Switchgrass (Panicum virgatum L.) pollen-mediated gene flow and transgene bioconfinement strategies

Switchgrass (Panicum virgatum L.) is a native North American grass under development as a bioenergy feedstock. Genetic engineering has been employed to introduce transgenic traits for economical biofuel production. However, potential transgene movement from switchgrass raises regulatory and environmental concerns. Experimental studies are needed to determine the potential for transgene flow from switchgrass. The aims of this research are to estimate pollen dispersal and pollination distances for transgenic switchgrass, and evaluate genetically engineered male sterility as a transgene bioconfinement strategy. A three-year field experiment was performed to estimate switchgrass pollen dispersal and pollination distances using a modified Nelder wheel design. Switchgrass (cv. ‘Alamo’) plants, with whole plant and pollen orange fluorescent protein expression (OFP), were planted as a pollen source. Pollen traps and pairs of nontransgenic Alamo 2 pollen-receptor plant clones were positioned every 10 m extending to 20 m, 30 m, 30 m, and 100 m respective to the north, south, west, and east (direction of the prevailing wind). The finding of the study suggested that switchgrass pollen dispersal is inversely related to distance, and pollination can readily occur up to 100 m (maximum distance measured). To examine transgene bioconfinement, a proof-of-concept study was designed and tested in the model plant tobacco (Nicotiana tabacum L.). Targeted expression of the EcoRI endonuclease was driven by the tomato pollen-specific LAT52 promoter leading to selective male sterility the elimination of viable transgenic pollen. Nine EcoRI transgenic events were produced with normal morphology, and EcoRI expression was specific to pollen and negligible in other tissues. Bioconfinement efficacy was examined in glasshouse experiments by hand-crossing transgenic plants to nontransgenic male-sterile and emasculated tobacco. The results demonstrated at or near 100% transgene bioconfinement for one or more generations. Transgenic lines were further examined in field trials where EcoRI tobacco plants were placed in plots with a male-sterile plants. Once again, transgene bioconfinement was observed at or near 100% in four EcoRI lines. These results suggest EcoRI-driven male sterility is a safe and effective approach for transgene bioconfinement, and this strategy should be translatable to switchgrass as well as other plant species