Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system

Incorporation of a selectable marker gene during transformation is essentia l to obtain transformed plastids. However, once transformation is accomplis hed, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-Iox site-specific recombination system for the el imination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented fox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in ti ssue culture cells was frequently accompanied by a large deletion of a plas tid genome segment which includes the tRNA-Val(UAC) gene. However, the larg e deletions were absent when cre was introduced by pollination. Thus pollin ation is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transfor mation. The nuclear cre gene could subsequently be removed by segregation i n the seed progeny. The modified CRE-Iox system described here will be a hi ghly efficient tool to obtain marker-free transplastomic plants.