Transposon tagging is a powerful technology for studying gene function; however, somatic transposition prior to gametogenesis may result in multiple derivatives bearing the same insertion. In order to attempt to increase germinal transposition, we synthesized two novel transposon-tagging constructs. We placed the transposase gene under control of either of two microspore-specific promoters identified in Arabidopsis or Antirrhinum within the Ds element that also harbored kanamycin resistance. Both transposase and NPTII gene sequences were optimized in silico based on codon usage bias data in dicot species. Agrobacterium-mediated transformation was used to insert the constructs into diploid potato. The transgenic... More
Transposon tagging is a powerful technology for studying gene function; however, somatic transposition prior to gametogenesis may result in multiple derivatives bearing the same insertion. In order to attempt to increase germinal transposition, we synthesized two novel transposon-tagging constructs. We placed the transposase gene under control of either of two microspore-specific promoters identified in Arabidopsis or Antirrhinum within the Ds element that also harbored kanamycin resistance. Both transposase and NPTII gene sequences were optimized in silico based on codon usage bias data in dicot species. Agrobacterium-mediated transformation was used to insert the constructs into diploid potato. The transgenic plants were subjected to both anther culture and outcrossing to wild type diploid potato to observe the opportunity for transposition in either gametophytic process. Green fluorescent protein screening was not efficient but kanamycin selection was able to eliminate 94 % of wild type progenies. Transposants were recovered following anther culture of two transgenic lines whereas no transposants were found among more than 250 T1 seedlings. Anther culture provided a unique venue for the observation of the influence of gametophye specific promoters on transposition in potato.
