Development of transgenic tobacco lines with pyramided insect resistant genes

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.