Relevant bibliographies by topics / Agrobacterium

Academic literature on the topic 'Agrobacterium'

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Published: 4 June 2021
Last updated: 7 July 2024

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Journal articles on the topic "Agrobacterium"

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Mougel, Christophe, Benoit Cournoyer, and Xavier Nesme. "Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2." Applied and Environmental Microbiology 67, no. 1 (January 1, 2001): 65–74. http://dx.doi.org/10.1128/aem.67.1.65-74.2001.

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ABSTRACT Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K2TeO3 was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K2TeO3 concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K2TeO3-amended medium. The bona fide agrobacterium colonies growing on media amended with K2TeO3 were confirmed to beAgrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed forAgrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 103 to 104agrobacteria · g of dry soil−1 in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.
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Dubrovna, O. V., and L. V. Slivka. "Optimization of Agrobacterium-mediated transformation of perspective winter wheat genotypes in vitro." Faktori eksperimental'noi evolucii organizmiv 26 (September 1, 2020): 190–95. http://dx.doi.org/10.7124/feeo.v26.1264.

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Aim. Optimization of conditions for genetic transformation of new perspective winter wheat genotypes. Methods. Agrobacterium-mediated transformation in culture in vitro. Results. The influence of the optical density of cells of the agrobacterial suspension, the concentration of the antibiotic cefotaxime, the duration of coculture on the frequency of obtaining kanamycin-resistant regenerants of new winter wheat genotypes by genetic transformation of callus cultures were investigated using LBA4404 and AGL0 strains. It is shown that depending on the strain the most optimal is the concentration of agrobacteria 0.2-03 OD, duration of coculture for 2-3 days and the use of cefotaxime at a concentration of 250-500 mg/L. Conclusions. The optimal parameters for conducting Agrobacterium-mediated transformation of callus cultures of new perspective winter wheat genotypes were selected. Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures.
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Denkovskienė, Erna, Šarūnas Paškevičius, Justina Stankevičiūtė, Yuri Gleba, and Aušra Ražanskienė. "Control of T-DNA Transfer from Agrobacterium tumefaciens to Plants Based on an Inducible Bacterial Toxin-Antitoxin System." Molecular Plant-Microbe Interactions® 33, no. 9 (September 2020): 1142–49. http://dx.doi.org/10.1094/mpmi-03-20-0067-r.

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High-value pharmaceutical products are already successfully produced in contained facilities using Agrobacterium-mediated transient transformation of plants. However, transfection methods suitable for open field applications are still desirable as a cheaper alternative. Biosafety concerns related to the use of recombinant agrobacteria in an industrial transfection process include possible transformation or transfection of unintended hosts or spread of the genetically modified agrobacteria in the environment. In this paper, we explored a novel biocontrol approach resulting in greater biosafety of the transient expression process in plants. Our proposed solution involves inducible expression of Agrobacterium tumefaciens toxin PemK and antitoxin PemI that provides for strictly regulated T-DNA transfer from agrobacteria to plants. We also identified several other toxins from putative Agrobacterium toxin-antitoxin modules and demonstrate their potential usefulness in the control of Agrobacterium tumefaciens as a DNA vector.
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Slivka, L. V., and O. V. Dubrovna. "Genetic transformation of new perspective winter wheat genotypes in vitro." Faktori eksperimental'noi evolucii organizmiv 26 (September 1, 2020): 270–75. http://dx.doi.org/10.7124/feeo.v26.1278.

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Aim. Optimization of conditions for genetic transformation of new perspective winter wheat genotypes and production of transgenic plants. Methods. Agrobacterium-mediated transformation in vitro culture using callus cultures. Results. The influence of the optical density of cells of agrobacterial suspension, the concentration of the antibiotic cefotaxime, the duration of coculture on the frequency of obtaining kanamycin-resistant regenerants of new winter wheat genotypes by genetic transformation of callus cultures were investigated. By Agrobacterium-mediated transformation of morphogenic calluses of new perspective winter wheat genotypes were obtained plant-regenerants in the genome of which revealed the complete incorporation of a genetic construct containing oat and nptII transgenes. Conclusions. Agrobacterium-mediated transformation of callus cultures of new perspective winter wheat genotypes was optimized, and transgenic plants with the target gene of ornithine-δ-aminotransferase were obtained. Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures, ornithine-δ-aminotransferase gene.
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Shaposhnikov, Anton D., and Tatiana V. Matveeva. "Homologues of octopine/vitopine synthase genes in natural GMOs." Ecological genetics 20, no. 1S (December 8, 2022): 38–39. http://dx.doi.org/10.17816/ecogen112343.

