Studies On Tissue Culture Ang Estadlishment Of Agrobacterium-Mediated Transformation Of Siraitia Grosvenorii (Swingle) C. Jeffery

In this experiment, two varieties of Siraitia grosvenorii (Swingle) C. Jeffery that named Qingpiguo and Yuanxinghongmaozi were used as the materials. The system of tissue culture and agrobacterium-mediated transformation of Siraitia grosvenorii was established and some GUS transgenic plants of Siraitia grosvenorii had been obtained successfully. The results of study were included in the following.t1. The pathway of adventitious bud differentiation may become the main pathway on tissue culture of the Siraitia grosvenorii. The differentiation rate of callus produced by the pathway of des-differentiation was low, which was disadvantage to tissue culture of Siraitia grosvenorii.2. The leaves and buds were the suitable material for tissue culture of Siraitia grosvenorii, which showed the high differentiation rate of adventitious buds. The buds differentiation rate of callus, which induced by the axillary buds, was higher comparatively.3. On the leaves culture, the optimal medium of the buds differentiation was MS medium supplemented with 2.0mg-L-1 BA and 0.5mg-L-1 IBA, the buds differentiation rate was 61.9%, and average number of buds was 5.48. On the axillary buds culture, the optimal medium of the buds differentiation was MS medium supplemented with 1.0mg-L-1 BA and 0.5mg-L-1 IBA, the bud differentiation rate was 98.9%, and average number of buds was 5.27.4. In 2-5 generation subculture, the capability of buds differentiation was decline with the leaves subculture time increasing.5. The differentiation rate of the callus was drop with the callus age increasing. When the callus age was five days, the differentiation rate was the highest, and the rate was 14%.6. The effects of the bud differentiation were difference in the different position of the leaves and axillary buds, which the leaves and axillary buds lied in 3-4 node showed the higher differentiation rate of buds. The rate was 96.9% and 48.7%.7. The leaves of subculture plant still had higher differentiation abilities. The bud differentiation rate of Qingpiguo and Yuanxinghongmaozi was 65% and 50%.8. In the culture of root inducement and sound seedling, the optimal medium was 1/2MS medium supplemented with 0.8-1.0mg-L-1 IBA and 0.4-0.6mg-L-1 MET. The root induce rate was 100%, the number of root was 5.5-6.03.9. The sensitive concentration of Km was 6mg/L on the leaf-fragment of Siraitia grosvenorii. The non-transgenic seedling couldn't induce roots on the medium contained Km.10. When the leaf-fragment of Siraitia grosvenorii pre-cultured for 4 days before being transformed, GUS gene expressed perfectly. The test of instantaneous expression indicated: the leaf-fragment that the whole dyed to navy blue was 21.7%, the leaf-fragment that the edge and inner dyed to deeper blue-spot was 69.6%, and the leaf-fragment that the edge dyed to blue was 8.7%. The rate of instantaneous expression was 100%.11. Leaf-fragment of Siraitia grosvenorii were dipped in agrobacteriumliquid for 5~60 seconds that concentration was OD600 0.1 ~0.5 and co-cultivated in 48 hours, then GUS gene expressed optimal, the expressed rate was 100%.12. A lot of fake-transgenic seedling was generated because of the selection after regeneration. The method, which the selection before regeneration combines with the filtration of root generation, could wipe off the fake-transgenic seedling efficiency.13. GUS gene had been expressed steadily in the Siraitia grosvenorii plants. The transgenic plants of Siraitia grosvenorii were gained in the experiment.