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Title: Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase

item SOMLEVA, MARIA - Metabolix, Inc
item CHANG, AI - Metabolix, Inc
item RYAN, KIERAN - Metabolix, Inc
item Thilmony, Roger
item PEOPLES, OLIVER - Metabolix, Inc
item SNELL, KRISTI - Metabolix, Inc
item Thomson, James - Jim

Submitted to: BMC Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2014
Publication Date: 8/22/2014
Citation: Somleva, M.N., Chang, A.X., Ryan, K.P., Thilmony, R.L., Peoples, O., Snell, K.D., Thomson, J.G. 2014. Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase. BioMed Central (BMC)Biotechnology. 14:79. doi: 10.1186/1472-6750-14-79.

Interpretive Summary: Switchgrass, a perennial grass native to North America, is an important biofuels crop and is a candidate for improvement using genetic modification. Since this crop is a wind-pollinated outcrossing species, commercialization of genetically engineered switchgrass will require the development of containment methods to reduce transgenic pollen dispersal. To address this issue, we have investigated the use of a rice pollen-specific promoter to express the Bxb1 recombinase mediating the removal of the introduced transgenes. The results indicate that this strategy can be successfully used to perform Bxb1-mediated excision of transgenes from switchgrass pollen. Although it is clear that the containment system will require optimization to achieve highly efficient commercial levels of transgene removal from pollen, this research is an important step towards developing a reliable biocontainment system in switchgrass.

Technical Abstract: Switchgrass (Panicum virgatum L.) has 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 development of this crop as a biorefinery feedstock through genetic engineering. 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, we have demonstrated the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1. A novel approach utilizing cotransformation 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 22-34% depending on the transformation event and assay used. The results presented here mark an important milestone towards the development of a reliable biocontainment system for switchgrass.