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The process of horizontal gene transfer causes the appearance of natural genetically modified organisms. At the moment, it is known that over 7% of dicotyledonous plant species are naturally transgenic, i.e. nGMO [1]. These plants contain the genes of agrobacteria, which are integrated in the nuclear genome during infection. In some species of naturally transgenic plants, agrobacterial genes have been preserved for millions of years of evolution. Among these genes, genes encoding octopine/vitopine synthase (ocs/vis) can be distinguished [2]. The study of homologues of octopin/vitopin synthase genes in naturally transgenic plants: their structures and diversity, products of encoded enzymes will allow us to establish the functions and evolutionary role of homologues in nGMO. Currently, bioinformatic and genetic engineering methods are used to solve these problems. ocs\vis-like were found in 7 species: Albizia julibrissin Durazz., Cenostigma pyramidale (Tul.) Gagnon G.P.Lewis, Paulownia fortunei (Seem.) Hemsl., Pterocarya stenoptera C.DC., Rehmannia glutinosa Steud., Santalum album L., Viscum album L. In total twenty one ocs/vis sequences are known in 17 nGMO species. Twenty sequences are intact. This may indicate the functional significance of these genes for nGMO. Phylogenetic analysis of currently known ocs/vis-like genes of Agrobacterium, Rhizobium and natural GMOs suggests that diversity of studied genes is wider, than it was estimated based on agrobacterial sequences. On the phylogenetic tree constructed by the neighbor-joining method, 6 clusters for ocs/vis can be distinguished. Three clusters contain nGMOs and agrobacteria, showing the relationship of the T-DNA sequences of nGMO with those of currently known strains of of Agrobacterium/Rhizobium. Three clusters contain only nGMOs. One of them consists of species that belong to the Cannabaceae family. Other clusters are heterogeneous. No significant ecological similarities were found among the studied species. The obtained results can be used to study the diversity of ancient and modern strains of agrobacteria, their host specificity and the possible role of their genes in plant evolution. The work was supported by the RSF, grant 21-14-00050 and Research Resource Center for molecular and cellular technologies of Saint Petersburg State University.
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Portier, Perrine, Marion Fischer-Le Saux, Christophe Mougel, Catherine Lerondelle, David Chapulliot, Jean Thioulouse, and Xavier Nesme. "Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers." Applied and Environmental Microbiology 72, no. 11 (August 25, 2006): 7123–31. http://dx.doi.org/10.1128/aem.00018-06.

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ABSTRACT Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely “species-specific” markers tagging the core genomes of the species.
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Bogomaz, F. D., and T. V. Matveeva. "Expression sequences of opine synthase genes in natural GMOs based on analysis of their transcriptomes." Plant Biotechnology and Breeding 5, no. 3 (October 29, 2022): 15–24. http://dx.doi.org/10.30901/2658-6266-2022-3-o2.

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Agrobacterium is a natural genetic material delivery system that humans use to produce genetically modified plants (GMO). In nature, GMOs also occur with the participation of agrobacteria. In 2019, the list of known natural GMOs was expanded by an order of magnitude, and facts were found in favor of the expression of agrobacterial genes in natural GMOs. The frequency of this phenomenon for dicotyledon plants has been estimated at 7 percent. Opine synthase genes turned out to be the predominant ones of agrobacterial origin in natural GMOs. They probably perform important functions in natural GMOs. In 2021, an article was published with an updated list of natural GMOs, but the list of genes expressed in natural GMOs has not been updated since 2019.The aim of this work is to update the list of opine synthase genes expressed in natural GMOs. The research methods included bioinformatic search using queries based on the sequences of opine synthase proteins from Agrobacterium rhizogenes, A. tumefaciens and A. vitis, their homologues from Ipomoea and Nicotiana plants, in the TSA database of the National Center for Biotechnology Information (NCBI) using the TBLASTN algorithm with default settings.The study resulted in the addition of another 18 species to the list of natural GMOs with expressed opine synthase genes, 12 of which belong to genera where natural GMOs were not previously described (Albizia, Cenostigma, Averrhoa, Gynostemma, Eurycoma, Gypsophila, Myosoton, Camptotheca, Gustavia, Eschweilera, Cestrum, Jasminum, and Paulownia).An analysis of the diversity of the detected sequences showed that homologues of cucumopine and mikimopine synthase predominate among them. The end products of these genes are optical isomers. In the future, it makes sense to start studying the functions of opine synthases in plants from these genes.
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Adnan, Mohd, Saif Khan, Mitesh Patel, Eyad Al-Shammari, and Ibraheem M. A. Ashankyty. "Agrobacterium." Reviews in Medical Microbiology 24, no. 4 (October 2013): 94–97. http://dx.doi.org/10.1097/mrm.0b013e3283642449.

