ISOLATION, MOLECULAR CHARACTERIZATION OF Bacillus thuringiensis AND DEVELOPMENT OF TRANSGENIC FIELD BEAN (Lablab purpureus L. Sweet) AGAINST POD BORER.
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Date
2007-09-19
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University of Agricultural Sciences GKVK, Bangalore
Abstract
Bacillus thuringiensis (Bt) produces a variety of insecticidal crystal proteins (ICPs) upon sporulation. These proteins, called δ-endotoxins, are highly toxic to lepidopteran, dipteran and coleopteran insects. Different ICP genes of Bt have been successfully engineered into many crop-plants to obtain resistance against lepidopteran insects. The field bean (Lablab purpureus L.) grown largely in dry land conditions, is an important pulse crop that suffers from heavy pest damage. The pod borers, Helicoverpa armigera and Adisura atkisoni are two important pests that cause enormous loss. The present investigation lays emphasis on isolation, molecular characterization of Bacillus thuringiensis strains and development of transgenic field bean (Lablab purpureus L.) with Cry 1F gene against pod borer (Helicoverpa armigera). The soil samples used for isolation of Bt were collected from different parts of south India. Thirty nine Bt isolates were obtained form the fifty soil samples. All these isolates were found to be gram positive, endospore forming and crystalliferous. PCR amplification of the isolates using different primers showed that twenty four isolates were lepidopteran-specific, ten specific to coleopteran and four active against nematode. The analysis of crystal proteins of these isolates by SDS-PAGE revealed that majority of the isolates had proteins of size 120 kDa and above. Twenty four of the isolates which proved to be lepidopteran-specific were observed to be lethal to silkworm. Attempts were made to standardize an efficient regeneration protocol for field bean. Five varieties were used for the study among which selection 321 was found to perform better. Transformation of field bean was carried out using Cry 1 F gene by Agrobacterium-mediated method. Confirmation of transgene in putatively transformed plants was done by PCR analysis using Cry 1F gene specific primer. Five out of twenty five transformed plants were found to be transgenics.
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