Construction of transposon tagging system in germinal cells for creating homozygous mutants

Transposon tagging is an effective tool of insertional mutagenesis for studying gene function. A higher number of independent insertion mutants can be obtained from a few transgenic launch pads via germinal transposition. Our lab previously developed a one-time inducible Ac/Ds transposable system, COKC, where the Ds element in the intron of the Ac transposase is driven by the salicylic acid inducible promoter PR-1a (Pathogenesis related-1a). In order to increase germinal transposition and generate homozygous mutants, we treated floral tissues at uninucleate stages of microspore in COKC transgenic rice with salicylic acid in two salicilylic acid treatments. The first approach was pot induction, where rice panicle was induced in the pot prior to anther culture. The other approach was culture induction, where the rice anther was isolated first and then simultaneously induced and incubated. The transposition efficiency of anther-derived regeneration shoots from the two sets were 5 % and 20 %, respectively. For pot induction, all of the regeneration calli were homozygotes. In contrast, for culture induction, most of the regeneration calli were heterozygotes, which may result from somatic transposition after embryogenesis of the callus. In order to increase germinal transposition, the present study aims to construct three novel germinal transposon tagging systems OsP1-0380PL, OsP41-0380PL and OsP128-0380PL. In these systems, the Ac transposase is driven by each of the three pollen specific promoters identified in rice cultivar TNG67. The above tagging constructs were transformed into rice and tobacco through Agrobacterium. After that, the transformants were subjected to anther culture to observe the frequency of transposition in gametophytic process, where more homozygotes could be derived. In conclusion, the aim of this thesis is appling to improve germinal transposition to create stable homozygous mutants in a single generation in plants.