Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system

Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre- loxP site- specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid- targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation ( Transformation Protocol), by pollination ( Pollination Protocol) or from a transient, non- integrated T- DNA ( Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10 - 16 months, depending on which protocol is used.