TAXI : a new vehicle for the transfer of genes into monocotyledonous plants

Doctoral Thesis


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University of Cape Town

The transfer of foreign genes into cereals followed by their correct expression in a tissue specific, developmentally regulated manner has become an important research focus. Based on the mechanism of Agrobacterium tumefaciens mediated transformation in dicots, a modified method to transform the monocotyledonous rye ( Secale cereale L. ) via A.tumefaciens mediated transformation was attempted. The induced bacterium culture was injected into rye seedlings, the transferred reporter gene, uid.A, was detected by PCR, and the expression of the gene was tested by histochemical assays. However, successful transformation and integration of the transgenes remained doubtful, because the frequency of kanamycin resistance in the progenies ( Rl , R2 and R3 ) did not increase. To achieve a real transformation and heritable transgenic rye, a new vehicle for gene transfer to plants was developed. A macromolecular complex, termed the TAXI, consisted of histone HI-protected single stranded DNA, containing a selectable marker gene (npt JI), linked either to a reporter gene (uidA) or a glutenin gene. The constructs were transferred by injection of rye seedlings. Molecular analyses demonstrated that all three genes were integrated and expressed in transformed rye and their progenies ( Rl and R2 ). TAXI mediated gene transfer to rye revealed an important advantage in that single or low numbers of transgenes were inserted into the transformed plant genome. However, the method of TAXI delivery to plants was not efficient. To improve this, a new approach, combining TAXI transformation and the biolistic process, was developed. A rapid regenerable callus line of a grass species, Digitaria sanguinalis, was established as a test system. TAXI coated gold particles, carrying a selectable marker gene (bar) and a reporter gene (uid.A), were used in bombardment experiments. The results of herbicide resistance and molecular analyses demonstrated that single copies or low numbers of the bar gene were inserted and expressed in regenerated transformed D. sanguinalis. Mendelian segregation in the Rl population was observed in four out of five transgenic lines.

Bibliography: leaves 90-103.