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    Development of transgenic tobacco lines with pyramided insect resistant genes
    (Scientific and Technical Research Council of Turkey, 2018) Bakhsh A.; Dınç T.; Hussain T.; Demırel U.; Aasim M.; Çalişkan M.E.
    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. © TÜBİTAK.
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    Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say)
    (Springer International Publishing, 2019) Hussain T.; Aksoy E.; Çalışkan M.E.; Bakhsh A.
    Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15–80% of CPB mortality. A significantly lower fold-change (0.87–4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87–6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world. © 2019, Springer Nature Switzerland AG.