Factors influencing Agrobacterium-mediated transformation of switchgrass cultivars

To optimize Agrobacterium tumefaciens-mediated transformation, factors influencing gene delivery, selection of transformed cells, and plant regeneration were investigated using two major switchgrass cultivars, including a lowland tetraploid cultivar Alamo and an upland octoploid cultivar Cave-in-Rock (CIR). Transient expression studies monitored by histochemical β-glucuronidase assay in seedling segments indicated that A. tumefaciens strain EHA105 was more effective in gene delivery than LBA4404 or GV3101. Of three major selectable genes, the bialaphos resistance (bar) gene and the hygromycin phosphotransferase (hpt) allowed effective selection of transformed cells using 2 mg l−1 glufosinate ammonium herbicide and 50 mg l−1 hygromycin, respectively; whereas the neomycin phosphotransferase II gene did not yield effective selection using 100 mg l−1 kanamycin. Herbicide- or hygromycin-resistant calluses were induced from seedling segments after 2–3 months of selection. Transformants of ‘Alamo’ with the bar or hpt were obtained 3–4 weeks after the resistant calluses were transferred onto regeneration medium; in contrast, no regenerant was produced from the calluses of ‘CIR’. Most of transformants showed normal growth in the greenhouse. Low percentages of mature seeds ranging from 1.7 to 8.7% of husks were obtained from open pollinated plants. Southern blot analysis confirmed stable integration of the bar in selected T0 transformants. Reverse transcription PCR and herbicide/hygromycin tolerance tests indicated expression of transgenes. The optimized transformation protocol using basal parts of seedling as explants shortened the process by 4–5 weeks, and it has potential use for transformation of other switchgrass cultivars.