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Kaur, Maninder, Pooja Manchanda, Anu Kalia, Farah K. Ahmed, Eugenie Nepovimova, Kamil Kuca, and Kamel A. Abd-Elsalam. "Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants." International Journal of Molecular Sciences 22, no. 19 (October 8, 2021): 10882. http://dx.doi.org/10.3390/ijms221910882.

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Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.
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Bell, C. R., and W. D. Ramey. "Hybrid biovars of Agrobacterium species isolated from conifer roots." Canadian Journal of Microbiology 37, no. 1 (January 1, 1991): 34–41. http://dx.doi.org/10.1139/m91-006.

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A total of 377 heterotrophic bacteria were isolated on nonselective medium from the rhizoplanes of five species of conifer. The species were western hemlock (Tsuga heterophylla), Douglas-fir (Pseudotsuga menziesii), lodgepole pine (Pinus contorta), white spruce (Picea glauca), and western red cedar (Thuja plicata). Twenty-eight strains from this population were identified as presumptive agrobacteria. All proved nontumourigenic. Principal-component analysis indicated that the strains, which had clustered into two discrete groups, had intermediate biovar characteristics. Cluster 1 was predominately biovar 3/2 in character, cluster 2 was predominately biovar 2/3. All the presumptive agrobacteria were distributed randomly with respect to the tree species. This study demonstrates that agrobacteria, although atypical, do occur in forest soils and attests to the ubiquity of the genus in soil. Key words: Agrobacterium, biovars, crown gall, conifers.
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Dissertations / Theses on the topic "Agrobacterium"

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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
Palancia
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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.
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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
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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
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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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Books on the topic "Agrobacterium"

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Gartland, Kevan M. A., and Michael R. Davey. Agrobacterium Protocols. New Jersey: Humana Press, 1995. http://dx.doi.org/10.1385/0896033023.

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Kan, Wang. Agrobacterium Protocols. New Jersey: Humana Press, 2006. http://dx.doi.org/10.1385/1597451304.

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Wang, Kan, ed. Agrobacterium Protocols. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-1658-0.

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Wang, Kan, ed. Agrobacterium Protocols. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-1695-5.

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Gelvin, Stanton B., ed. Agrobacterium Biology. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-030-03257-9.

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A, Gartland K. M., and Davey M. R. 1944-, eds. Agrobacterium protocols. Totowa, N.J: Humana Press, 1995.

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Wang, Kan, ed. Agrobacterium Protocols Volume 2. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1385/1597451312.

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Tzfira, Tzvi, and Vitaly Citovsky, eds. Agrobacterium: From Biology to Biotechnology. New York, NY: Springer New York, 2008. http://dx.doi.org/10.1007/978-0-387-72290-0.

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Tzvi, Tzfira, and Citovsky Vitaly, eds. Agrobacterium: From biology to biotechnology. New York: Springer, 2008.

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Brightwell, Gale. Molecular genetics of exopolysaccharide synthesis in agrobacterium radiobacter. Norwich: University ofEast Anglia, 1993.

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Book chapters on the topic "Agrobacterium"

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Mangena, Phetole. "Agrobacterium tumefaciens." In Plant Transformation via Agrobacterium tumefaciens, 25–38. Boca Raton: CRC Press, 2022. http://dx.doi.org/10.1201/b22829-3.

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Britton, Monica T., Matthew A. Escobar, and Abhaya M. Dandekar. "The Oncogenes of Agrobacterium Tumefaciens and Agrobacterium Rhizogenes." In Agrobacterium: From Biology to Biotechnology, 523–63. New York, NY: Springer New York, 2008. http://dx.doi.org/10.1007/978-0-387-72290-0_14.

