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Öğe An insight into cotton genetic engineering (Gossypium hirsutum L.): current endeavors and prospects(SPRINGER HEIDELBERG, 2015) Bakhsh, Allah; Anayol, Emine; Ozcan, Sancar Fatih; Hussain, Tahira; Aasim, Muhammad; Khawar, Khalid Mahmood; Ozcan, SebahattinCotton (Gossypium hirsutum L.) is the most significant cash crop and backbone of global textile industry. The importance of cotton can hardly be over emphasized in the economy of cotton-growing countries as cotton and cotton products contribute significantly to the foreign exchange earnings. Cotton breeders have continuously sought to improve cotton's quality through conventional breeding in the past centuries; however, due to limited availability of germplasm with resistant to particular insects, pests and diseases, further advancements in cotton breeding have been challenging. The progress in transformation systems in cotton paved the way for the genetic improvement by enabling the researchers to transfer specific genes among the species and to incorporate them in cotton genome. With the development of first genetically engineered cotton plant in 1987, several characteristics such as biotic (insects, viruses, bacteria and fungi) resistance, abiotic (drought, chilling, heat, salt), herbicide tolerance, manipulation of oil and fiber traits have been reported to date. Genetic engineering has emerged as a necessary tool in cotton breeding programs, strengthening classical strategies to improve yield and yield contributing factors. The current review highlights the advances and endeavors in cotton genetic engineering achieved by researchers worldwide utilizing modern biotechnological approaches. Future prospects of the transgenic cotton are also discussed.Öğe 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, SebahattinIn 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.Öğe 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, SebahattinThe 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.Öğe Molecular Characterization of Snowdrop Lectin (GNA) and its Comparison with Reported Lectin Sequences of Amaryllidaceae(CZECH ACADEMY AGRICULTURAL SCIENCES, 2016) Khabbazi, Saber Delpasand; Bakhsh, Allah; Sancak, Cengiz; Ozcan, SebahattinPlant lectins have become efficient sources of insect resistance in crops. The present study was conducted to identify, amplify, clone and characterize the plant lectin gene GNA. The lectin, present in Galanthus nivalis (snowdrop), is an agglutinin toxic to hemiptera. The attempt was made to elucidate the relationship of the lectin gene trGNA (GNA isolated and characterized from Turkey) with other previously cloned lectins having insecticidal activity and to ensure the presence of the conserved mannose-binding region/site in the gene sequence. The full-length cDNA of trGNA was 477 bp that contained a 333 bp open reading frame encoding 157 amino acid proteins with 23 amino acids of signal peptide. BLAST results showed that trGNA has 89-97% similarity with previously reported GNA sequences while it has 84-96% similarity with earlier reported GNA protein sequences. No intron was detected within the region of genomic sequence corresponding to trGNA full-length cDNA. According to the search results from the NCBI (National Centrer for Biotechnology Information database), trGNA from Galanthus nivalis is most similar to the previously reported lectin sequences of Narcissus tazetta with a similarity percentage of 87%. The obtained results are useful for engineering of plants with enhanced insecticidal activity against chewing and sucking insects, causing crop pests. In addition, medical application of lectins may also be considered.Öğe 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, SebahattinMost 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.