Bacillus thuringiensis CryIA Protein Levels in Raw and Processed Seed of Transgenic Cotton: Determination Using Insect Bioassay and ELISA

The levels of the Bacillus thuringiensis Berliner variety kurstaki CryIA(b) and CryIA(c) insecticidal proteins in unprocessed (raw) and processed seed of transgenic cotton were evaluated using insect bioassay and enzyme-linked immunosorbent assay (ELISA) procedures. Larvae of the tobacco budworm, Heliothis virescens (F.), were exposed to sublethal concentrations of ground cottonseed or purified CryIA proteins incorporated into artificial insect diet. The dose-response function of treatments was fit using nonlinear regression analysis to calculate EC50 (dose reducing larval growth by 50%) values and 95% CIs. The bioassay detection limit for both CryIA(b) and CryIA(c) proteins was 0.5 ng/ml insect diet. At 0.5 ng/ml, H. virescens larval weight was consistently reduced by ≥9% compared with the associated control weight. ELISA analyses of CryIA(b) and CryIA(c) protein in cotton seed provided data similar to bioassay results. Cotton line 81 [CrylA(b)] contained 11.28 µg/g (bioassay) and 14.00 µg/g (ELISA) active protein in raw seed. Line 531 [CryIA(c)] contained 2.89 µg/g (bioassay) and 2.22 µg/g(ELISA) active protein in raw seed. Processing eliminated ≥97% of the active CrylA(b) and CryIA(c) proteins in the transgenic cottonseed.