Protoplast Culture Of Siraitia Grosvenorii

Siraitia grosvenorii is an important economic plant grown in Guangxi locally. Its fruit can be used as Chinese traditional medicine directly. There are plentiful fructose, glucose, protein and variety of vitamin in its fruit. The fruit has been used as a folk medicine for sore throats, cough, minor stomach and other troubles. Now, it has been used for drinking and condiment. The sweet triterpenoid glycosides in its fruits may serve as a sweet substance for those who suffered from diabetes, adiposity and hypertension. At present, S.grosvenorii varieties are facing problems of degeneration and hybridization and needed improvement. It has been known that cultivating and breeding protoplast is an effective way of improving variety qualities. So far no one have studies the culture of the protoplast of S.grosvenorii. We discuss about isolate the protoplast of varied materials in different levels of concentration of ferment and research on the influence of types of sugar and hormone and the concentration on the formation of callus. The thesis uses the leaves of S.grosvenorii cultivated outdoor, the leaves of aspectic tube plantlet and the branch of aspectic-tube plantlet as materials, and studies the conditions of isolation and culture of protoplast. And the results are as followed. The cultured leaves of S.grosvenorii. are sterilized and then the skin is stripped. The branch of aspectic-tube plantlet is cut into small pieces which are soaked in 0.4M mannitol. In 2% cellulase and 1% pectolyase and other ferments fluid stabilizer, under the condition of 28±1℃and in dark, ferment has been resolved for 3~4 hours; the leaf of aspectic-tube plantlet is cut into a stripe which is 2mm in length and 1mm width and is soaked in 0.4m ferment fluid stabilizer. In 4% cellulase, 1% pectolyase and ferment fluid of other elements, under the condition of 28±1℃and in dark, 50rpm vibrating ferment and 6-7hr can resolve lots of vigorous protoplast. Adding 50mg/L ascorbic acid into ferment fluid can raise the living percentage of protoplast by 12.5%. Concerning the division time and frequency of culture protoplast, the leaves of cultivated S.grosvenorii is better than the aspectic tube plantlet for protoplast isolation, the solid-liquid dual layer culture is better than thin layer culture and LMET embedding culture. Because the protoplast of S.grosvenorii is sensitive to NH4+, protoplasts could be cultured in MS basic culture medium in which NH4NO3 is eliminated. The suitable plating density is 1×105个/mL. Medum supplemented with 2mg/L 2,4-D, 5mg/LBA, 1mg/LKT and 2% coconut juice, was suitable for the protoplast culture. The protoplast isolated from mesophyll of cultivated leaf begin to divide in two or three days, small groups of cells appear in night or ten days, and callus will come into being in two weeks.