Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by Subject "Agrobacterium tumefaciens"

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    Development of an Agrobacterium vitis transformation system for grapevine
    (Stellenbosch : Stellenbosch University, 2000-03) Joubert, Dirk Albert, 1973-; Pretorius, I. S.; Vivier, Melane A.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    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.
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    Strains of the crown gall pathogen, Agrobacterium tumefaciens, and their biological control in South Africa
    (Stellenbosch : Stellenbosch University, 1989-03) Van Zyl, F. G. H. (Frederik Gerhardus Heremias); Loos, M. A.; Stellenbosch University. Faculty of Science. Department of Microbiology.
    No abstract available.