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. 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.

Assessment of Pigeonpea (Cajanus cajan L.) transgenics expressing Bt ICPs, Cry2Aa and Cry1AcF under nethouse containment implicated an effective control against herbivory by Helicoverpa armigera (Hübner)

“…45,46 A major impetus for the mitigation of this pest in pigeonpea has been transgenesis due to the lack of availability of resistance sources in the cultivated germplasm. 47,48 Studies accruing from the literature, [14][15][16][17][18][19] including the work from our group, 15 have demonstrated the feasibility of curtailing this pest in pigeonpea using different Bt ICPs under laboratory conditions. However, there has not been a single report demonstrating the performance of these transgenic events under soil conditions.…”

“…The overall leaf damage ranged between 5 and 20% and demonstrated that most of the transgenic plants were effective in resisting the insect pest compared to wild type plants. Based on stringent evaluation of the transgenic plants for their efficacy against H. armigera under two different situations in the nethouse, ten transgenic pigeonpea events (cry2Aa events-7, 10,11,13,14;22,24,25,6) were shortlisted and selected as superior. Comparison of efficacy against herbivory versus the amount of cry protein in each of the events in both the experiments showed that leaf damage ranged between 9% and 27% in the soil experiment and 3% and 26.7% in the pot experiment ( Fig.…”

“…Bacillus thuringiensis ( Bt ) insecticidal proteins (ICPs) that encode a delta endotoxin have proved to be the most suitable and safe alternative to chemical pesticides. The major emphasis of our group has been in the development of transgenics in pigeonpea for mitigation of the pod borer H. armigera using a tissue culture‐independent in planta transformation strategy . Several other research groups have also produced transgenic pigeonpea with the development of diverse Bt genes .…”

“…The major emphasis of our group has been in the development of transgenics in pigeonpea for mitigation of the pod borer H. armigera using a tissue culture-independent in planta transformation strategy. [11][12][13][14][15] Several other research groups have also produced transgenic pigeonpea with the development of diverse Bt genes. [14][15][16][17][18][19] The ability of transformed plants to perform against insect pressure might vary between laboratory and soil conditions.…”

An efficient in vitro regeneration protocol to generate stable transgenic lines of Black gram (Vigna mungo)

“…Prolonged incubation of shoots in root induction media resulted in the ageing which had a negative effect on normal growth and seed yield (Ghosh et al 2017). Though there are reports of various direct rooting protocols in black gram (Saini et al 2002;Muruganatham et al 2005;Harisaranraj 2008;Adlinge et al 2014), those protocols lack detailed information about the number of roots produced per explant and root length.…”