| Anemone flaccida Fr. Schmidt is a species of Anemone genus. The dried rhizome of A.flaccida locally named as "Diwu", is used as a Chinese traditional herb medicine with functions of expelling rheumatism detumescence, relieving pain, and being conducive to the physical. However, in the natural condition, the growth period of this species is very short, its biomass accumulation is small, and seed sprouts difficultly, which result in poor natural resource. Therefore, it is very important to realize cultivation of the species. In this study, the technological systems of micro-propagation from leaf explant in vitro was studied for fast propagation of seedlings and the preliminary polyploidy was induced to resolve the problem of the germplasm shortage. The main results are as follows:(1) The buds of rhizome were treated with wrapping cotton soaked with different concentrations of colchicine as mutagen and then transplanted in soil after treatment. Morphological observation showed that, the treatment with 0.2% colchicine for 8 days got the highest mutation rate of 81.67%, and fatality rate was the lowest. It was a feasible method for polyploidy induction in this species.(2) The sterilized buds from rhizome were cultivated initially on solid medium of MS+2.0 mg 6-BA/L+0.2 mg NAA/L+0.8% agar+3% sucrose and then subcultured on the medium of MS+1.0 mg 6-BA/L+0.3 mg NAA/L+0.5 mg KT/L+3% sucrose +0.8% agar. The buds regenerated from explants were treated with dripping or immersing different concentration of colchicine solution for several hours. The results showed that, the mutation rate of adventitious buds was lower than that of buds from rhizome, only about 13%, but the fatality rate was higher, reaching about 81.7%.(3) The leaves of regenerating buds were used as explants and cultured on MS basic medium containing different concentration of 6-BA and NAA under light or dark condition. Observation showed that, there was no callus induced in any case, but adventitious buds were regenerated directly. The optimum medium for adventitious buds regeneration was MS+1.5 mg 6-BA/L+0.2 mg NAA/L+0.8% agar+3% sucrose under dark, with the highest regenerating rate of 21.67%. Light condition affected the initial regeneration rate, but did not affect the number of adventitious buds that regenerated from a single leaf explant.(4) On the MS basic medium added different concentrations of 2,4-D and 6-BA and cultured under light or dark condition, the leaf explant was not induced to generate callus either, but generate adventitious buds directly. The appropriate medium was MS+2.5 mg 6-BA/L+0.2 mg 2,4-D/L+0.8% agar+3% sucrose under dark, with the highest regeneration rate of 28.33% in this system. Similarly, the generation rate under light was lower than that under dark condition, and light did not affect the number of adventitious buds that regenerated from a single leaf explant.(5) On the basis of the above, on the MS basic medium added the combination of different concentration of 6-BA with 0.1 mg 2,4-D/L and 0.1 mg NAA/L, and cultured under light or dark condition, observation showed that, regeneration rate of adventitious buds from leaf explant was the highest, reaching 32.78%, on the medium of MS+1.5 mg 6-BA/L+0.1 mg 2,4-D/L+0.1 mg NAA/L+0.8% agar+3% sucrose under dark. The initial regeneration rate was affected by light, but number of adventitious bud regeneration was not affected from a single leaf explant.(6) After subculturing for four times, the adventitious buds with only new leaves were cultured in room temperature (19±2℃) under light or dark, or in refrigerator under 4℃on the rooting medium of 1/2 MS+1.0 mg NAA/L+0.8% agar+3% sucrose. The results showed that, the optimum condition for rooting was in refrigerator under 4℃with 70% rooting rate. When roots grew to 0.5 cm in length, the plantlets were cultured under light to produce healthy seedlings. |
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