A Small Serine Recombination System CinH for Precise Excision of Plastid DNA

Selectable markers are necessary for transgenic plant selection. However, once stable transformants have been isolated, the marker gene is no longer needed. Development of marker removal applications from plastid is an essential step for both environmental safety and recycling of limited markers. In this study, we report plastid marker-gene excision using the small serine recombination system, CinH. Plastid marker precise excision was tested in tobacco plants transformed with the pTCH-RS2 vector, containing the visual marker mCherry gene flanked by directly oriented non-homologous RS2 recognition sites. We demonstrated the precise excision of RS2-flanked mCherry gene from the plastid genome mediated by the recombinase, CinH. Nuclear-transformed transgenic tobacco plants expressing a plastid-targeted CinH recombinase were crossed with transplastomic pTCH-RS2 plants, and the T1 hybrids exhibited 100% efficient excision of the mCherry marker gene. PCR and Southern blot identified approximately 30% of the regenerated T2 plants were recombinase free. In the absence of CinH, transformed plastid genomes were stable and excision events heritable. Thus, the CinH recombination system should prove to be a useful tool for site-specific manipulation of the plastid genome and for generating marker-free plants.