| Salt stress negatively impacts agricultural yield throughout the world affecting production whether it is for subsistence or economic gain. The United Nations Environment Program estimates that approximately 20 % of agricultural land and 50 % of cropland in the world is salt-stressed. Modern molecular genetics and plant transformation advances have made salt stress feasible to assess biotechnological strategies based on engineered biosynthetic pathways, targeted gene or protein expression or alteration of the natural stress responsiveness of genes for development of salt tolerant crops. Previous works with model transgenic plants have demonstrated that cellular accumulation of mannitol can alleviate salt stress. The rationale behind this strategy is that mannitol can stabilize proteins and cellular structures and can increase the osmotic pressure of the plant cell under salt stress.In order to test this hypothesis in peanut, transgenic peanut (Arachis hypogaea L. cv. Shanyou 523) to express the mtlD gene (encoding mannitol-1-phosphate dehydrogenase) for the biosynthesis of mannitol via genetic transformation mediated by Agrobacterium tumefaciens and the pollen-tube pathway were engineered in this reasearch. Furthermore, the exogenous total DNA ofKandelia candel L. Druce and Oenothera litlaralis Schlect were also introduced into the peanut by the pollen-tube pathway technique.The main results and achievements contained in this thesis are as follows: 1. Established a highly efficient genetic transformation system using the peanut leaf as starting material. In this system, leaf explants were preconditioned for 3 days on BM medium [Murashige and Skoog (MS) salts basal medium supplemented with 30 g/L sucrose, 2 mg/L AgNO3, 6 mg/L Glutamine and a hormone combination of 0.8 mg/L onaphthaleneacetic acid (NAA) and 3 mg/L 6-benzyladenine (BA) solidified with 0.7 % agar, pH 5.8], followed by co-cultivation with A. tumefaciens EHA105 carrying binary vector pBIMTL, the latter of which contains a mtlD gene and a neomycin phosphotransferase (ATT/7) gene, on BM medium for 2 days in the dark. Then the explants were washed three times with steriled water and placed onto BM mediumcontaining 80 mg/L neomycin and 500 mg/L carbenicillin. After 5-6 weeks, the explants with buds were transferred to EM medium [MS basal medium supplemented with 30 g/1 sucrose, 2 mg/L AgN03 and 3 mg/L BA solidified with 0.7 % agar, pH 5.8] with 80 mg/L neomycin and 500 mg/L carbenicillin for the elongation of buds. 3-4 weeks after cultured, 2- to 3-cm-high elongated shoots were excised and planted on RM medium [MS basal medium supplemented with 30 g/L sucrose, 2 mg/L AgNOa , and 0.8 mg/L NAA solidified with 0.7 % agar, pH 5.8] with 40 mg/L neomycin for rooting. When the roots occurred, about 3 weeks later, the plantlets were planted in sand for further growth.2. The transformed T0 generations obtained through A. tumefaciens-medialed transformation above, were tested positive for mtlD genes by polymerase chain reaction (PCR) analysis. Results of PCR-Southern blot also confirmed that the mtlD gene was integrated into the peanut genome. The transformation rate was 5.3 % in this research.3. Plasmid pBlMTL, which contained mtlD gene and NPTH gene, was also employed as donor and introduced into peanut after self-pollination by pollen-tube pathway. PCR analysis and PCR-Southern blot of the transformed TO, TI, T2 generations also indicated the mtlD gene v/as integrated into the peanut genome successfully. The transformation rate was 5.9 % through this method. The germination rates of transgenic mtlD peanut were increased 21.9 % compared with control plants.4. The exogenous total DNA of Kandelia candel L. Druce and Oenothera littaralis Schlect were also introduced into the peanut through the pollen-tube pathway technique. The salt tolerance experiment results showed that the seeds from transgenic plants have better germination than control plant in the 140 mmol/L NaCl medium. The germination rates of transgenic peanut were e... |
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