In vitro shoot grafting on rootstock: An effective tool for Agrobacterium-mediated transformation of pigeonpea (Cajanus cajan (L.) Millsp.)

Lack of reproducible in vitro transformation method in pigeonpea limits the application of biotechnological breeding approaches for its genetic improvement. The present study describes a transformation method using novel in vitro shoot grafting technique for two cultivars ICPL87 and ICPL87119. Modified Murashige and Skoog (MS) medium with 1 mg l(-1) 6-benzylaminopurine and 0.2 mg l(-1) alpha-naphthaleneacetic acid induced an average of 25 shoots from decapitated embryonic axis explants after six weeks of culture. These shoots were further elongated in a modified MS medium containing 0.5 mg l(-1) 6-benzylaminopurine along with 0.5 mg l(-1) gibberellic acid for another four weeks. Grafting of pigeonpea shoots to seedling rootstock allowed 95% recovery of shoots. The whole regeneration process, starting from explant preparation to complete plant development, took 12-13 weeks. Further, the explants were infected with Agrobacterium tumefaciens harboring a binary vector pBI121. Transient and constitutive beta-glucuronidase expressions were obtained in putative transgenic shoots selected at 100 mg l(-1) kanamycin. The selected shoots were grafted on non-transgenic root stock to establish putative transformants. T-0 and T-1 transformants were conformed through polymerase chain reaction for presence of neomycin phosphotransferase gene. An overall 9% of transformation efficiency was recorded in both cultivars.