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Schilperoort, R. A. "Plant-Agrobacterium Interaction." In Recognition in Microbe-Plant Symbiotic and Pathogenic Interactions, 431–32. Berlin, Heidelberg: Springer Berlin Heidelberg, 1986. http://dx.doi.org/10.1007/978-3-642-71652-2_44.

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Hooykaas, Paul J. J. "Agrobacterium molecular genetics." In Plant Molecular Biology Manual, 65–77. Dordrecht: Springer Netherlands, 1988. http://dx.doi.org/10.1007/978-94-017-5294-7_4.

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Hooykaas, Paul J. J. "Agrobacterium molecular genetics." In Plant Molecular Biology Manual, 49–61. Dordrecht: Springer Netherlands, 1989. http://dx.doi.org/10.1007/978-94-009-0951-9_4.

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Hooykaas, Paul J. J. "Agrobacterium molecular genetics." In Plant Molecular Biology, 83–87. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-017-6951-8_5.

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Hooykaas, Paul J. J., and Teresa Mozo. "Agrobacterium molecular genetics." In Plant Molecular Biology Manual, 75–83. Dordrecht: Springer Netherlands, 1994. http://dx.doi.org/10.1007/978-94-011-0511-8_5.

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Matthysse, Ann G. "The Genus Agrobacterium." In The Prokaryotes, 91–114. New York, NY: Springer New York, 2006. http://dx.doi.org/10.1007/0-387-30745-1_5.

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Binns, Andrew N., and Paolo Costantino. "The Agrobacterium Oncogenes." In The Rhizobiaceae, 251–66. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-5060-6_12.

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Firoozabady, Ebrahim, and Adelheid R. Kuehnle. "Agrobacterium-Mediated Transformation." In Plant Cell, Tissue and Organ Culture, 181–95. Berlin, Heidelberg: Springer Berlin Heidelberg, 1995. http://dx.doi.org/10.1007/978-3-642-79048-5_15.

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Conference papers on the topic "Agrobacterium"

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Toplaghaltsyan, Anna, Zhaneta Karapetyan, Susanna Keleshyan, G. Avetisova, L. Melkonyan, G. Tsarukyan, and V. Ghochikyan. "Enzymatic activity of nitrogen-fixing soil bacteria." In 5th International Scientific Conference on Microbial Biotechnology. Institute of Microbiology and Biotechnology, Republic of Moldova, 2022. http://dx.doi.org/10.52757/imb22.37.

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Soil is a medium for more than 100 enzymes. During soil deterioration the change of enzymes occurs much sooner than of other parameters in the soil so they are considered the best indicators of soil health. These enzymes play a vital role in supporting soil ecology and health by direct agents of the biological catabolism of soil organic and mineral components. Еnzymatic activities in the soil are mainly of microbial origin. In a number of potential bacterial enzymes that play an important role in maintaining soil health, some of the important ones are protease, lipase, cellulase, amylase and urease [1]. The aim of this work was the study of enzymatic activity in nitrogen-fixing bacteria, such as protease, lipase, cellulose, amylase and urease. The bacteria used in the experiments were Agrobacterium sp. strain M-1 (MN717167) and Agrobacterium sp. strain Y-2 (MN721294), previously isolated by us from saline soils of the villages Mrgashat and Yeghegnut of Armenia, respectively [2]. Proteolytic activity of nitrogen-fixing strains was determined using Skim Milk Agar, lipolytic activity - by Burk's Agar with 2% Tween 80, cellulolytic activity - by Cellulose Congo Red Agar. Bacterial suspension (107-108 CFU/ml) was added into punch holes in the agar and plates were incubated at 30 °C during 3-7 days. Enzymatic activity was detected by clear zones around holes. In case of amylolytic activity Starch Agar was used. A fresh colony of bacteria was streaked on the surface of the agar by double streak and was incubated for 3-5 days at 30 °C. Then the surface of the agar was flooded with Gram’s iodine solution. A clear zone surrounding the bacterial growth confirmed the hydrolysis of starch. Urease activity was investigated by using Urea broth. The cultures were inoculated separately into test tubes and incubated at 30 °C for 4 days. The appearance of a deep pink color indicated a positive result. The results of the experiments presented in Table 1 show the presence of a fairly wide range of enzymes in cultures Agrobacterium sp. strain M-1 and Agrobacterium sp. strain Y-2. Table 1. Enzymatic activity of nitrogen-fixing bacteria Nitrogen-fixing bacteria Enzymatic activity Protease Lipase Cellulase Amylase Urease Agrobacterium sp. strain M-1 - + + + + Agrobacterium sp. strain Y-2 + + + + + Therefore, it can be assumed that the use of Agrobacterium sp. strain M-1 and Agrobacterium sp. strain Y-2 with a variety of enzymatic activities can provide the soil with the enzymes necessary for the normal course of global carbon and nutrient cycles.
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Yakubovskaya, A. I., I. A. Kameneva, T. N. Melnichuk, T. V. Matveeva, M. V. Gritchin, and S. F. Abdyrashitov. "Agrobacterium tumefaciens - associative microsymbionts of grain crop." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.281.

