| It is one of the most famous and valuable Chinese herbal medicine in Anhui Province of China, and it is regarded and protected for its value by government. It has the advantage of diminishing inflammation, resisting senescence and improving immunity because of its content of amylose and alkaloid. But the wild resources of D. Huoshanense are reducing as the reason of the poor living conditions, low productivity, overexploitation and so on. At present, the main breeding method using in D. Huoshanense is vitro propagation technology, but that cannot satisfy with the needs of market because of the low content of alkaloid in plants. However, the liquid culture can produce alkaloid in a short period with slow development of cells except for low output, that is the very reason for restricting industrial produce.The experiments were used 60Co-γ,SNP,PA,SA and direct current magnetic field on D.huoshanense protocorm like-bodies(PLB) to research the living situation of PLB and the accumulating of alkaloids, so that we can provide technical parameters for establishing large-scale liquid culture and producing higher content alkaloids, and finally provide theoretical basis for the synthesis of alkaloids.The main results were following:1. The optimum irradiation dose for PLBs was 10Gy, the growth curve was similar as"S"type in the 44days subculture period. And the biomass reached peak at the 36th day, which was 26.54g/DW·flask, 22.47% higher than the CK; the alkaloids quantity reached peak at the 40th day, which was 0.035g/DW·flask, 81.76% higher than the CK. The pH of culture and the electrical conductivity changed slightly, the medium environment fit PLBs to be cultured continuously. 60Co-γirradiation could increase the activities of POD, SOD, CAT, PAL and decrease the activities of PPO to improve cell growth and the accumulation of alkaloid of PLBs.2. High concentration of SNP decreased the biomass of PLBs, but enhanced alkaloids production. The optimum concentration was 0.1mmol·L-1, the biomass reached peak at the 32th day in the 44 days subculture period, which was 14.51g/DW·flask, lower than the CK; the alkaloids production reached peak at the 36th day, which was 0.0395 g/DW·flask, 101.53% higher than the CK. By adding SNP could cause the medium acidification and increase the relative conductivity of PLBs. So, high concentration of SNP hurt PLBs but could increase the activities of POD,PAL to improve the accumulation of alkaloid of PLBs.3. PA could improve the biomass and alkaloid content of suspension-cultured D.huoshanense PLBs, 7.5g·L-1 was the optimum concentration, the fresh weight was 17.26g/flask, 39.31% higher than the CK; the alkaloid content was 0.026g/DW·flask, 83.8% higher than the CK. PA could increase the activities of PAL and decrease the activities of POD to improve cell growth and the accumulation of alkaloid of PLBs.4. The optimum concentration of SA for PLBs was 100μmol·L-1, the fresh weight was 13.60g/flask, 17.18% lower than the CK; the alkaloid content was 0.022g/DW·flask, 1.62-fold higher than the CK. By adding SA could cause the medium acidification, and increase the relative conductivity of PLBs, but increase the activities of POD,PAL to improve the accumulation of alkaloid of PLBs.5. With the direct current magnetic field on D.huoshanense PLBs, the biomass was obviously enhanced. Under the condition of magnetic field and SA on PLBs, the fresh weight was 8.65g/flask, 41.57% higher than the CK; the alkaloid content was 0.0135g/DW·flask, 11.18% lower than the CK. The results showed that Magnetic field associated with SA could not increase the accumulation of alkaloid, decrease the activities of POD and PAL and the secondary metabolites of PLBs.6. Through observing of PLBs by paraffin slice technique, we found that alkaloid of PLBs mainly distribute in the cytoplasm.
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