Regeneration of glyphosate-tolerant Nicotiana tabacum after plastid transformation with a mutated variant of bacterial aroA gene

Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics faces a major hurdle for finding efficient and safe candidates. Until now، herbicide tolerance genes seem to be attractive alternatives. In this study، a variant form of 5-enoylpyruvyl shikimate-3-phosphate synthase (EPSPS) gene that harbors G96A and A183T substitutions and confers higher resistance against broad-spectrum herbicide، glyphosate، was replaced with spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. A previous study showed that، glyphosate herbicide imposes lethal effects on the structure and integrity of plastids membrane، even in low concentration. In order to overcome this problem، we used an altered procedure for selection of transplastomic cells. Long preculture incubation followed by slowly increased glyphosate concentration let sufficient expression of the transgene and tolerant calli were regenerated through direct selection of transformed plastids in the presence of the glyphosate.