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    Antifeedant and stomach poison effects of different plant extracts to Spodoptera littoralis (Lepidoptera: Noctuidae)
    (ENTOMOLOGICAL SOC TURKEY, EGE UNIV, 2013) Karakoc, Omer Cem; Gokce, Ayhan
    Antifeedant and stomach poison effects of 9 plant extracts were tested on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Antifeedant effects of plant extracts varied from 22.44% to 68.96% and the most active plant extracts were Delphinium consolida (%68.96), Chrysanthemum segetum (62.67%), Artemisia vulgaris (61.09%) and Tanacetum mucroniferum (55.87%). Dose-response studies with the extracts showed that all extracts produced strong antifeedant effects ranging from 68.91% to 82.20% at 2 mg/cm(2) dose, D. consolida (48.92%) and T. mucroniferum (45.02%) also exhibited antifeedant properties at dose as low as 0.5 mg/cm(2). In stomach posion effects studies, C. segetum was the most toxic extract with %74.24 mortality. It was followed by D. consolida and T. mucroniferum with 70.53% and 67.04% mortality, respectively. Dose-mortality studies with C. segetum showed that it was the most toxic extract with 0.35 mg/cm(2) LC50 and 3.85 mg/cm(2) LC90 values.
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    DEVELOPMENT OF INSECT-RESISTANT COTTON LINES WITH TARGETED EXPRESSION OF INSECTICIDAL GENE
    (INST BIOLOSKA ISTRAZIVANJA SINISA STANKOVIC, 2016) Bakhsh, Allah; Anayol, Emine; Khabbazi, Saber Delpasand; Karakoc, Omer Cem; Sancak, Cengiz; Ozcan, Sebahattin
    In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T-0 progeny were further raised under greenhouse conditions to obtain progeny (T-1). The introduced gene was properly inherited and expressed in T-1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized.
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    Inducing osmotic stress leads to better genetic transformation efficiency in cotton (Gossypium hirsutum L.)
    (TUBITAK SCIENTIFIC & TECHNICAL RESEARCH COUNCIL TURKEY, 2016) Barpete, Surendra; Bakhsh, Allah; Anayol, Emine; Ozcan, Sancar Fatih; Oguz, Muhammet Cagri; Karakoc, Omer Cem; Ozcan, Sebahattin
    The present study investigated the effect of different salts on cotton shoot regeneration and transformation efficiency. Two-day-old germinating embryos of a local cotton cultivar (SG-125) were pretreated with 50 mM each of NaCl, CaCl2, and KCl for 60 min. The embryo explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pTF101.1 that carried the insecticidal gene (cry1Ac) under control of wound-inducible promoter (AoPR1) and bilanafos acetyl reductase (bar) gene for plant selection. The salt-pretreated embryos showed maximum response on regeneration MS medium containing 0.50 mg/L 6-benzylaminopurine (BAP) and 0.10 mg/L indole-3-butyric acid (IBA), also supplemented with 5 mg/L bialaphos for in vitro screening of the transformed plantlets. The primary transformants were further screened by molecular techniques for integration and expression of the introduced gene. Maximum transformation efficiency (1.10%) was noted on KCl-treated explants compared to nontreated (control) explants. In conclusion, pretreatment of explants with 50 mM KCl for 60 min induced positive effects and triggered shoot regeneration in primary cotton transformants.
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    Targeted expression of insecticidal hybrid SN19 gene in potato leads to enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata Say) and tomato leafminer (Tuta absoluta Meyrick)
    (Springer, 2017) Ahmed, Hussein Abdullah Ahmed; Onarici, Selma; Bakhsh, Allah; Akdogan, Guray; Karakoc, Omer Cem; Ozcan, Sancar Fatih; Aydin, Gulsum
    The expression of insecticidal genes must be induced at appropriate time and in sufficient amount to confer protection against targeted pests. However, the increased scientific reports of resistance development in insect pest against insecticidal delta-endotoxins, produced by Bacillus thuringiensis, provide impetus for the development of alternative insect management strategies. The present study was conducted to investigate the importance of targeted expression of a hybrid insecticidal gene (SN19) in potatoes. For this purpose, two plant expression vectors were constructed by cloning hybrid SN19 gene (cry1Ba-domain I-III and cry1Ia-domain II) under the control of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1) and CaMV 35S promoter, and were transferred to Agrobacterium tumefaciens strain EHA 105. Four potato genotypes (Marabel, Innovator, Tokat 10/1 and Tokat 6/24) were transformed with EHA 105 strain harboring pTF101.1 35S-SN19 and pTF101.1 AoPR1-SN19 constructs. Phosphinothricin (PPT) was used at concentration of 1 mg/l for selection of primary transformants. PCR results showed the presence of both introduced SN19 and bar genes in 43 plants out of total 154 putative transgenics. Expression of SN19 protein in primary transformants was confirmed by Western blot assays. The mechanical wounding of transgenic plants exhibited more accumulated levels of SN19 proteins during post wounding period. Leaf biotoxicity assays with Colorado potato beetle (Coleoptera) and tomato leafminer (Lepidoptera) exhibited 100% mortality of the pests in primary transformants. Based on our mortality results with both constructs, we concluded that the potato transgenic lines exhibited targeted expression of insecticidal gene under the control of AoPR1 promoter upon insect wounding with eliminated toxicity of Cry protein and hence can be further used effectively in potato breeding programme.
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    Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton
    (SPRINGER, 2016) Anayol, Emine; Bakhsh, Allah; Karakoc, Omer Cem; Onarici, Selma; Kom, Deniz; Aasim, Muhammad; Ozcan, Sebahattin
    Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept 'less intrusive' expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L-1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T (1) transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.