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The installed absence of Ti-plasmids in the genome structure of strains of diazotrophic bacteria Agrobacterium tumefaciens 32 and A. tumefaciens P3 was found, which characterizes the strains as non-pathogenic. Bacterization of grain seeds by associative bacterial strains stimulates root development by up to 40%.
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Kuluev, B. R., A. V. Knyazev, Z. R. Vershinina, Al K. Baymiev, and A. V. Chemeris. "AGROBACTERIUM-MEDIATED TRANSFORMATION OF TRANSPLASTOMIC TOBACCO PLANTS." In The Second All-Russian Scientific Conference with international participation "Regulation Mechanisms of Eukariotic Cell Organelle Functions". SIPPB SB RAS, 2018. http://dx.doi.org/10.31255/978-5-94797-318-1-61-62.

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Taipova, R. M., and B. R. Kuluev. "Agrobacterium-mediated transformation of Amaranthus cruentus L. epicotyls by the ARGOS-LIKE transgene." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.244.

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The present study describes the results of our research in Agrobacterium-mediated transformation of epicotyl segments of Amaranthus cruentus variety “Bagryanyi” by the ARGOS-LIKE transgene of Arabidopsis thaliana controlled by the 35S promoter. For shoot regeneration from epicotyl segments after Agrobacterium-mediated transformation, Murashige-Skoog (MS) medium containing 13 μM 6-benzylaminopurine and 1 μM α-naphthylacetic acid was used. For the selection of transgenic shoots, 10 mg/L of hygromycin B was added to the MS medium.
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Toplaghaltsyan, Anna, Zhaneta Karapetyan, Susanna Keleshyan, G. Avetisova, L. Melkonyan, G. Tsarukyan, and V. Ghochikyan. "Enzymatic activity of nitrogen-fixing bacteria isolated from Armenian saline soils." In 5th International Scientific Conference on Microbial Biotechnology. Institute of Microbiology and Biotechnology, Republic of Moldova, 2022. http://dx.doi.org/10.52757/imb22.38.

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Nitrogen-fixing bacteria with phytostimulating potential have great interest in the modern world. The aim of this work was to study the enzymatic activity of nitrogen-fixing strains Agrobacterium sp. M-1 and Agrobacterium sp. Y-2. Studies have shown that these strains are capable to produce proteases, lipases, cellulases, amylases and ureases, enzymes that play a vital role in maintaining soil health. In addition, these bacteria have several significant characteristics: salt tolerance, pH stability, maintaining of viability at low and high temperatures. Therefore, the above strains have every chance to become the basis for the creation of a multifunctional biofertilizer in the future
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Panfilova, M. A., E. V. Mikhaylova, Kh G. Musin, and B. R. Kuluev. "In vitro cultivation, aquaculture and methods of transformation of water caltrop Trapa L." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.190.

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In vitro and aquarium cultures of water caltrop were obtained. Optimal conditions of cultivation were determined. The cultures were subjected to Agrobacterium rhizogenes-mediated and biolistic transformation.
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Hyejin Cho and Hui-Hsien Chou. "E. coli and Agrobacterium non-coding regulatory RNA discovery." In 2011 IEEE International Conference on Bioinformatics and Biomedicine Workshops (BIBMW). IEEE, 2011. http://dx.doi.org/10.1109/bibmw.2011.6112545.

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Guo, Minliang, Yujuan Xu, Dawei Gao, and Nan Xu. "Signal Crosstalk between Two Different Agrobacterium Two-Component Systems." In The 5th World Congress on New Technologies. Avestia Publishing, 2019. http://dx.doi.org/10.11159/icbb19.110.

