Method for Bxb1-Mediated Site-Specific Integration In Planta

Gene targeting in plants through homologous recombination has been sparsely reported, although notable breakthroughs have been achieved in recent years (1 ). In particular, the use of zinc finger nucleases to promote homologous end joining has revived the promise that homologous gene targeting could someday become practical for plant genetic engineering (2, 3 ). An alternative and complementary approach that has progressed steadily over the years has been recombinase-mediated site-specific integration (4 ). In this approach, a first recombination site is introduced into the genome to serve as a target for inserting subsequent DNA. Here, we describe the method for generating the chromosomal target and the subsequent insertion of new DNA into the chromosomal target by Bxb1-mediated site-specific integration. This method would permit the comparison of different molecular constructs at the same genomic locations.