Development and characterization of Bt pigeonpea (Cajanus cajan L. Millspaugh) plants for resistance against Helicoverpa armigera (Hübner)
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Date
2014
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CCSHAU
Abstract
Pigeonpea [Cajanus cajan (L) Millsp.] is an important protein rich legume crop grown in many semi arid tropical regions of the world. Development of transgenics in pigeonpea remains dogged by poor plant regeneration in vitro from transformed tissue and low frequency transformation protocols. Transgenic pigeon pea plants of variety Manak (H 77-216) expressing cry1Ac gene were generated by tissue culture independent, rapid and efficient, transformation method using Agrobacterium tumefaciens strain LBA4404,
harboring pBin-35Scry1Ac (Kharb et al., 2012). Putative transformants were screened at early stage by subjecting small leaf pieces to PCR amplification using plant direct PCR kit and transformation frequency of 45.0 % was obtained. Quantitative ELISA of 60 days old T0, transgenic plants showed expression of Cry protein ranging from 153.5ng/g – 562.5ng/g FW of leaf. Of the 374 seeds harvested from all the T0 plants, 324 were sown to obtain progeny (T1) plants and 233 of these were randomly analyzed to determine their transgenic nature. Out of the 233 T1 plants 125 amplified cry1Ac gene, substantiating the integration and
transmission of the transgene in the transgenic pigeon pea plants. Gene integration was also checked in T2 plants by direct plant PCR The transformed (T1) plants genomic DNA showed single copy of cry1Ac gene integration by Southern hybridization and different transformation events were observed. Single copy for each transgenic event was also confirmed by real time PCR. Insect bioassay proved its usefulness over control plants for pod borer resistance. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and offers immense potential for generating transgenic plants of difficult-to-regenerate pigeon pea.
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Keywords
planting, transgenics, pigeonpeas, genes, genetic processes, dna, pcr, transgenic plants, genetics, animal developmental stages