A bacterial mannitol-1-phosphate dehydrogenase (mtlD) gene driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter was transferred into tomato plants using an Agrobacterium tumefaciens-mediated transformation protocol in an attempt to improve abiotic stress tolerance in the transformed plants. Transgene integration was confirmed by PCR analysis and Southern blot analysis, and transgene expression was confirmed by reverse transcription (RT)-PCR and direct mtlD (EC 1.1.1.17) activity. Upon exposure to low temperature stress (4°C) in a cold chamber, transgenic plants survived up to 48 h, while nontransformed plants were unable to survive and gradually died. Transgenic plants subjected to the chilling stress showed a significant decrease in electrolyte leakage and increased lipid peroxidation, as assessed by measuring malondialdehyde (MDA) content. Under the cold condition, transgenic plants also showed a significant increase in the activities of antioxidant enzymes (superoxide dismutase and catalase) and in relative water content (RWC) in comparison to non-transformed plants. Drought (polyethylene glycol in medium) and salinity (sodium chloride in medium) tolerance tests revealed that transgenic lines exhibited a higher tolerance for abiotic stresses than non-transformed plants. These findings indicate that the introduction of a bacterial mtlD gene into tomato conferred tolerance to abiotic stresses to the transformed tomato plants.

Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore, experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic cotransformation. 6-benzylaminopurine at 0.05 mg l -l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets in indole-3-aceticacid 0.5 mg l -l . Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent.