Agrobacterium tumefaciens-mediated transformation of big bluestem (Andropogon gerardii Vitman)

Big bluestem (Andropogon gerardii Vitman) is native to the Great Plains of North America and is an important warm-season forage and ornamental grass. Recently it has been proposed as one of the more ideal biomass producers for cellulosic ethanol production due to its high biomass yield and low input needs. A robust genetic transformation protocol is needed for germplasm improvement or functional genomics studies in big bluestem. In the present study we report, for the first time the establishment of a reproducible Agrobacterium-mediated transformation protocol for big bluestem. Mature seeds of a big bluestem cultivar Bison were cultured for producing embryogenic calli, which were then used for infection by the Agrobacterium tumefaciens strain AGL1 harboring either the binary vector pTF101.1, which carries the glufosinate resistance (bar) gene or a modified binary vector pMDC32 which carries the hygromycin resistance gene (hpt). Glufosinate ammonium at 6 mg L-1 or hygromycin B at 250 mg L-1 was used effectively to select for resistant callus followed by lowered concentrations of both during regeneration and rooting. Based on PCR test alone or in combination with herbicide leaf painting, the overall transformation frequency was 2.8-3.6 % by using the pTF101.1 vector and 2.4-3.3 % by using the modified pMDC32 vector, both of which are comparable to transformation frequencies reported for other grasses. PCR test showed that all Basta(A (R))-resistant plants following the leaf painting contain the bar gene and subsequent Southern blot analysis confirmed that the all tested transgenic plants resulted from independent transgenic events.