Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas

Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg's B-5 medium containing 3 % (w/v) sucrose, 3 mg/l kinetin and 1 mg/l indole-3-butyric acid. More than 90 % of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2 d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200 mu M acetosyringone. Putative transformants were selected using media containing 15 mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56 % of the explants were transformed with an average of 3.4 +/- 0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.