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Kolomiec, A., and A. Pungin. "Efficiency of Agrobacterium Transformation of Skullcap (Scutellaria galericulata L.)." In ChemBioSeasons 2022. Kemerovo State University, 2022. http://dx.doi.org/10.21603/chembioseasons2022-19.

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Ilina, E. L., A. S. Kiryushkin, E. D. Guseva, and K. N. Demchenko. "Methodological approaches to agrobacterium-mediated transformation of buckwheat (Fagopyrum esculentum Moench)." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.106.

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The method of Agrobacterium rhizogenes-mediated transformation of buckwheat has been established; composite plants have been obtained. The distribution of the cellular response to auxin by reporter proteins with different maturation times coincides.
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Reports on the topic "Agrobacterium"

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Alan R. White and Ann G. Matthysse. Cellulose Synthesis in Agrobacterium tumefaciens. Office of Scientific and Technical Information (OSTI), July 2004. http://dx.doi.org/10.2172/840242.

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Ron, Eliora, and Eugene Eugene Nester. Global functional genomics of plant cell transformation by agrobacterium. United States Department of Agriculture, March 2009. http://dx.doi.org/10.32747/2009.7695860.bard.

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The aim of this study was to carry out a global functional genomics analysis of plant cell transformation by Agrobacterium in order to define and characterize the physiology of Agrobacterium in the acidic environment of a wounded plant. We planed to study the proteome and transcriptome of Agrobacterium in response to a change in pH, from 7.2 to 5.5 and identify genes and circuits directly involved in this change. Bacteria-plant interactions involve a large number of global regulatory systems, which are essential for protection against new stressful conditions. The interaction of bacteria with their hosts has been previously studied by genetic-physiological methods. We wanted to make use of the new capabilities to study these interactions on a global scale, using transcription analysis (transcriptomics, microarrays) and proteomics (2D gel electrophoresis and mass spectrometry). The results provided extensive data on the functional genomics under conditions that partially mimic plant infection and – in addition - revealed some surprising and significant data. Thus, we identified the genes whose expression is modulated when Agrobacterium is grown under the acidic conditions found in the rhizosphere (pH 5.5), an essential environmental factor in Agrobacterium – plant interactions essential for induction of the virulence program by plant signal molecules. Among the 45 genes whose expression was significantly elevated, of special interest is the two-component chromosomally encoded system, ChvG/I which is involved in regulating acid inducible genes. A second exciting system under acid and ChvG/Icontrol is a secretion system for proteins, T6SS, encoded by 14 genes which appears to be important for Rhizobium leguminosarum nodule formation and nitrogen fixation and for virulence of Agrobacterium. The proteome analysis revealed that gamma aminobutyric acid (GABA), a metabolite secreted by wounded plants, induces the synthesis of an Agrobacterium lactonase which degrades the quorum sensing signal, N-acyl homoserine lactone (AHL), resulting in attenuation of virulence. In addition, through a transcriptomic analysis of Agrobacterium growing at the pH of the rhizosphere (pH=5.5), we demonstrated that salicylic acid (SA) a well-studied plant signal molecule important in plant defense, attenuates Agrobacterium virulence in two distinct ways - by down regulating the synthesis of the virulence (vir) genes required for the processing and transfer of the T-DNA and by inducing the same lactonase, which in turn degrades the AHL. Thus, GABA and SA with different molecular structures, induce the expression of these same genes. The identification of genes whose expression is modulated by conditions that mimic plant infection, as well as the identification of regulatory molecules that help control the early stages of infection, advance our understanding of this complex bacterial-plant interaction and has immediate potential applications to modify it. We expect that the data generated by our research will be used to develop novel strategies for the control of crown gall disease. Moreover, these results will also provide the basis for future biotechnological approaches that will use genetic manipulations to improve bacterial-plant interactions, leading to more efficient DNA transfer to recalcitrant plants and robust symbiosis. These advances will, in turn, contribute to plant protection by introducing genes for resistance against other bacteria, pests and environmental stress.
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Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.

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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.
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Masters, Alicia. Agrobacterium-mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes. Ames (Iowa): Iowa State University, January 2020. http://dx.doi.org/10.31274/cc-20240624-137.

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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.

