Transformation Of Citrate Synthetase Gene From Tobacco Into Medicago Sativa L. Mediate By Agrobacterium Tumefaciens

Alfalfa (Medicago sativa L.) is one of perennial leguminious forage. It's awarded the best grazing grass for its high yield, good quality, abundant nutrition, high economic value. In china, alfalfa mainly grows in the north. With the quick development of pasturage industry in the south of China today, the need for alfalfa becomes more and more. Accordingly, there has a wide perspective for developing alfalfa production in the south. However, there were some bad factors such as high temperature, rainy climate and acid soil, which hinder the growth of alfalfa in the south. The toxicity of aluminum to alfalfa is a main problem in acid soil. Throwing lime and organic fertilizer as traditional paths to relieve the toxicity of aluminum to plant, but they cost too much to manipulate effectively. Therefore, cultivation of a new breed with resistant aluminum is the key to introduce alfalfa in the south. However, It's not notable that the effect of traditional cultivation technology on improving the aluminum tolerance of alfalfa. In recent years, the development of molecular biology brings along a novel thought for solving this problem.This research intend to transfer the citrate Synthetase gene of being tobacco into alfalfa by agrbacterium-mediated genetic transgenic method. The expression of the exgenetic gene could strengthen the synthesis and exudation of citrate in alfalfa accordingly. Exudation of citrate from root of alfalfa would relieve aluminum toxicity in acid soil, which could enhance the adaptability of alfalfa growth in the south, and make beneficial search for introducing. At present, there has been acquired main results as follows.Optimization of regeneration system on alfalfa in the south China. The experimental material was a new line which has been bred in the south for many years and possessed good adaptability in local area. For decrease abnormal shooting in used regeneration system, we adjusted the concentration rate between auxins in callus inducement phase, and the suitable culture medium for callus inducement was determined as SH+2,4-D 3.0mg/L +KT 0.6mg/L, which reduced the formation of abnormal shooting. In addition, we found lamina excel to petiole in phases of callus inducement and differentiation after different explants be taken as experimental material.Determination of the appropriate stress for resistant Km and filtration way to Km in culture medium. Prophase filtration to Km as appropriate way was determined by examination. Besides, we determined 10mg/L and 25mg/L Km as optimal stress in callus inducement phase and differentiation phase respectively.Establishment of genetic transformation system on the alfalfa. At the base of the agrobacterium mediated genetic transformation, we optimized every approach of genetic transformation to the alfalfa. The optimal preculture time of explant was 2 days. The optimal culture time of agrobacterium tumefaciens was 20-22 hours. The suitable OD₆₀₀ of engineering bacterial suspension was 0.4.The suitable time for explants to be infected by agrobacterium was 10-15 mins. The effective result could be obtained when co-culture time was 2 days. In addition, we found a way that axenic water including 1000mg/L Car wash the explant after the phase of co-culture could reduce pollution of explant caused by agrobacterium tumefaciens in the experience.Obtain of genetic transformation plant and identification. After filtrated culture, we obtained 23 resistant lines. It was proved by PCR examination that target gene CS has been transferred into the alfalfa. One positive line was obtained. It was displayed further by RT-PCR examination that the CS gene was of a certain expression level in the positive plant. After enzyme activity analysis for lamina and tolerance examination for root in transgenic line respectively, the CS activity of lamina in the transgenic line was higher than in the comparison. Besides, it was showed that the tolerance of the transgenic plant to aluminum toxicity was higher than the comparison by examination after dealing their roots with 10μmol/L and 15μmol/L AlCl₃.