title: Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.
abstract: 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. T0transformants were selected on the basis of PCR and protein expression profile. T1events 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 T1transgenic 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 T2generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3plants 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 (T0, T1,and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.
authors.affiliation: Department of Biotechnology, St. Xavier's College (Autonomous), 30, Park Street, Kolkata, 700016, West Bengal, India.
authors.forename: Gourab
authors.initials: G
authors.lastname: Ghosh
authors.affiliation: Department of Biotechnology, St. Xavier's College (Autonomous), 30, Park Street, Kolkata, 700016, West Bengal, India.
authors.forename: Shreeparna
authors.initials: S
authors.lastname: Ganguly
authors.affiliation: Department of Biotechnology, St. Xavier's College (Autonomous), 30, Park Street, Kolkata, 700016, West Bengal, India.
authors.forename: Arnab
authors.initials: A
authors.lastname: Purohit
authors.affiliation: Department of Botany, Krishnagar Govt. College, Krishnagar, 741101, West Bengal, India.
authors.forename: Rituparna Kundu
authors.initials: RK
authors.lastname: Chaudhuri
authors.affiliation: Division of Plant Biology, Bose Institute, P1/12 C.I.T. Scheme VII M, Kankurgachi, Kolkata, 700054, West Bengal, India.
authors.forename: Sampa
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Biotechnology, St. Xavier's College (Autonomous), 30, Park Street, Kolkata, 700016, West Bengal, India. dipankar_12@yahoo.co.in.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chakraborti
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D036561:Cajanus / Q000235:genetics / Q000378:metabolism* / Q000469:parasitology*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D009036:Moths / Q000472:pathogenicity*; D010572:Pest Control, Biological / Q000379:methods
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Gene pyramiding; In vitro shoot grafting; Insect bioassay; Multiple shooting; Pigeonpea transformation