Tryptophan and indole analog mediated plastid transformation.

A nonantibiotic/herbicide-resistance selection system for plastid transformation is described here in technical detail. This system is based on the feedback-insensitive anthranilate synthase (AS) α-subunit gene of tobacco (ASA2) as a selective marker and tryptophan (Trp) or indole analogs as selection agents. AS catalyzes the first reaction in the Trp biosynthetic pathway, naturally compartmentalized in the plastids, by converting chorismate to anthranilate and is subjected to feedback inhibition by Trp. In addition to Trp, various Trp analogs and indole compounds that can be converted to Trp analogs can also inhibit AS activity and therefore are toxic to cells. When cells are made to express the feedback-insensitive ASA2, they acquire resistance to these analogs and can be selected for during transformation process. We have demonstrated the feasibility of this selection system in tobacco (Nicotiana tabacum L. cv. Petit Havana). ASA2-expressing transplastomic plants were obtained on medium supplemented with either 7-methyl-DL-tryptophan (7-MT) or 4-methylindole (4-MI). These plants show normal phenotype and fertility and transmit the resistance to the selection agents strictly maternally.