STUDIES ON THE DEVELOPMENT OF OSMOTIC STRESS RESISTANT WHEAT (Triticum aestivum L.) BY INTRODUCING ALDOSE REDUCTASE GENE THROUGH PARTICLE BOMBARDMENT

Ertugrul (Setenci), Fahriye
Ph.D., Department of Biotechnology
Supervisor: Prof. Dr. Meral YUCEL
Co-supervisor: Prof. Dr. Denés DUDITS

 January 2002, 176 pages

Environmental stresses come in many forms; the most prevalent stresses commonly exert their effects by changing the plant water status. In response to the decrease in water potential caused by drought, low temperature, or high salinity, certain osmoprotectant compounds such as proline, other amino acids and polyamines and hydroxy compounds (sucrose, polyols and oligosaccharides) are accumulated in vascular and nonvascular plants.
Pathways involving these compounds may be exploited to produce stress-tolerant plants by genetic engineering techniques.
Aldose reductase belongs to the aldo-keto reductase superfamily, monomeric NADPH-dependent oxidoreductase with broad substrate specificity, ranging from aldose sugar to aromatic aldehydes. It catalyses the first step of the polyol reaction route; the reduction of D-glucose to sorbitol. It also works as detoxificant for the reduction of toxic aldehydes, generated from the reactions of free radicals and membrane lipids, by converting them into alcohols.
Aldose reductase gene, ALR, isolated from alfalfa (MsALR), was constructed into a plant expression vector, and then named as pAHALR. Wheat immature embryos and suspension cultures were bombarded with this plasmid and transformed immature embryos were cultured on medium containing 10 mg/L Bialaphos or PPT as selective agent. For the molecular analysis of putative transgenic samples, PCR, Southern and Western blots were carried out. Also aldose reductase enzyme activity was shown by HPLC only in transgenic samples by the accumulation of sorbitol, which is the end product of aldose reductase catalyzed glucose reduction reaction. Protective function of ALR was verified by MDA analysis under stress conditions.
Regeneration of transformed calli, which were proven by molecular analysis, is brought to a certain point and still in progress. In the future, we are planning to complete regeneration studies and obtain fully fertile plants. The activity of the gene will also be investigated on these plants by biochemical and physiological analysis.

Keywords: Aldose reductase, osmotic stress, osmotolerance, particle bombardment, wheat tissue culture, sorbitol.