Establishment Of Suspension Culture System Of Sanchi Ginseng Cells And Accumulation Pattern Of Sapenins

Panax notoginseng(Burk.)F.H.Chen belongs to genus of Ginseng,Araliaceae family and has a perfect pharmacologic action,which is extensively applied to the treatment of diseases such as hypertension,hyperlipidemia and cerebrovascular disease and so on.It is also a medical plant in extremely great market demand.However,influenced by the natural environment,biological climate,arable area and other factors,the extensive cultivation of Panax notoginseng is seriously limited.The application of the tissue culture technology not only can avoid these problems and but also can intensively produce the effective secondary metabolite so as to relieve the shortage of Panax notoginseng market and the problem of the wild resource protection.Selecting the annual seeding of Panax notoginseng from Wenshan Yunnan Province as the explant material,this research conducted the callus inducement,callus subculture,cells suspension cultivation and the secondary metabolite accumulation on the basis of the tissue culture technology in order to optimize a suitable cultural condition and establish an efficient suspension cultivation system,which provided a theoretical foundation to the subsequent enlarging cultivation and the industrial production.It also has a significant importance to the sustainable development of the rare and endangered medical plant resources.The main results of this experiment are shown as follows:1.For the stage of callus inducement,this research focused on the effect of the different explants,culture mediums and hormone combinations on the induction and subculture of Panax notoginseng callus for screening out the best cultural condition.The result indicated that the subculture had a best effect when the callus inducement was conducted with the leaves or roots and inoculated to culture medium of MS+2.0 mg·mL-1 2,4-D+0.5 mg·mL-16-BA.2.For the stage of the establishment of suspension cultivation system,the research took the loose and fragile embryonic callus that grew vigorously as the initial material,optimizing the growth condition from the type of basic culture medium,kind and concentration of hormone,initial inoculum size,shaking speed,concentration of sucrose and pH.Then measuring the growth curve of the cell in suspension culture.The result showed that the most suitable conditions were MS+2.0 mg·mL-1 2,4-D+0.5 mg·mL-1 6-BA+1.0 mg·mL-1 NAA+1.0 mg·mL"1 KT+30 g·L-1 sucrose with 60 g·L-1 initial inoculum size and 110 rpm rotate speed of shaker.The original pH was 5.8.Under the above environment,the cells had the best growth which presents a "S" curve,the biomass achieved the maximum at 20d after inoculating.3.For the stage of the secondary metabolite accumulation,the research mainly analyzed the effects of the total nitrogen,NO3-/NH4+,methyl jasmonate,salicylic acid on the accumulation of the Panax notoginseng saponins.The result demonstrated that the saponins reached the highest when the total nitrogen was 60 mmol·L-1 and 30 mmol·L-1 KNO3+15 mmol·L-1 NH4NO3 which equaled to NO3-/NH4+=3:1;Low concentration of methyl jasmonate can promote the accumulation of Panax notoginseng saponins.The saponins reached the highest when the concentration was 50 μmol·L-1;Salicylic acid have no significant effect on the accumulation of Panax notoginseng saponins.The saponins reached the highest when the concentration was 100 μmol·L-1.