Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA

Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH-MRS and pTCH-RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear-genome transformed tobacco plants expressing plastid-targeted ParA and CinH recombinases, respectively. One hundred per cent of both types of F₁ hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F₂ plants showed that approximately 30% (CinH-RS2) or 40% (ParA-MRS) had lost the recombinase genes by segregation. The postexcision transformed plastid genomes were stable and the excision events heritable. The ParA-MRS and CinH-RS2 recombination systems will be useful tools for site-specific manipulation of the plastid genome and for generating marker-free plants, an essential step for reuse of SMG and for addressing concerns about the presence of antibiotic resistance genes in transgenic plants.