PJB-2023-270
GENE TRANSFORMATION PROTOCOL TO DEVELOP GENETICALLY ENGINEERED FODDER BY USING BETA-GLUCURONIDASE GENE
Saima Shafique
Abstract
Switchgrass (Panicum virgatum L.) is used as fodder for livestock due to high nutritional value and it has potential to be grown in cool and warm conditions. Switchgrass breeding is difficult due to the high degree of self-incompatibility hence, grass production can be increased through gene transformation. The research focused to develop protocols for the regeneration and gene transformation of forage grass. To enhance the callus and regeneration efficiency of switchgrass through Agrobacterium-mediated transformation. Switchgrass callus was transformed with 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 and to enhance the process, 100 µM acetosyringone was added in co-cultivation media. Pre-selection of transformed callus were carried for seven days with 150 mg/L of cefixime. The switchgrass transgenic callus was shifted to selection media containing hygromycin (50 mg/L). Histochemical staining is used to evaluate the presence of the Gus gene in the hygromycin-resistant transgenic plants. The successful transformation of switchgrass, opens up opportunities for gene function analysis and germplasm improvement through the application of biotechnology.
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