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

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

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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 α-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 β-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. T0 and T1 transformants were confirmed through polymerase chain reaction for presence of neomycin phosphotransferase gene. An overall 9% of transformation efficiency was recorded in both cultivars.

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