was completely sequenced (GenBank Accession No. AF044495), in both directions, and found to contain an 8kb chemotaxis cluster. The cluster begins with orf1, followed by orf2, cheYl, cheA, cheR, cheB, cheY2, orf9 and orflO. All of the identified homologues showed a high degree of sequence conservation with their counterparts in the chemosensory regions of the related bacteria S. meliloti and R. sphaeroides, and were arranged in a similar order. A homologue of the flagellar gene fliF was identified directly downstream of the che cluster. This arrangement is similar to that seen in S. meliloti, where the che operon is followed by a large region containing flagellar/motility-related genes. It was therefore postulated that the region identified in this work could be linked to the cluster of flagellar/motihty-related genes previously identified in A. tumefaciens. Mutant strains were created by in-frame deletion of cheA and orflO, and insertion of a neomycin resistance cassette in orfl, cheA and fliF. The oifl and cheA mutants showed wild type motility, but impaired chemotactic capabilities. Deletion of orflO appeared to have no effect on either motility or chemotaxis, under the conditions studied. Mutation of fliF resulted in a non-motile, non-flagellate phenotype. A "gutted" strain was created by deletion of the entire che cluster. As with the orfl and cheA mutant strains, the gutted strain showed severely impaired chemotaxis, but wild type patterns of motility. Preliminary work was conducted on the construction of a selectively-infective phage (SIP) system for studying bimolecular interactions within, and between, the che and vir systems of A. tumefaciens. A phage vector was constructed, which following further testing, should allow such work to begin. Probing with a fragment coding for the conserved region of an MCP recently identified m R. leguminosarum, suggested that A. tumefaciens could contain a number of proteins resembling the classical MCPs of E. coli. A putative MCP homologue was also identified in pDUB1911, downstream of the main che cluster. Although the che cluster was found not to contain a homologue of cheW, heterologous probing and PGR using consensus primers indicated that c/ieWmaps elsewhere in the A. tumefaciens genome.
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Curtis, Ian Scott. "Genetic improvement of lettuce (Lactuca sativa L.) using Agrobacterium tumefaciens." Thesis, University of Nottingham, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363565.
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Lowe, Jeffrey Michael. "Studies on flower senescene and Agrobacterium transformation in Chrysanthemum morifolium." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334925.
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Depierreux, Durand Christiane. "Mecanismes de reconnaissance plantes-microorganismes. Lectine associee chez agrobacterium tumefaciens." Orléans, 1989. http://www.theses.fr/1989ORLE2035.
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L'infection du peuplier par agrobacterium tumefaciens debute par la fixation de cette bacterie sur le tissu vegetal. Si les pilis sont classiquement impliques dans l'attachement des bacteries sur un tissu cible, la souche etudiee ne presente que de rares pilis observes en microscopie electronique. La synthese d'un derive fluorescent a permis d'etudier ces bacteries par cytofluorimetrie en flux et de visualiser la presence de structures glycanniques par l'emploi de lectines fluoresceinylees. Etudiee en spectrofluorimetrie, la souche d'a. Tumefaciens fixe une neoglycoproteine fluorescence fucosylee a ph 5. Cette fixation est un phenomene saturable pouvant etre inhibe par la molecule non marquee. L'extrait bacterien obtenu par lavage des bacteries a ph 9, presente une activite hemagglutinante optimale a ph 5. Divers polysaccharides sont inhibiteurs et en particulier la fucoidine. La preparation d'une matrice d'agarose substituee par la fucoidine a permis la purification partielle de cette lectine par chromatographie d'affinite. L'etude de l'interaction a. Tumefaciens-cellules de peuplier a ete abordee. Une fraction oligosaccharidique isolee a partir d'un surnageant de digestion enzymatique de feuilles de peuplier s'est revelee tres bonne inhibitrice de l'hemagglutination par la lectine bacterienne purifiee et de la fixation des bacteries sur les cellules en culture. Ces interactions glycanne-proteines peuvent etre a l'origine de l'attachement d'a. Tumefaciens sur le tissu vegetal
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Picard, Christine. "Caractérisation et détection dans le sol des Agrobacterium du pommier." Lyon 1, 1993. http://www.theses.fr/1993LYO10163.
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Le crown gall est une maladie des vegetaux particulierement genante en pepinieres. Une bonne prophylaxie peut etre obtenue par plantation de plants sains. Afin d'eviter la plantation en sol contamine, il est necessaire de s'assurer de l'absence d'agrobacterium pathogenes dans les sols. Dans le cas du pommier, les tumeurs de crown gall sont induites par des souches bien particulieres. Les agrobacterium isoles de crown gall de pommier sont bien pathogenes. Mais ils ont de nombreuses particularites par rapport aux souches d'agrobacterium pathogenes generalement decrites: 1) ils sont tres proches genetiquement l'un de l'autre; 2) ils ont une gamme d'hotes tres etroite; 3) ils induisent la synthese de traces de nopaline, mais ils sont incapables de cataboliser la nopaline; 4) les genes de pathogenie qui ont ete detectes dans ces souches presentent peu d'homologie avec ceux de souches a large gamme d'hotes. Cependant, les agrobacterium de pommier semblent se rapprocher des agrobacterium a nopaline: ils sont capables d'induire la synthese de nopaline, et ils renferment des sequences nucleotidiques homologues au gene nos. De plus, des sequences nucleotidiques du gene tmr et de l'intergene virb-virg sont totalement homologues a celles decrites chez les agrobacterium a nopaline. La methode de detection dans les sols des agrobacterium pathogenes est basee sur l'amplification enzymatique (pcr) avec les amorces specifiques situees dans l'intergene virb-virg. Chaque etape du protocole a ete developpee pour permettre de detecter le plus petit nombre possible de sequences cibles. Experimentalement le protocole permet de detecter une seule sequence cible. Ceci permet de detecter de 10#3 a 10#8 cellules par gramme de sol. Lors de son application en conditions agronomiques, la methode a permis d'estimer a 410#4 la population d'agrobacterium pathogenes d'un sol de pepiniere de pommier naturellement contamine
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Hamel, Andre Louis Carleton University Dissertation Biology. "Construction of plasmids for studying genetic events in Agrobacterium tumefaciens." Ottawa, 1987.
