Expression profiling of Cry1Aabc gene and insect bioassay for Helicoverpa armigera in transgenic pigeonpea

Abstract

The major devastating insect pest of pigeonpea is <em>Helicoverpa armigera</em> which is highly susceptible to the insecticidal proteins of <em>Bacillus thuringiensis </em>(<em>Bt</em>). A codon-optimized chimeric <em>Cry1Aabc </em>gene of <em>Bt</em> driven by a constitutive promoter was introduced in pigeonpea genotype PKV Tara through embryo infection to germinating seeds and floral bud with <em>Agrobacterium</em> strain EHA 105 harboring <em>Cry1Aabc</em> gene. The present investigation of <em>in planta</em> transformation technique was undertaken to confirm and select transformants with higher level of Cry protein expression from segregating population and to study the expression of Cry endotoxin through qualitative and quantitative enzyme linked immunosorbent assay (ELISA) assay. Polymerase chain reaction (PCR) screening with gene specific markers and ELISA assay of 15 different events in T₂, T₃ and T₄ generations was carried out. Out of 220 progenies tested, 209 were positive in PCR and 208 in qualitative ELISA assay. Quantitative ELISA was performed in the selected plant on the basis of higher optical density (OD) and qualitative ELISA performed on different targeted tissue. Concentration of Cry1Ab and Cry1Ac endotoxin was recorded up to 1.347 ng/mg and 0.186 ng/mg of leaf tissue, respectively. Leaf feeding assays with neonate larvae of <em>H. armigera </em>recorded significantly higher mortality rate (89.33%) in positive plants as compared to control. In insect bioassay study it was found that expression level of Cry protein in positive plants was able to reduce the leaf damage to minimal along with mortality of larvae. The positive plants reported in present investigation had potential for providing tolerance against <em>H. armigera</em>.