| Alfalfa (Medicago saliva L), known as "king of forage", is a highly valued forage legume that is cultivated on more than 35million hectares in the warm temperate and cool subtropical regions all over the world. Alfalfa most likely produces a greater yield of protein and essential amino acid per hectare than any other crop. The most fining area to the growth of Alfalfa in china'is the Yellow River regions and northwest of china. Alfalfa is not adapted to south of china because of the rainy weather and acidic soil. Many people want to migrate Alfalfa to south of china from the cool subtropical region, because of its important feeding values to animals. Aluminum toxicity is the main problem in acidic soil. So it is very important for Alfalfa to improve its aluminum tolerance ability in order that it can grow better in south of china. It has been achieved in aboard to improve plant aluminum tolerance ability by gene engineering technique. In this experiment, we want to improve Alfalfa aluminum tolerance ability via introducing aluminum simulated protein gene into Alfalfa. First we established a highly effective regeneration and transformation system and introduced one gene associated to aluminum resistant to improve the Alfalfa aluminum tolerance ability, which will be benefit to immigrate Alfalfa to south of china. The main results are as follows: 1 .Establishing the highly effective regeneration systemThe highly regenerative genotype was screened from the four Alfalfa varieties, SHEGNSH1804, CUF101. BADDING and WL52540. The effective regeneration protocol was established. BAODING has the highest regenerative ability of the four varieties. The cotyledon of BAODING was incubated on UM media to induce embryogenic callus, and then the embrogenic callus were transferred to UM liquid media to suspend 1-2 weeks, embryogenic callus suspension were transferred to SHE embryo developmental medium, the mature embryoies were selected and incubated on the 1/2SHGN medium to recover intoplantlet. The whole regeneration progress can be achieved within 3-4 months. 2.Establishing the highly effective transformation system.The genotypes apt to transformation were screened from the six Alfalfa varieties. BAODING, CLIF 101, WL52540 are the genotypes apt to transformation. The factors affecting transformation rate, including the genotype of explant, the state of the explant, the state of the Agrobacterium tumefaciens , co-cultural time, were investigated using GUS transient expression technique. In this way, the Agrobacterium-mediated transformation system was established taken leaves as acceptor via vacuum infiltration. And a highly effective transformation system was also established taken embryogenic callus as acceptor. The transient transformation rates can be achieved almost 50%, via transforming embryogenic callus suspension mediated by Agrobacterium tumefaciens. The transgenic plant can be obtained for about four month.3.Introducing one aluminum tolerance associated gene into Alfalfa and doing molecular analysis to the transgenic plantThe GhAlin gene, which was cloned from cotton, was introduced into Alfalfa mediated by Agrobacterium tumefaciens. The leaves and roots of transgenic plant can be stained into blue employing GUS historical stain technique. The same size fragment of interested gene can be emplicated from the transgenic plant genome by PCR analysis, which shows the target gene has transferred into plant genome. 4.Doing the aluminum tolerance experiment to analyze the role of the geneThe two-week-old cuttings were placed in hydroponic chamber filled with 3L of 0.5 mM CaCl2 in deionized water, pH4.3. After the roots reached 3~5cm, the solution was replaced with 3L of preconditioning solution containing 0, 25, 50, 100umol/L AlCl3 and 0.5 mM CaCl2, at pH.4.3. Root length was measured before and after 1d and 7d of AICl3 treatment, respectively. We used the relative root growth as a parameter to elicit the aluminum tolerance ability of transgenic plant. It shows the transgenic plant roots treated with...
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