Studies On Agrobacterium-Mediated Transformation Of Apple Rootstock M.26 With MdNHX1 Gene

Stress from environment, especially from the salinization, is the most important limited factor for plant growth. Recently, several transgenic plants, which exhibit remarkably enhanced salt resistance by constitutive expression Na+/H+ antiporter genes, raised the glory perspective to obtain salt resistance crop plants by using this tactic. The function of NHX gene isolated from fruit tree plants and the NHX gene transgenic fruit tree plants had not been reported up to date. In present research, MdNHX1, a vacuolar Na+/H+ antiporter gene (MdNHX1) was isolated from Luo-2, a salt tolerant apple rootstock(Malus domestica Borkh.), and was introduced into apple rootstock M.26 by Agrobacterium-mediated transformation. The MdNHX1 gene expression, copy number and several physiological events variation of transgenic apple rootstock M.26 lines under salt stress were analyzed. All results mentioned above were useful for the utilization of MdNHX1 gene to transform other fruit tree plants. The main results were as follows:1. Leaf of apple rootstock'M.26'was used in this study. Different combinations of plant growth regulators were studied to induce the adventitious buds. The result showed that the best medium for inducing adventitious buds was MS+4.0 mg/L6-BA+0.2mg/L NAA +30 g/L sucrose +7.0 g/L agar (pH5.8). And the regeneration efficiency on this medium was 96%.2. Agrobacterium-mediated transformation of leaves from apple rootstock'M.26'was conducted and 15 kanamycin-resistant plantlets were obtained.3.15 kanamycin-resistant lines obtained in this research were assayed by PCR, of which 11 lines were MdNHX1-positive and nptII-positive. When the transgenic plants were assayed by Southern blot,6 of the 11 MdNHX1-positive lines were positive. The Results demonstrated that exogenous gene had successfully integrated into the genomic DNA of apple rootstock M.26.4. Real Time PCR analyse results unfolded the existence of difference expression level among separated transgenic lines, and these differences were more obviously when the salt stress treating time was prolonged. N7 line had the highest and stable expression level of MdNHXl gene.5. Under different NaCl stress conditions, several physiological events variation of transgenic plants were studied. The results showed that the salt tolerance ability was enhanced at different degree in transgenic plants. Compared to control plants, Na+ and K+ contents were significantly increased in roots of transgenic plants; Na+ content was slightly decreased in leaves of transgenic plants. These demonstrated that the salt tolerance ablility was enhanced by genitic transformation.