| Siraitia grosvenorii is an unique medicinal and sweetener economic crops in China,the mogroside V, the effective ingredients of its fruit, is one of the most sweet non-sugar sweetners in the world. Cucurbitadienol Synthase (CS) is the key enzyme of Mogrosides V biosynthesis. It will be a way to increase the content of Mogrosides V by using genetic engineering technology to construct CS-overexpressed vector and transferring it into Siraitia grosvenorii by Agrobacterium-mediated transformation. Siraitia grosvenorii is a kind of dioecious plants. A large number of male plants always cause a waste of land and capital because of a small female proportion in the sexual offspring population. On the other hand, germplasm resources of Siraitia grosvenorii are scarce so that the traditional cross-breeding work hard. In this study, the vitro regeneration system of Siraitia grosvenorii was optimized by using leaves of female plant as explants. Meanwhile, with the method of orthogonal experimental design, a genetic transformation system was established by using Agrobacterium-mediated transformation to transfer CS-overexpressed vector into Siraitia grosvenorii. These provided scientific basis for transgenic breeding of Siraitia grosvenorii. The major findings are as follows:1. In aseptic seedling tissue culture, the best hormone combination for cultivating strong seedlings was 0.3 mg/L 6-BA and 0.05 mg/L IBA, which made the plant height was up to 7.67 cm, and the number of newly increased leaves number was 4.90, the largest leaf area was up to 8.00 cm2.2. The best hormone combination for leaves inducement was 0.7 mg/L TDZ and 0.2 mg/L IBA. The callus induction time was 12.7 days, and the callus induction rate was 89.43%, and the size of callus was 0.73 cm,3. The best concentration of 6-BA for inducing differentiation of bud in callus was 0.5 mg/L The differentiation rate was up to 73.33%, and the average number of buds was 4.11.4. The best screening concentration of Km for genetic transformation was 10 mg/L.5. The best bacteriostasis concentration of Cef for leaf genetic transformation was 300 mg/L.6. The best bacteriostasis concentration of Cef for callus genetic transformation was 600 mg/L.7. In the study of leaf genetic transformation, the results showed that the optimal combination of orthogonal test was A3B1C2D1, which meant that one day was the best pre-culture time,0.5 of OD600 of Agrobacterium strain liquid was optimal, the best infection time was 5 minutes, and the optimal time of co-culture was 3 days. After delay screening,224 resistant seedlings were detected by PCR. As the result, there were three positive seedlings, to which CS-overexpressed vector was transferred among them and the total transgenic rate was 1.34%.8. There were no positive seedlings as a result of PCR detection in the study of callus genetic transformation. But according to the statistical results of other indicators besides the transgenic rate, the optimal combination of orthogonal test was A3B1C2D1, which meant that the pre-culture time was 2 days, the OD600 of Agrobacterium strain liquid was 0.4, the time for Agrobacterium infection to cotyledon was 15 minutes, and the reasonable time of co-culture was one day.9. The results showed that the leaf as a receptor might be more suitable for genetic transformation of Siraitia grosvenorii than callus. |
PDF Full Text Request