PHAGE RECOMBINASES TO OBTAIN MERKER-FREE TRANSPLASTOMIC CROPS

Natural containment due to the lack of pollen transmission makes plastid localization of transgenes an attractive alternative to nuclear gene transformation. Genetically stable transplastomic crops can be obtained only by selection for antibiotic resistance genes. Associated yield penalty and consumer opposition dictates that the marker gene is removed from future transplastomic crops. We developed a two-stage protocol for the removal of marker genes using site-specific recombinases. First we transform the plastid genome with marker genes flanked with site-specific recombinase target sites. When transformation of the plastid genome is accomplished, we introduce the gene of a site-specific recombinase into the nucleus. This nuclear gene encodes a plastid-targeted recombinase that efficiently excises the plastid marker gene. While the plastid marker gene is efficiently excised, the recombinase gene is introduced into the nucleus and has to be segregated away in the seed progeny. We now test the feasibility of plastid marker excision by direct export of the Cre recombinase enzyme from Agrobacterium into chloroplasts. When the recombinase enzyme rather than its gene is transferred from Agrobacterium to the plants cell, there is no opportunity for the incorporation of recombinase gene in the nucleus. Direct export of proteins for marker gene excision will facilitate the applications of plastid transformation in vegetatively propagated crops (potato, apple trees) in which seed propagation is incompatible with variety preservation.