Transformation of Tobacco cpDNA with FusionE7GGG/GUSGene and Homologous Recombination Mediated Elimination of the Marker Gene

A transformation vector harboring the marker gene aadA conferring spectinomycin resistance and the fusion E7GGG/gus gene of interest was employed for the transformation of tobacco cpDNA via biolistics. The gene of interest consisted of the oncogenic E7GGG gene from human papillomavirus strain 16 fused with the reporter gus gene. Both transgenes were equipped with the same promoter and termination sequences arranged as direct repeats. Biolistics with circular vector yielded 20 shoots rooted in spectinomycin containing medium; with linear vector no rooted plants were obtained. After 4 months of in vitro selection the plants were burst into flower in a greenhouse and pollinated with non-transgenic plant pollen. Seeds were harvested individually from each plant capsule and planted onto selection medium. Rare white seedlings were recovered by transfer onto medium without spectinomycin and self-pollinated. GUS activity of 15 seedlings from each self-pollinated plant was measured and 26 plants with highe...