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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Marton, L. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995. Office of Scientific and Technical Information (OSTI), February 1996. http://dx.doi.org/10.2172/184286.

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Marton, L. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994]. Office of Scientific and Technical Information (OSTI), December 1994. http://dx.doi.org/10.2172/197759.

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Reisch, Bruce, Avichai Perl, Julie Kikkert, Ruth Ben-Arie, and Rachel Gollop. Use of Anti-Fungal Gene Synergisms for Improved Foliar and Fruit Disease Tolerance in Transgenic Grapes. United States Department of Agriculture, August 2002. http://dx.doi.org/10.32747/2002.7575292.bard.

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Original objectives . 1. Test anti-fungal gene products for activity against Uncinula necator, Aspergillus niger, Rhizopus stolonifer and Botrytis cinerea. 2. For Agrobacterium transformation, design appropriate vectors with gene combinations. 3. Use biolistic bombardment and Agrobacterium for transformation of important cultivars. 4. Characterize gene expression in transformants, as well as level of powdery mildew and Botrytis resistance in foliage of transformed plants. Background The production of new grape cultivars by conventional breeding is a complex and time-consuming process. Transferring individual traits via single genes into elite cultivars was proposed as a viable strategy, especially for vegetatively propagated crops such as grapevines. The availability of effective genetic transformation procedures, the existence of genes able to reduce pathogen stress, and improved in vitro culture methods for grapes, were combined to serve the objective of this proposal. Effective deployment of resistance genes would reduce production costs and increase crop quality, and several such genes and combinations were used in this project. Progress The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. All pairwise interactions were additive but not synergistic. Per objective 2, suitable vectors with important gene combinations for Agrobacterium transformation were designed. In addition, multiple gene co-transformation by particle bombardment was also tested successfully. In New York, transformation work focused on cultivars Chardonnay and Merlot, while the technology in Israel was extended to 41B, R. 110, Prime, Italia, Gamay, Chardonnay and Velika. Transgenic plant production is summarized in the appendix. Among plants developed in Israel, endochitinase expression was assayed via the MuchT assay using material just 1-5 days after co-cultivation. Plants of cv. Sugraone carrying the gene coding for ESF12, a short anti-fungal lytic peptide under the control of the double 358 promoter, were produced. Leaf extracts of two plants showed inhibition zones that developed within 48 h indicating the inhibitory effect of the leaf extracts on the six species of bacteria. X fastidiosa, the causal organism of Pierce's disease, was very sensitive to leaf extracts from ESF12 transformed plants. Further work is needed to verify the agricultural utility of ESF12 transformants. In New York, some transformants were resistant to powdery mildew and Botrytis fruit rot. Major conclusions, solutions, achievements and implications The following scientific achievements resulted from this cooperative BARD project: 1. Development and improvement of embryogenesis and tissue culture manipulation in grape, while extending these procedures to several agriculturally important cultivars both in Israel and USA. 2. Development and improvement of novel transformation procedures while developing transformation techniques for grape and other recalcitrant species. 3. Production of transgenic grapevines, characterization of transformed vines while studying the expression patterns of a marker gene under the control of different promoter as the 35S CaMV in different part of the plants including flowers and fruits. 4. Expression of anti-fungal genes in grape: establishment of transgenic plants and evaluation of gene expression. Development of techniques to insert multiple genes. 5. Isolation of novel grape specific promoter to control the expression of future antimicrobial genes. It is of great importance to report that significant progress was made in not only the development of transgenic grapevines, but also in the evaluation of their potential for increased resistance to disease as compared with the non engineered cultivar. In several cases, increased disease resistance was observed. More research and development is still needed before a product can be commercialized, yet our project lays a framework for further investigations.
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9

Porat, Ron, Doron Holland, and Linda Walling. Identification of Citrus Fruit-Specific and Pathogen-Induced Promoters and Their Use in Molecular Engineering. United States Department of Agriculture, January 2001. http://dx.doi.org/10.32747/2001.7585202.bard.

