FLP-mediated site-specific recombination for genome modification in turfgrass

To develop molecular strategies for gene containment in genetically modified (GM) turfgrass, we have studied the feasibility of using the FLP/FRT site-specific DNA recombination system from yeast for controlled genome modification in turfgrass. Suspension cell cultures of creeping bentgrass (Agrostis stolonifera L.) and Kentucky bluegrass (Poa pratensis) were co-transformed with a FLP recombinase expression vector and a recombination-reporter test plasmid containing β-glucuronidase (gusA) gene which was separated from the maize ubiquitin (ubi) promoter by an FRT-flanked blocking DNA sequence to prevent its transcription. GUS activity was observed in co-transformed cells, in which molecular analyses indicated that FLP-mediated excision of the blocking sequence had brought into proximity the upstream promoter and the downstream reporter gene, resulting in GUS expression. Functional evaluation of the FLP/FRT system using transgenic creeping bentgrass stably expressing FLP recombinase confirmed the observation in suspension cell culture. Our results indicate that FLP/FRT system is a useful tool for genetic manipulation of turfgrass, pointing to the great potential of exploiting the system to develop molecular strategies for transgene containment in perennials.