Marker Free Plants using Bxb1-Mediated Site-specific Recombination Driven by a Seed-specific Promoter

An important tool for the production of GM crops is the selectable marker gene (SMG), which allows for the identification of a few transformed plants from among the bulk of non-transformed plants. The SMG, usually an antibiotic or herbicide-resistance gene, remains in the genome of GM crops. Several strategies have been employed in plant genetic transformation to remove SMGs, including site-specific recombination (SSR) systems. The mycobacteriophage Bxb1 SSR system has been used in plant transgenesis to excise SMGs. The objective of this research is to use Bxb1, a uni-directional SSR system, to excise the SMG and render it unable to reinsert into the genome of the tobacco plant. The Bxb1 recombinase is codon-optimized to express in plants and is driven by a tissue-specific seed promoter. The binary vector was designed to allow the SSR system to delete both the SMG and the recombinase-coding region from the genome of the tobacco plant. The vector was transformed into tobacco, and T0 putative transgenic plants were obtained. GUS-positive T0 lines were transferred to soil for setting T1 seeds and used for excision analysis. Bxb1-mediated excision was preliminarily identified in T1 seeds, and T1 plants through junction PCR analysis. Sequencing has confirmed successful excision results.