Browsing by Author "O'Kennedy, Martha Margaretha"

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    Genetic enhancement of pearl millet
    (Stellenbosch : Stellenbosch University, 2004-03) O'Kennedy, Martha Margaretha; Botha, F. C.; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).
    ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola.