Effect of hygromycin on tomato cultures and its application in Agrobacterium- mediated tomato transformation

摘要

Development of an efficient protocol for genetic transformation in plants requires effective shoot regeneration and antibiotic selection systems. Hygromycin and cefotaxime sensitivity studies were conducted using leaf and petiole explants of tomato (Solanum lycopersicum cv. Solan Vajr) to explore the aptness of hygromycin resistance as a selectable marker and cefotaxime in controlling excessive bacterial growth during genetic transformation studies. A decrease in fresh weight of explants was observedwith increase in the concentration of hygromycin resulting in full or partial inhibition of shoot regeneration. The concentration of 25mg/l resulted in complete necrosis of the explants within two weeks of culturing which was further used for genetic transformation studies of tomato for the selection of transformed cells using Agrobacterium- mediated gene transfer system. Transformed callus lines along with the nontransformed callus were analyzed by the pcr amplification, which demonstrated 100 % hygromycin selection efficiency,'whereas, only 40.0% of transformants contained both the hpt and chi 11 (chitinase) genes, indicating that chil 1 gene was eliminated in 60.0% hpt- positive lines during T-DNA transfer and integeration into the plant genome.