Regeneration and transformation of an elite inbred line of maize (Zea mays L.), with a gene from Bacillus thuringiensis

AbstractTissue culture conditions for regeneration of fertile plants from immature embryos were optimized using four local inbred lines of Zea mays. Immature embryos were cultured on five different media. The highest regeneration efficiency of 86.40% was observed for BR-6 at N6 modified media (supplemented with 1.0 mg L−1 2,4-D, 25 mM L-Proline, and 100 mg L−1 Casein Hydrolysate) while B-46, EX-285 and EX-295 gave a regeneration percentage of 57%, 77%, and 63%, respectively. A total number of 1646 immature embryos were bombarded by a gene gun using tungsten micro projectiles coated with a multi-gene plasmid pCMAC, containing gusA (reporter gene), hpt (hygromycin phosphotransferase), and cry1Ac genes driven by maize ubiquitin promoter. Regenerates were selected on a medium containing 40 μg/L hygromycin and then screened with GUS assay. Thirty-five transgenic lines were obtained with a transformation frequency of 2.21%. The integration and expression of cry1Ac was confirmed with PCR, Southern Blot analysis, ELISA, and Western Blot analysis. The studies confirmed that cry1Ac gene has been stably transformed into the local inbred line (BR-6) and caused 80–100% mortality to armyworm (Spodoptera frugiperda) in insect feeding bioassay