Master of Science (MS)


Plant Pathology and Crop Physiology

Document Type



I investigated the effect of co-cultivation temperature and binary Ti vectors on the fresh weight yield of leaf disks of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith) after Agrobacterium tumefaciens-mediated transformation. I concluded that the temperature at 20ºC during co-cultivation of leaf disks with A. tumefaciens was the most critical factor on the reproducible enhancement of fresh weight yield. The highest fresh weight increase and presumably transformation efficiency was achieved under the following experimental conditions: A. tumefaciens strain LBA4404 containing pLSU binary Ti vector was grown overnight at 28oC in liquid Agrobacterium medium and the concentration was adjusted to 3x107 cells/ml (0.3 A600 units/ml). Leaf disks were co-cultured at room temperature with bacteria for 60 min in the liquid MS medium containing 200 M acetosyringone and 0.001% (w/v) Silwet L-77 without antibiotics. Pretreated disks were co-cultivated for 4 days under constant light at 20C in solid MS shoot medium containing 200 M acetosyringone without antibiotics. Co-cultured leaf disks were selected for 14 days under constant light at 25C on MS shoot selection medium containing 300 mg/l kanamycin and 500 mg/l carbenicillin. Selected leaf disks were transferred to freshly prepared selection medium for an additional 14 days under constant light at 25C. Fresh weight yields were measured at the end of a 28 day growth period. Of the four tobacco cultivars tested, Xanthi (nn Smith) was found to be sensitive to kanamycin concentrations at 100 mg/L and above, and hygromycin concentrations at 25 mg/L and above. I compared five co-cultivation temperatures from 15, 18, 20, and 22 to 25oC during the 4-day period and found the temperature at 20oC promoted the highest increase in fresh weight yield. I also tested four new binary Ti pLSU vectors containing the plant-expressible kanamycin resistance gene, and four new pLSU vectors containing the hygromycin resistance gene with or without the β-1, 4-Glucuronidase (GUS) gene. New pLSU vectors were similar to conventional pCAMBIA vectors in promoting the increase in fresh weight yield.



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