| Anemone flaccida Fr. Schmidt belongs to Anemone. The dry rhizoma of A. flaccida is called as "Diwu" in the native medicine so that this plant is also called as Diwu simply. It was used to confront inflammatory and pain. According to this object, we have established the system of rapid propagation and identified the way of morphogenesis in this research.(1) The result showed that it can prevent fungal contamination effectively by dipping the explant in 1.5% Duojunling solution for 2 h in tissue culture process, and then dip the explant in 0.2% HgCl2 for 9 min to prevent the contamination and the explants were not injuried obviously. The antibiotics added to the medium prevented endophyte contamination effectively in the tissue culture process and the explants were normal. Endophyte contamination was prevented effectively when the concentration of cefotaxime sodium was increased to 300 mg/L which we used in the medium, and there was no side-effects for explant.(2) The regeneration and induction capacity of explants were impacted by its physiological state. It was easy to induce callus from bud, which was hardly from petiole and root. So the best explant was healthy buds.(3) Different kinds of hormones influenced the dedifferentiation of explant evidently. The explants of buds did not induced callus on the medium without hormones. The explant cultured on the medium with combination of 6-BA and auxin was better than that with the combination of ZT and auxin, and the combination of 6-BA and NAA was better than the combination of 6-BA and IBA. However, the rate of induction declined when we used the two types of cytokinin compound.(4) The ways of regeneration were impacted by different ratio of hormones. There was no adventitious buds and Corm-like structure (CLS) induced on the medium without hormones. There were only adventitious buds induced on the medium that just contained 6-BA. The induction rate of CLS raised when the medium contained 0.2 mg/L NAA and 6-BA. The induction rate of CLS was highest in the R5 (MS+0.2 mg/L NAA+2.0 mg/L 6-BA) group. All the induction rates of CLS and adventitious buds declined in the R6 (MS+0.5 mg/L NAA+1.0 mg/L 6-BA) and R7 (MS+0.5 mg/L NAA+2.0 mg/L 6-BA) group. There was no CLS induced when the concentration of NAA increased to 1 mg/L in the R8 group, but a few of adventitious buds were induced in the same condition in R8 (MS+1 mg/L NAA+1.0 mg/L 6-BA)and R9 (MS+1 mg/L NAA+2.0 mg/L 6-BA)group. There was few of CLS induced in R10 (MS+1 mg/L NAA+3.0 mg/L 6-BA) group.(5) The explants which grew on the MS medium were best, The B5 medium was worse than MS medium. The explant was worst cultured on the White and Nitsch medium, the leaf was yellow and the explant browned seriously.(6) Percentage of shoots rooted and number of roots per shoots reduced in the conditions of 18°C. Low temperature was conducive for rooting. In the conditions of 5°C, percentage of shoots rooted and number of roots per shoots were highest when the medium contained 0.2 mg/L NAA, but the roots were slender. When the medium contained 0.5 mg/L IBA, the number of roots per shoots reduced but the root was thick. In the conditions of 5°C, when the medium contained 0.2 mg/L NAA, that was conducive for rooting.(7) We took the samples incubated on the medium I (MS+2.0 mg/L 6-BA+0.2 mg/L NAA) and II (MS+2.0 mg/L 6-BA+1.0 mg/L NAA) every four days, to observe how the morphogenesis happened. Purple calli were induced on the medium I after incubated 24 d, but white calli were induced on the medium II after 28 d incubation. There were two types of meristem center in the samples incubated on the medium I . The meristem of type A differentiated early and had less meristem tissue. The meristem of type B differentiated later and had more meristem tissue than type A. We have observed mature somatic embryogenesis in the materials incubated on the medium II. The white calli changed to green. |
