| Changium smyrnioides Wolff.is an endemic monotypic species genus plant which belongs to the Umbelliferae family and it's a famous Chinese material medicine in east China.Changium smyrnioides become a rare and endangered plant,due to the extensive use of Changium smyrnioides and good medicinal value which induce the excessive exploitation of wild resources and self-recovery ability of Changium smyrnioides is poor.Modern researchs found that the root bark of Changium smyrnioides contains abundant furanocoumarin,such as isoimperatorin,imperatorin,xanthotol,phellopterin,psoralen,bergapten and so on,and furanocoumarin has several pharmacological effects,such as anti-tumor,photochemical effect and anti-HIV.This study aim to produce coumarin by plant cell suspension culture and regulate the yield of coumarin in suspension cells.The research contents and results are as follows.1 The relevant literature was summarized,and a systematic review was given about advance in studies on production of secondary metabolites by medical plant tissue culture and Changium smyrnioides material medicine.Part 1 summarized methods of producing different kinds of secondary metabolites,regulation of the yield and improve the production by medicinal plant tissue culture.Part 2 mainly introduced the biological characteristics,endangered causes and research progress in tissue culture of of Changium smyrnioides.This review provides theoretical basis for the following experiment.2 Three explants(Changium smyrnioides's leaves,petioles and stems)were choosed to callus induction on the basis of literature research and to screen the callus which is Suitable for suspension culture.We choose the cells induced by Changium smyrnioides's leaves to suspension culture,using yield of biomass and total coumarin as the index,optimized the culture conditions,such as medium,inoculation quantity,sugar adding quantity,culture temperature,and establish the Changium smyrnioides cell suspension culture system.The results indicated that the best culture condition is 3%Changium smyrnioides cells were inoculated into MS basic culture medium contains 3%sucrose(hormones NAA1.5 mg·L-1+2,4-D0.5 mg·L-1+KT0.2 mg·L-1+6-BA0.5 mg·L-1),25?,speed 120 r·min-1,co-culture for 20 days.3 On the basis of the last chapter,the suspension cells of Changium smyrnioides were cultured by two-step method.Through suspension cell biomass,the accumulation of total coumarin and two kinds of furocoumarin(bergaptol,bergapten)content as an index,examining the best training mode of Changium smyrnioides suspension cell.The result showed that accumulation of Changium smyrnioides suspension cell which was cultivated for 20 days biomass reached the maximum 179 per/?L.The biomass increased 12.6%was more than that by one-step method.After cultivating by two-step method for 24 days,the accumulation of total coumarin reached the maximum 0.2882 mg-L-1.Compared to one-step method,it was increased 60.7%.The two kinds of furocoumarin(bergaptol,bergapten)content reached 1.79%?0.36%.increased by 1.75 times and 28.6%than step method.4 To study the effects of browning inhibitors on Changium smyrnioides Wolff suspension cells growth and secondary metabolites production.Different concentrations of AsA,AC,AHC,Na2S2O3 and PVP were added to the light brown suspension cells,and the contents of phenols,coumarin,bergaptol and bergapten were determinated by UV-Vis and HPLC.PVP with low concentration and AsA improved the growth of the suspension cells in different degrees.After using 2 mg-mL-1 ASA,coumarin content of ASA 2 group increased 60.90%compared with the control group.The browning phenomenon caused by salicylic acid were inhibited by adding 2 mg/ml of AsA into suspension culture system(with salicylic acid as the inducer).At the same time,the content of bergaptol and bergapten were increased 20.61%and 25.00%.AsA was the best anti-browning agent in this study.It can inhibit browning,promote cell growth and accumulation of secondary metabolites of Changium smyrnioides Wolff suspension cells.5 To investigate effects of 3 elicitors(Salicylic Acid,Methyl Jasmonate,yeast extract)and precursor(L-Phenylalanine)on cell growth and furocoumarin production in Changium smyrnioides suspension cells.Stable-phase cell suspension cultures were treated with elicitors and precursor.To study the optimum concentration and time of the best elicitor that take Changium Smyrnioides cells growth and accumulation of furocoumarin in suspension cells and culture medium as an indicator.The result shows that SA and MeJA significantly improved the production of bergaptol,bergapten and xanthotoxin in Changium Smyrnioides suspension cells and the optimum conditions were establishd.The 200 umol·L-1 SA induced three kinds of Furanocoumarin content in suspension cultures of Changium smyrnioides to reache 0.654%,0.293%,0.060%and biomass was improved by 42.14%compared with CK.It is an effective way to improve furocoumarin contents in suspension cells and culture medium that addition of SA and MeJA in Changium Smyrnioides suspension cells. |
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