Development of transgenic pigeonpea using high throughput plumular meristem transformation method

Abstract

Tissue culture based poor regeneration along with restricted rooting responses are considered to be major hindrances for in vitro transgenic pigeonpea development. Present study was designed to establish a novel method of Agrobacterium tumefaciens mediated plumular meristem transformation in pigeonpea for improvement of transgenic development frequency. Three days old decapitated seedlings of pigeonpea cultivar ICPL 87119 were pricked at plumular meristem region under in vitro conditions. After infecting with Agrobacterium binary vector pBI121, the explants were co-cultivated in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified- Murashige and Skoog medium. Transformed seedling with well-developed tap root system were established in soil. GUS activity as well as PCR based confirmation of transgene presence was demonstrated in transgenic events. Transformation frequency of 72% was achieved for the first time in pigeonpea. Further, kanamycin mediated stringent selection was used for the screening of T1 seeds. Established T1 progenies were analysed by PCR and Southern blot, to confirm transgene integration and copy number, respectively. This is the first report of transgenic pigeonpea development, where the combination of culture based Agrobacterium-infection and culture independent plant establishment, coupled with PCR based selection method was found to be most preferable for faster and frequent establishment of transgenic plants. This method will contribute to large scale transgenic pigeonpea development for its improvement and satisfy the requirement of routine transformation experiments for T-DNA insertion mutagenesis.