Marker-free site-specific gene integration in plants

For nearly 15 years, the use of site-specific recombination systems in plants has focused on the deletion or integration of DNA. Each of these applications offers practical solutions to two problems in biotechnology: the presence of unneeded DNA in the transgene locus and variation in the locus structure among independent transgenic lines. Given that each of these separate applications is becoming more practical for commercial use, it is time to consider combining them into an integrated technology. Here we propose a strategy for a "combined step" method that makes use of two site-specific recombination systems: one for integrating the DNA and a second for removing sequences that are not needed after DNA transfer. This strategy is based on published data and exemplifies the use of the Cre-lox, FLP-FRT and R-RS inducible systems.