Production of Marker-free Transgenic Tobacco Plants by FLP/ frt Recombination System

Abstract

Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/ frt site-specific system of 2-μm plasmid from Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target ( frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/ frt site-specific recombination system was constructed, which included the frt-containing vector pCAMBIA1300- betA-frt-als-frt and the FLP expression vector pCAMBIA1300- hsp-FLP- hpt. The FLP recombinase gene was introduced into transgenic ( betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat-shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41 % re-transgenic tobacco plants, which indicated that this system could make a great contribution obtaining the marker-free transgenic plants.