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Reis, Maria Cecília dos. "Transformação genética do fungo entomopatogênico Beauveria bassiana via Agrobacterium tumefaciens." Universidade Estadual de Londrina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Microbiologia, 2004. http://www.bibliotecadigital.uel.br/document/?code=vtls000102880.
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A metodologia de transformação genética mediada pela bactéria Agrobacterium tumefaciens (agro-transformação) foi aplicada com sucesso para o fungo entomopatogênico Beauveria bassiana. Conídios de B. bassiana foram transformados utilizando-se o gene hph de Escherichia coli que confere resistência ao antibiótico higromicina B como marcador seletivo, sob controle de promotores heterólogos e a seqüência de término trpC de Aspergillus nidulans. A eficiência de transformação foi de até 28 e 96 transformantes por 104 e 105 conídios alvos, respectivamente, utilizando três vetores binários distintos. Alta estabilidade mitótica dos transformantes (80-100%) foi observada após 5 passagens sucessivas em meio não seletivo. A ocorrência de transformantes abortivos foi observada para todos os vetores binários utilizados. Os transformantes foram analisados quanto a presença do gene hph por PCR e Southern blot. A análise de Southern revelou a ocorrência de integração de mais de uma cópia do gene hph no genoma dos transformantes. O método de agro-transformação foi efetivo na obtenção de transformantes de B. bassiana resistentes à higromicina, possibilitando seu emprego em estudos de mutagênese insersional neste fungo.Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomopathogenic fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 104 and 105 target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on non-selective media. Abortive transformants were observed for all the hphr vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.
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Mata, Marcia Magalhães. "Transformação do fungo ocratoxigênico Aspergillus westerdijkiae medida por Agrobacterium tumefaciens." Universidade Estadual de Londrina. Centro de Ciências Biológicas. Programa de Pós-Graduação em Microbiologia, 2006. http://www.bibliotecadigital.uel.br/document/?code=vtls000114068.
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Aspergillus westerdijkiae é um produtor de ocratoxina A que tem sido freqüentemente encontrado em grãos de café. A ocratoxina A é conhecida por ter efeitos nefrotóxicos e potencial carcinogênico para espécies animais. Neste trabalho é reportada pela primeira vez a transformação genética de A. westerdijkiae mediada por Agrobacterium tumefaciens. Conídios foram transformados para resistência a higromicina B usando a linhagem AGL-1 de A. tumefaciens. A condição mais favorável de transformação permitiu a obtenção de 36 transformantes para cada 106 conídios alvo. Dentre 600 transformantes, um total de 30 (5%) mostraram variações morfológicas. Dois mutantes (T10 e TS18) com significativa redução da capacidade de produção de ocratoxina A foram obtidos. Ambos mostraram significativa redução na conidiogênese quando comparados à linhagem selvagem. As seqüências que flanqueiam os sítios de integração do T-DNA foram amplificadas pela técnica denominada Thermal Asymmetric Interlaced PCR (TAIL-PCR). A seqüência obtida a partir do mutante TS18 mostrou homologia com uma possível monooxygenase de A. fumigatus. Para o mutante T10 foi identificada uma seqüência homóloga a um fator de transcrição da família jumonji de A. fumigatus.Aspergillus westerdijkiae is a potent ochratoxin A producer fequently associated to coffee beans. Ochratoxin A is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report the first Agrobacterium-mediated transformation of A. westerdijkiae. Conidia were transformed to hygromicin B resistance using AGL-1 strain of Agrobacterium tumefaciens. The most favourable conditions allowed a mean transformation frequency of 36 transformants per 106 target conidia. Among 600 transformants 30 (5%) showed morphological alterations. Two transformants (T10 and TS18) with consistently reduced ochratoxin A production were obtained. Both of then also showed significant reduction in conidiogenesis compared with the wild type. Fungal sequences flanking the insertion site could be amplified by Thermal Asymmetric Interlaced PCR. The sequence obtained from tarnsformant TS18 had similarity to a monooxygenase putative from A. fumigatus. Transformant T10 had a sequence with similarity to a jumonji family of transcription factor from A. fumigatus.
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Joshi, B. "Physicochemical studies on lectins from agrobacterium radiobacter and xanthomonas campestris." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 1996. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2966.
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