Transformation of Pigeonpea with Different Cry Genes to Impart Pod Borer Resistance

Abstract

The present investigation was carried out to generate transgenic pigeonpea newlinecarrying different cry genes, characterization and identification of superior transgenic newlineevents and pyramid different cry genes to increase the pod borer resistance. In planta newlinetransformation using embryonic axis as explants resulted in 65 putative transformants newlinecarrying cry1Ac and 17 carrying cry2Aa. For quick identification of transgenics in T1 newlinegeneration, optimization of kanamycin concentration for sand based screening was newlineattempted using seeds from non transgenic plants and kanamycin concentration of 100 newlineppm was found to inhibit the seedling growth in non transgenic plants. Screening of newlineseeds from primary transformants on 100 ppm kanamycin did not show direct newlinecorrelation between the presence of the transgene and normal growth of the seedlings. newlineTherefore progenies of primary transformants were screened individually for the newlinepresence of transgene through PCR assay. newlineIn transgenic plants carrying cry1Ac larval mortality ranged from 0 to 92.3 per newlinecent and it was 20 to 70 per cent in plants with cry2Aa. In the transgenic plants Cry1Ac newlinecontent ranged from 0.54 to 0.93and#956;g/g and Cry2Aa ranged from 0.04 to 1.45and#956;g/g. There newlinewas significant reduction in both larval weight and larval length when fed with newlinetransgenic plants compared to non transgenic plants. Southern hybridization revealed newlinesingle copy integration in selected transgenic events. Site of integration of cry1Ac was newlineconfirmed in two events using Thermal Asymmetric Interlaced (TAIL) PCR which newlineindicated that for event number 191-5 integration is between 175604-175605bp and for newline190-26 is between 8775857-8775858 bp. newlinePyramiding of cry genes in different combinations viz., cry1Ac + cry2Aa, newlinecry1Ac + cry1F, cry1F + cry2Aa and cry1F + cry1Acm resulted in increased larval newlinemortality compared to the plants carrying single cry genes. Of the four different newlinecombinations tried cry1F + cry1Acm gave highest mortality of 86.67 per cent. newline