Target Lines for in Planta Gene Stacking in Japonica Rice

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded similar to 4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

Automated summary

Target sites were generated in the Japonica rice genome using the Mycobacteriophage Bxb1 site-specific integration system. Single copy lines with target sites that were not close to a centromere, known genes or repetitive DNA, and were able to express the reporter gene were successfully produced. New traits were stacked into Japonica rice through gene transfer and site-specific integration.