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This one year BARD project was funded to develop methods to monitor promoter activity a gene expression patterns in citrus fruit. To fulfill this goal, we divided the research tasks between both labs so that the Israeli side evaluated the use of microprojectile bombardment ; a tool to evaluate transient gene expression in various citrus fruit tissues, and the US side optimized technical parameters required for Agrobacterium-mediated transformation of various citrus cultivars. Microprojectile bombardment appeared to be a very efficient method for transient gene expression analysis in citrus leaf tissues but was somewhat less applicable in fruit tissues. Nevertheless, we did succeeded to achieve significant levels of 35S-GUS gene expression in young green flavedo tissue. However, only single random spots of 35S-GUS gene expression were detected mature flavedo and in juice sacs and albedo tissue. Overall, we assume that following some more technical improvements particle bombardment could provide a useful technique to rapidly analyze promoter activity at least in the flavedo tissue. For Agrobacterium-mediated transformation, we found that shoot cultures of 'Washington' navel oranges,'Fairchild' mandarins,'Eureca' lemons,'Troyer' citrange and various grapefruits provided a more reliable and consistent source of tissue for transformation than germinated seedlings. Moreover, various growth media's (McCown, Quoirin & Lepoivre, DCR) further improved shoot and root growth relative to MS mineral media, which is commonly used. Also pure white light (using bulbs which do not emit UV or blue light) improved shoot growth in various citrus varieties, and paromomycin appeared to be a more efficient antibiotic for the selection of transgenic plants than Kanamycin. Overall, these optimizations improve transformation efficacy and shoot growth and rooting capacity. In addition to the development of transformation methods, both Israeli and US labs achieved progress in the identification of citrus fruit-specific promoters. In Israel, we isolated a 3.6 kb promoter fragment of the thiamine biosynthesis c-thi gene, which is highly expressed in fruit peel tissue, whereas in the US we isolated a 1.5 kb promoter fragment of the citrus seed-specific cDNA CssH. The identification of more fruit-specific cDNAs and their corresponding promoter regions is currently in progress.
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10

Watad, Abed A., Paul Michael Hasegawa, Ray A. Bressan, Alexander Vainstein, and Yigal Elad. Osmotin and Osmotin-Like Proteins as a Novel Source for Phytopathogenic Fungal Resistance in Transgenic Carnation and Tomato Plants. United States Department of Agriculture, January 2000. http://dx.doi.org/10.32747/2000.7573992.bard.

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The goal of this project is to enhance fungal resistance of carnation and tomato through the ectopic expression of osmotin and other pathogenesis-related (PR) proteins. The research objectives were to evaluate in vitro antifungal activity of osmotin and osmotin and other PR protein combinations against phytopathogens (including Fusarium oxysporum, Verticillium dahliae, Botrytus cinerea or Phytophthora infestans), develop protocols for efficient transformation of carnation and tomato, express PR proteins in transgenic carnation and tomato and evaluate fungal resistance of transgenic plants. Protocols for microprojectile bombardment and Agrobacterium-mediated transformation of carnation were developed that are applicable for the biotechnology of numerous commercial cultivars. Research established an efficient organogenetic regeneration system, optimized gene delivery and transgene expression and defined parameters requisite to the high frequency recovery of transgenic plants. Additionally, an efficient Agrobacterium-mediated transformation protocol was developed for tomato that is applicable for use with numerous commercial varieties. Rigorous selection and reducing the cytokinin level in medium immediately after shoot induction resulted in substantially greater frequency of adventitious shoots that developed defined stems suitable for rooting and reconstitution of transgenic plants. Transformation vectors were constructed for co-expression of genes encoding osmotin and tobacco chitinase Ia or PR-1b. Expression of osmotin, PR-1 and/or chitinase in transgenic carnation mediated a high level resistance of cv. White Sim (susceptible variety) to F. oxysporum f. sp. dianthi, race 2 in greenhouse assays. These plants are being evaluated in field tests. Comprehensive analysis (12 to 17 experiments) indicated that germination of B. cinerea conidia was unaffected by PR protein expression but germ tube elongation was reduced substantially. The disease severity was significantly attenuated by PR protein expression. Constitutive expression of osmotin in transgenic tomato increased resistance to B. cinerea, and P. infestans. Grey mold and late blight resistance was stable through the third selfed generation. The research accomplished in this project will have profound effects on the use of biotechnology to improve carnation and tomato. Transformation protocols that are applicable for efficient stable gene transfer to numerous commercial varieties of carnation and tomato are the foundation for the capacity to bioengineer these crops. The research further establishes that PR proteins provide a measure of enhanced disease resistance. However, considerations of PR protein combinations and conditional regulation and targeting are likely required to achieve; sustained level of resistance.
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