An open-source system for in planta gene stacking by Bxb1 and Cre recombinases

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the 'x' number of transgenic loci into a single genome may seem trivial, (¼) (x) for a diploid species, but given the 'y' number of other nontransgenic traits that breeders also need to assemble into the same genome, the (¼) (x+y) probability for a 'breeding stack' could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific integration was mediated by the mycobacteriophage Bxb1 integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of lox sites. Site-specific integration occurred in ~10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of ~3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since neither the Bxb1-att nor the Cre-lox system is under patent, there is freedom to operate.