Generation of marker-free plastid transformants using a transiently cointegrated selection gene

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Abstract
Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops1, the possibility of marker transfer to wild relatives2 or microorganisms3 cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain4. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination5, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system6,7, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation8 in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker9. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.