Studies On The Conservation Of Cell And Tissue In Glycyrrhiza Inflata Batal

Glycyrrhiza inflata Batal is a very significant officinal plant in Chinese traditional medicine. For the past few years, the resource of this valuable plant has been reduced sharply because of the excess exploiting. With the advanced biotechnologies, large-scaled culture of Glycyrrhiza inflata Batal cells has showed its advantages, such as no restrict of climate or locations, etc. But this proved method has its defect, in the process of cultural passage there is some aberrance which might reduce its effective components. The solution for the problem is cell and tissue conservation. This paper focuses on the method research about the conservation and cryopreservation of cell and tissue of Glycyrrhiza inflata Batal, and on the research of the vitrification reagent mechanism. The results are as follow:1. 2, 3, 5-triphenyl chloride tetrazolylazo is the appropriate regent of determine the activity of dehydrogenase. The reaction is in the phosphate buffer condition, pH 7.6, the appropriate extraction regent of TF is acetone. This optimized method is precise and repeatple.2. The proportion of culture media and the conservation temperature are key facts to conservation process. With 1/2 MS+30 g/L sucrose as the culture medium, the Glycyrrhiza inflata Batal. culture cell can conserve 6 months under 4℃away form light. After 6 months, the culture is no longer keep on growing and its effective components are reduced.3. The only proper method of the long-term cell conservation is cryopreservation. The most efficient cryopreservation methods is vitrification. The results are: the material of the experiment is 14 d culture cell, preculture 2 d in MS+1.2 g/L sucrose culture medium, treated with 25% PVS2 at room temperature, then treated with 100% PVS2 at 0℃, launch into LN, 37℃bath thawing rapidly after 48 h, washed with MS+60 mol/L sucrose culture medium thrice, determine the activity of dehydrogenase with 2, 3, 5-triphenyl chloride tetrazolylazo, the survival rate of Glycyrrhiza inflata Batal suspension culture cell was 82.9% by TTC examination, and regeneration rate reached 80.4%.4. The research has confirmed the mechanism of the vitrification reagent. After treated with vitrification reagent, some changes will take place in membrane permeability, the content of unsaturated fatty acids, soluble protein and thermal stable protein. These factors have some associate with the survival rate.