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Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l⁻¹ kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.
Hairy root disease is caused by the infection of wounded higher plants with Agrobacterium rhizogenes. Transformation of tissues or plants with A. rhizogenes, and with rol genes, as well as hairy roots may produce alterations in the plant secondary metabolism. H₂O₂ and other ROS are involved as a signal in secondary metabolite production pathway and play a key role in plant defensive reactions. In this work, the effect of A. rhizogenes T-DNA on nicotine content, antioxidant enzymes activity, H₂O₂ production, pattern of peroxidase (POX) and superoxide dismutase (SOD) isozymes in hairy roots and regenerated plants were studied. Rise in SOD and POX activities in the transformed lines of TRa and TRb and in the resultant regenerated plants, also the decreased level of H₂O₂ in them, compared with the untransformed lines indicates that, the T-DNA genes expression of A. rhizogenes probably decreases H₂O₂ level by increasing the production of antioxidant enzymes. Decrees the level of H₂O₂ content in TRc line in spite of the similarity of antioxidant enzyme activity in comparison with normal root, indicate that A. rhizogenes activate other mechanisms except SOD and POX enzyme for reducing H₂O₂ level.
To understand alfalfa (Medicago sativa L.) reactions to osmotic stress, solutions with -0.5, -1 and -1.5 MPa osmotic potentials using PEG (Poly ethyleneglycol) and distilled water as control were prepared. In a germination test, eleven alfalfa cultivar seeds were allowed to germinate in these solutions. M. sativa cv. Yazdi and M. sativa cv. Gharayonje, selected as tolerant and sensitive cultivars, respectively, and were used for further studies. In all PEG solutions, root and shoot dry weights decreased in both cultivars. Under different levels of osmotic stress, root to shoot ratio increased significantly in Yazdi, whereas this parameter showed no significant differences in Gharayonje. Yazdi cultivar also showed higher activities of SOD (Superoxide dismutase), APX (Ascorbate peroxidase), CAT (Catalase), POD (Peroxidase), and higher reducing sugar contents of leaves in comparison with Gharayonje. These higher antioxidant activities help the tolerant cultivar to decrease oxidative damages of osmotic stress to membrane lipids as compared with its sensitive counterpart. As a result, electrolyte leakage and the amounts of MDA (Malondialdehyde), were higher in Gharayonje. This study highlights the importance of enzymatic and non-enzymatic antioxidant systems in scavenging reactive oxygen species which is caused by osmotic stress. It is seems that antioxidant systems are more active in tolerant cultivars than those of sensitive ones.
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