Bxb1-att Site-Specific Recombination System-Mediated Autoexcision to Prevent Environmental Transgene Escape

Transgenic plants are obtained experimentally with low frequency. Scientists use selectable marker genes (SMGs) and selection agents to select transformed cells from mostly untransformed cells in the production of transgenic plants. The SMG is usually an antibiotic or herbicide-resistance gene. However, the presence of SMGs in GM plants and subsequently in food, feed, and the environment has raised concerns from regulatory agencies and the public. In recent years, several strategies have been deployed to remove SMGs from GM plants. This chapter describes a case study highlighting the Bxb1-att site-specific recombination system for SMG removal in tobacco plants. Case study includes development-induced autoexcision cassette designed to delete both the SMG and the bxb1 gene with a seed promoter derived from common bean Phaseolus vulgaris phaseolin (phas) gene to drive bxb1 expression. GUS-positive T0 lines transferred to soil for setting T1 seeds. T1 progeny and their T2 generation are also obtained for study. Bxb1-mediated autoexcision events are identified in T1 seeds and T1 and T2 plants through junction PCR analysis. Sequencing confirmed successful excision events. Chimeric plants containing both excised and intact T-DNA were observed in both T1 and T2 independent lines. However, two homogenous SMG- and bxb1-free T2 lines were also obtained.