Isolation, characterization of nac transcription factor induced, during drought stress from rangpur lime and geneti̇c transformation to citrus aurantium rootsrock

Environmental stress factors are known to be the most important factors limiting the growth, production and distribution of all plants in the world. Drought, which is one of the abiotic stress factors, adversely affects the natural balance. The drought, which increases its effect on the world, negatively affects the citrus culivation as it is in other fruit species in Turkey and in the world. Sour orange (Citrus aurantium) rootstock is widely used rootstock in the culivation of citrus in Turkey and the Mediterranean Basin and is only moderately tolerant to drought. Therefore, the development of drought-resistant rootstock is one of the main aims of citrus breeding in Turkey. A partial sequence of a NAC gene induced by drought stress was previously identified form Rangpur lime (Citrus x limonia) rootstock. In this study, the 5' and 3' ends of this NAC gene were determined by RACE method and the full-length gene was isolated from the leaves of drought treated Rangpur lime rootstock. The full-length NAC gene was designated as ClNAC and its expression in response to drought was analyzed in different citrus rootstocks. The expression analysis showed that while the expression of ClNAC gene was increased in Rangpur lime, it was not induced at all in sour orange during 14-day drought treatment. Therefore, the ClNAC gene was transferred to sour orange by Agrobacterium tumefaciens-mediated genetic transformation method to improve drought tolerance of sour orange rootstock. For this, the full-length ClNAC gene was amplified by RT-PCR and cloned between Cauliflower mosaic virus (CaMV) 35S promoter and terminator regions. The gene casset was then cloned into the T-DNA region of pCAMBIA-1304 binary plasmid and transferred to Agrobacterium tumefaciens. The ClNAC gene was transferred to the sour orange (C. aurantium) epicotyl segments grown from seeds using the Agrobacterium-mediated transformation method previously developed and commonly used in citrus transformation. Potential transgenic shoots were regenerated from transformed epicotyl segments by direct organogenesis. The transgenic nature of regenerated shoots were tested by histochemical GUS staining and PCR methods. However, all tested shoots were negative for GUS and PCR tests indicating that no transgenic shoots were obtained. Alhough no transgenic plants were obtained in this study, a drought induced NAC gene was isolated, its expression was analyzed, and the gene was cloned into gene transformation vector. In addition, genetic transformation and regeneration of transgenic sour orange was optimized in this study.