Precise excision of plastid DNA by the large serine recombinase Bxb1

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP- and attB-flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH-PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear-transformed transgenic tobacco plants expressing a plastid-targeted Bxb1 recombinase were crossed with transplastomic pTCH-PB plants, and the T₁ hybrids exhibited efficient excision of the target sequence. The Bxb1-att system should prove to be a useful tool for site-specifically manipulating the plastid genome and generating marker-free transplastomic plants.