REGENERATION AND AGROBACTERIUM MEDIATED TRANSFORMATION STUDIES IN DIFFERENT TOMATO (Lycopersicon esculentum) CULTIVARS

BÜLBÜL, Yeter

M.Sc., Department of Biology

Supervisor: Assoc. Prof. Dr. Hüseyin Avni ÖKTEM

Co-Supervisor: Prof. Dr. Meral YÜCEL
August 1998, 78 pages

An optimized regeneration protocol and a suitable transformation technique is a necessity for obtaining transgenic plants in a given plant species. This study aims to establish a regeneration and Agrobacterium based transformation system in cultivated tomato (Lycopersicon esculentum). Regeneration studies were carried out on four different tomato cultivars namely WC156, Pearson, H2274 and SC2121. Three different explants, namely aseptically grown 10-20 days old cotyledons, hypocotyls and leaf disks from 2-3 months old soil grown tomato plants were analyzed for their regeneration potencies. All explant types were capable of forming callus in MS based media (MS salts, Gamborg's vitamins, 3% sucrose (w/v), 0.7% agar, pH 5.7) supplemented with 2.5 mg/L benzyl adenine (BA) and 0.2 mg/L indoleacetic acid (IAA). Cotyledon explants from all four types of tomato cultivars formed well-developed shoots in 30-45 days. Shooting response in other explants, on the other hand, showed cultivar dependent variations. Shoots regenerated from cotyledon tissue of all cultivars showed root development in root inducing media (MS salts and vitamins, 3% sucrose (w/v), 0.7% w/v agar and 0.1 mg/L IAA, pH 5.7). Rooted plants transferred to soil and grown to maturity under greenhouse conditions. The entire regeneration process takes about 5-6 months. Transformation studies were carried out on leaf disks, cotyledon, and hypocotyl explants by using Agrobacterium tumefaciens EHA105 strain harbouring the pGUSINT binary vector carrying coding sequences for npt-II (kanamycin resistance) and uida (GUS) genes. Compared to controls, Agrobacterium treated explants exhibited slower callus formation and poor shooting response in the presence of 50 mg/L kanamycin. Only cotyledon explants gave rise to kanamycin resistant callus and positive GUS activity determined by histochemical staining assays.

Keywords: Tomato, Regeneration, Agrobacterium, Transformation, neomycin phosphotransferase-II (npt-II), GUS,