Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T transformants were selected on the basis of PCR and protein expression profile. T events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T, T and T). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

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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. T 0 and T 1 transformants were confirmed through polymerase chain reaction for presence of neomycin phosphotransferase gene. An overall 9% of transformation efficiency was recorded in both cultivars.

Vascular wilt caused by Fusarium udum Butler is the most important disease of pigeonpea throughout the world. F. udum isolate MTCC 2204 (M1) inoculated pigeonpea plants of susceptible (ICP 2376) and resistant (ICP 8863) cultivars were taken at invasion stage of pathogenesis process for transcriptomic profiling to understand defense signaling reactions that interplay at early stage of this plant–pathogen encounter. Differential transcriptomic profiles were generated through cDNA-AFLP from M1 inoculated resistant and susceptible pigeonpea root tissues. Twenty five percent of transcript derived fragments (TDFs) were found to be pathogen induced. Among them 73 TDFs were re-amplified and sequenced. Homology search of the TDFs in available databases and thorough study of scientific literature identified several pathways, which could play crucial role in defense responses of the F. udum inoculated resistant plants. Some of the defense responsive pathways identified to be active during this interaction are, jasmonic acid and salicylic acid mediated defense responses, cell wall remodeling, vascular development and pattering, abscisic acid mediated responses, effector triggered immunity, and reactive oxygen species mediated signaling. This study identified important wilt responsive regulatory pathways in pigeonpea which will be helpful for further exploration of these resistant components for pigeonpea improvement.

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