| Anoectochilus formosanus Hayata is a rare and endangered medicinal plant. In order to better protect and exploit and utilize this traditional precious materials, we induced multiple shoot and further carried out seedling from wild plants. Also the stable system of inducing multiple shoot and seedling were established, research the survival rate in 4 kinds of culture medium and its enzymatic activity. We investigated the fresh weight, dry weight, proliferation multiples, total flavonoid content and yield by adding fungal elicitors to the culture medium in later stage, then made sure of the fungal elicitor which could promot the biomass and total flavonoids for further experiments. Under current conditions, the role of analgesic activity of the vitro seedling added elicitor was initially investigated.It was found that the suitable culture medium on multiple shoot of A.formosanus is H medium with 6-BA 1.0 mg·L-1, NAA0.8mg·L-1, sucrose 20 g·L-1, pH5.4, litht period:12 h·d-1, light intensity:1400μmol·m-2·s-1, culture temperature:25±1℃, and cultured for 27 days.The optimal culture conditions on seedling cultured were as follows:B5+NAA 0.2 mg·L-1+6-BA 2.0 mg·L-1+sucrose 20 g·L-1+activated carbon 2.0 g·L-1+agar 6.0 g·L-1, pH5.8, litht period:12h·d-1, light intensity:1400μmol·m-2·s-1, culture temperature:25±1℃. The results showed that after the explant in the seedling culture medium in 56 days, the plant height was 3-4cm, possessed 4-5 leaves and 3-4 roots. At this time the test-tube seedlings could be used for the transplanting.The results showed that the maximum survival rate of the test-tube seedlings was in the sawdust+perlite+humus (1:1:1) culture medium which was higher than any other medium. In early transplanting stage, the active of PL A, PPO and POD were increased in varying degrees. Tending to stabilize after 12 days, this demonstrated the plant had adapted to the new environment at this time. The transplanted plants were abserved using microscope, and we found out the the plant had no change of plant morphology, which proved that the possibility of regeneration was obtained by use of tissue culture methods.The six kinds of endogeny fungus as the elicitors were isolated from the culture plant of Anoectochilus formosanus. The elicitor AF5 played a significant role in promoting the biomass and total flavonoids accumulation. Based on the plant Growth curve, we chose the 9th week as the time of adding AF5. and the optimal concentration was 200 mL·L-1.We also studied on the analgesic activity of plantlet in vitro of A formosanus. When the alcohol extract of A formosanus was 6.0 g·kg-1 and 12.0 g·kg-1 by intragastric administration, the writhing reaction induced by acetic acid in mice was prominently inhibited. We also researched the effects of different ethanol extract of A formosanus by organic solvent on writhing reaction induced by acetic acid in mice. With the dosage of the n-butanol and the acetic ether part 6.0 g·kg-1 respectively, it presented lower writhing number of times in duced by acetic acid in mice significantly. While effect of the butanol part was better than acetic acid one. The results showed that A formosanus had good analgesic action. |
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