Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations

The efficient production of stable transgenic plants is important for both crop improvement and functional genomics. Site-specific integration of foreign genes into a designated genomic position is an attractive tool for minimizing expression variability between transgenic lines. Here, we studied the utility of a Cre-mediated, site-specific integration approach, facilitated by particle bombardment, for streamlining the production of stable transgenic plants, using rice as a model species. Using this method, we generated 18 different transgenic lines containing a precise integration of a single copy of beta-glucuronidase gene (gusA) into a designated genomic location. Eleven of these lines contained no illegitimate integration in the background (single-copy lines), and seven contained illegitimate integrations in addition to the site-specific integration (multicopy lines). We monitored gusA expression in these lines up to three to four successive generations. Each of the single-copy lines expressed the gusA gene at consistent levels and nearly doubled the expression level in the homozygous state. In contrast, multicopy lines displayed expression variation and gene silencing. In about half of the multicopy lines, however, expression of the site-specific integration locus could be reactivated and stabilized on segregation of the illegitimate integrations, whereas, in the remaining half, expression could not be restored, as they contained genetically linked illegitimate integrations. This study demonstrates that biolistic-mediated, site-specific gene integration is an efficient and reliable tool for streamlining the production of stable transgenic plants.