Evaluation of parameters affecting the genetic engineering of recalcitrant switchgrass (Panicum virgatum Roxb. ex Steud.) using GUS reporter gene system

Switchgrass (Panicum virgatum ) is used as a fodder for livestock due to its high nutritional value; it has potential to grow in cool and warm conditions as well as in low fertile soil. Switchgrass breeding is difficult due to its high degree of self-incompatibility; hence, grass production can be increased through cell and tissue culture. The present research focused on developing protocols for the regeneration and gene transformation of the forage grass to enhance the callus and regeneration efficiency through Agrobacterium -mediated transformation. The switchgrass callus was transformed with the pCAMBIA1302 vector carrying beta-glucuronidase (GUS) reporter gene, and 42% transformation efficiency was attained. To ensure a successful transformation, explants were infected with Agrobacterium for five minutes, following co-cultivation for two days. To enhance the process, 100 µM acetosyringone was added in co-cultivation media. Pre-selection of transformed callus was carried out for seven days with 150 mg/L of cefixime. The switchgrass transgenic callus was shifted to a selection medium containing hygromycin (50 mg/L). Histochemical staining was used to evaluate the presence of the GUS gene in the hygromycin-resistant transgenic plants. The successful transformation of switchgrass opens opportunities for gene function analysis and germplasm improvement through the application of advanced biotechnological tools.