Two approaches for induction and isolation of starch mutants in potato (Solanum tuberosum L.) : random versus gene targeted mutagenesis

In this thesis two approaches were used to induce structural mutations in potato starch biosynthesis genes in potato. First production of new monoploid amf genotypes through parthenogenesis made it possible to initiate mutation breeding for amfae double mutants. Two amf monoploids were selected which fulfilled most of the prerequisites. By inducing a mutation in one of the branching enzymes in an amf -mutant it is possible to select a double mutant having less branched amylopectin. This mutation can easily be identified by iodine staining. Amylose-free starch will stain red and less branched amylopectin will stain blue, like amylose containing starch. Mutations were induced by X-ray irradiation of leaf explants followed by adventitious shoot regeneration and microtuber induction or followed by several rounds of multiplication of axillary buds and microtuber induction. In both cases the starch of microtubers was stained with iodine to screen for aberrant types. In 56 tuber samples blue or otherwise aberrant starch granules were found. With this kind of observations, the concept of mutation breeding for starch variants in monoploid potatoes is proven. A second way to induce structural mutations was the use of the Ac ( Activator )/ Ds ( Dissociation ) transposase system of maize in potato where the Ds transposon is activated by a transposase source. In this study the Ds element was linked to the GBSS gene, of which the phenotypic effect of deactivation is known i.e. red staining starch after iodine staining. The known amf mutation was used as a model system to gather more information about the transposition frequency of the Ds transposable elements in potato and to test the tagging of the wildtype GBSS gene. To activate the Ds element four Ds transposon containing plants were combined with the Ac transposase via cross combination or double transformation. Excision rates ranged from 14.8-48.4 %. Three phenotypic starch mutants were found after screening by iodine staining of tuber cut surfaces. These amylose-free mutants were analyzed by in vitro tests, Southern blot hybridization and sequencing. Strong indications were found that inactivation of the GBSS gene was caused by a transposable element