Transformation of tobacco with a NAC type transcription factor, tanac69-1 and characterization of transgenic plants via molecular and physiological techniques

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2015
Eroğlu, Ayten
Environmental stresses such as drought and salinity greatly affect plant production leading to reduction in yield and quality. Stress tolerant plants can be rapidly produced by transferring stress related genes or transcription factors to crop plants. In this study, TaNAC69-1 gene, a NAC type transcription factor, was isolated from Triticum aestivum L. cv. Yüreğir-89 and cloned into a Gateway compatible overexpression vector, pEarleyGate 100. Transformation of Nicotiana tabacum L. cv. Samsun leaves was carried out via Agrobacterium-mediated transformation method. A total of fourty five T0 putative transgenic tobacco lines were obtained at the end of independent transformation experiments. Stable expression and integration of TaNAC69-1 gene into the tobacco genome were evaluated by molecular, biochemical and physiological analysis. Traditional PCR was made by using gene specific and bar gene specific primers to screen transgenes. Stable integration of TaNAC69-1 gene into the genome was also confirmed by Southern blot analysis. vi Expression of TaNAC69-1 gene was examined by qRT-PCR with different time periods under salt and drought stress conditions using actin as a reference gene. It was found that drought and salt treatments led to significant up-regulation of TaNAC69-1 gene expression. Physiological assays such as germination, root and shoot lengths, fresh and dry weights were achieved to compare stress tolerance of wild type and transgenic tobacco lines under salt and osmotic stress conditions. Germination and survival rates of transgenic tobacco seeds were significantly higher than wild type seeds when subjected to NaCl and LiCl treatment. Root and shoot lengths, fresh and dry weights of transgenic plants significantly increased when compared to wild type plants with application of NaCl and LiCl. On the other hand there was no difference between growth parameters of wild type and transgenic lines with mannitol treatment. According to molecular and physiological analysis, overexpression of TaNAC69-1 gene increased salt and ionic stress tolerance of transgenic tobacco plants. Further experiments should be done with cold and heat stress treatments to better elucidate the function of TaNAC69-1gene under abiotic stress conditions.
Citation Formats
A. Eroğlu, “Transformation of tobacco with a NAC type transcription factor, tanac69-1 and characterization of transgenic plants via molecular and physiological techniques,” Ph.D. - Doctoral Program, Middle East Technical University, 2015.