Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase

Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated.

A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used.

The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.