GENETIC MANIPULATION OF TOBACCO AGAINST OSMOTIC STRESS VIA PROLINE OVERPRODUCTION

Eyidogan Inci, Fusun
Ph.D., Department of Biotechnology
Supervisor: Prof. Dr. Hüseyin Avni Öktem
Co-Supervisor: Dr. Laszlo Szabados

June 2001, 184 pages

Most plants grow in environmental conditions that are to a considerable degree, unfavourable for plant growth. One of the most commonly induced responses in all organisms undergoing water deficit is the production and/or accumulation of so called compatible osmolytes. In plants proline accumulation has been well correlated with tolerance to salinity and drought. Therefore, genetic manipulation of plants towards proline overproduction might enhance their tolerance against salt and drought stress. The aim of the present study was to develop osmotolerant transgenic tobacco plants via metabolic engineering for proline overproduction. For this purpose, coding sequences of Arabidopsis thaliana D1-pyrolline-5- carboxylate synthetase (AtP5CS) the key enzyme of proline biosynthetic pathway in plants, and/or a proline feedback insensitive mutant of the same gene that was obtained by random mutagenesis experiments was transferred to tobacco via Agrobacterium tumefaciens mediated gene transfer system. T0, T1, and T2 progeny of transgenic tobacco plants carrying the wild type AtP5CS gene were analysed for stable integration of transgenes, segregation patterns and osmotolerance under salt and drought stresses at seedling level and under greenhouse conditions. Compared to the controls most transgenic lines exhibited a higher proline accumulation, germination rate, lowered MDA content, better root development and capsule formation under salt and drought stress conditions. Furthermore, a remarkably higher proline accumulation (up to 8 fold) with respect to control was observed in transgenic tobacco plants that were transformed with the mutated AtP5CS gene. Taken all together, our results demonstrate an enhanced osmotolerance in transgenic plants engineered for proline overproduction.

Keywords: Proline, D1-pyrolline-5-carboxylate synthetase, P5CS, Osmotic Stress, Osmotolerance, Transgenic tobacco.

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