Target lines for recombinase-mediated gene stacking in soybean

Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118 Agrobacterium-mediated transgenic plants that were positive for gus expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1 kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a gfp-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a DsRed-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.