| Anemone flaccida Fr. Schmidt belongs to Anemone. Generally, the plant is called as Diwu simply, and also the dry rhizoma of A.flaccida is called as'Diwu'in the native medicine, which was used to confront inflammatory and pain. To optimize the rapid-proliferation in vitro of A.flaccida, this thesis focused on the effect of factors which affected the tissue culture growth stages, such as adventitious bud proliferation, CLS induction, rooting and acclimatization and transplants.The quantitative and qualitative analysis of soluble sugar, soluble protein, total saponins, chemical composition and active ingredients in DIWU's cultivated, wild and tissue products were carried out by the methods of spectrophotometry, FTIR and HPLC.The results provided a theoretical basis and technical support for seelings industrial production and artificial cultivation of A. flaccida. The main results were as follows:(1)The best medium for adventitious bud proliferation was 1/2 MS (220 mg/L CaCl2·2H2O)+6-BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.4 mg/L+750 mg/L CH, in which the multiplication coefficient was 4.11 and the shoot was orderliness and haleness.(2) The optimal culture medium for CLS induction was MS (440 mg/L CaCl2·2H2O) +6-BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.4 mg/L+500 mg/L CH, in which the induction rate was 80% with a lager diameter of bulb base and haleness shoot.(3) The appropriate inoculum density (4～5 explants/bottle) and subculture period (30 d) were beneficial to subculture proliferation. Edible sugar and tap water could instead of sucrose and distilled water to prepare subculture medium, which could be greatly reduce costs.(4) Adding 100 mg/L Vc to subculture medium could effectively inhibit the browning of explants.After culture for 40 d, the browning rate was only 58% while adding 100 mg/L Vc to subculture medium, which decrease 27 percentage points than control, and the browning were both slight.(5) 1/2MS+ NAA 0.5 mg/L+IBA 0.5 mg/L+AC 1.0 g/L was suitable for rooting of A.flaccida. In this medium, the rooting rate, root number (per explant) and the root length (cm) were 93.9%,10.5 and 1.73,respectiviely. The root was much thicker and relieved browning phenomenon after culturing for 55 d. (6) The optimal acclimatization method for rooted seedlings was in sterilized alluvial soil matrix on 5～6℃environment for 7～10 d;The survival rate could be 96% and germination rate was 86%, while using eight part alluvial soil, two part sand planting matrix; The survival and germination rate were both higher than 95% and 92%, whether the plantlets cultivated in local or transplanted to origin of A.flaccida.(7) The content of soluble sugar, soluble protein and total saponin in'DIWU'were assayed by spectrophotometry, the results as follows:Soluble sugar content:cultivated product>tissue product>wild product. Soluble protein content:tissue product>wild product>cultivated product. Cultivated product's total saponins content was highest while wild and tissue products have no significant difference.(8) The peak shape and position in FTIR fingerprint of the cultivated, wild and tissue product were consistent, which inferred that different sources DIWU contained the same chemical composition.(9) The peak of oleanic acid in HPLC fingerprint of DIWU's wild product and tissue product were distinct and the retention time was 6.69 min. Meanwhile, it was evident that the peak area of wild product was much larger, which indicating that the oleanolic acid content was higher.
|
PDF Full Text Request