Studied On Tissue And Cell Culture And Metabolic Regulation Of Effective Components Of Acanthopanax Senticosus

Acanthopanax senticosus,an important medicinal woody plant belonging to the family of Araliaceae,distributed mainly in northeastern Asia,which contains eleutherosides including eleutheroside A,B,C,D,E,F,G etc.,and complex polysaccharides as active ingredients in roots and leaves.A.senticosus is listed as threatened species because of over-collection from the natural habitat.Propagation of A.senticosus through conventional methods is difficult due to long period of cold,warm stratification and slow germination rate.Plant tissue culture is an easy and convenient process for efficient,potential bulk propagation of this plant.In the current study, regeneration of A.senticosus was carded out directly through somatic cell embryogenesis and indirectly through embryogenic callus.But cell suspension cultures of A.senticosus have not been reported as source of medicinally important secondary metabolites.In this experiment,using tissue and cell culture technology,for the purpose of inducing callus from A.senticosus and establishing the method for cell suspension culture with high ability of producing effective components,we investigated the callus induction conditions and cell state regulation by growth homogenization,established the cell suspension culture system,and inspected the growth laws of cell suspension system of A.senticosus,accumulation of glycosides, polysaccharides and the flavonoids using high concentration of sucrose induced osmotic stress and elicitors made from different fungi.During the callus induction,we used leaf and petiole of A.senticosus as explants and compared the effects of three different hormone combination of dedifferentiation medium(2,4-D 1.0 mg.L^-1;NAA 2.0 mg.L^-1+6-BA 2.0mg.L^-1;2,4-D 1.0 mg.L^-1+6-BA0.5 mg.L^-1and found the callus induced under the condition of NAA 2.0 mg.L^-1+6-BA 2.0 mg.L^-1was most suitable for the following suspension culture,and the callus induction ratio of the leaf and petiole were 93.3%and 100%respectively.We optimized the subculture conditions according to the callus status mainly on the different exogenous hormones combination and light period.The result showed that the callus grew better under the condition of NAA 1.0 mg.L^-1+6-BA 1.0 mg.L^-1and with the light period as 12/12 h (Light/Dark).After optimization of subculture condition,we compared different callus induced from different tissues and of different status and selected the cell lines that were most suitable for the following cell suspension culture:Ab line.We homogenized the cell groups by cell size screening and starvation culture which would produce the simultaneous growing callus and ensure that the cell groups in the following culture system stable and homogeneous,results stable and reliable.Taking consider of the characteristics of A.senticosus and the property of cell suspension culture,we initially established a suspension culture system to determine the optimum medium (MS+Sucrose 40 g.L^-1+NAA 1.0 mg.L^-1+6-BA 1.0 mg.L^-1,pH=5.8)at 25±1℃under darkness and found that the optimal inoculation density(60 g.L^-1);optimal initial concentration of sucrose(40 g.L^-1);optimum rotary speed(110 rpm).Under the conditions mentioned above,the growth cycle of A.senticosus cell suspension culture was 40 days and the maximum cell dry weight during the growth cycle was 18.82 g.L-1 DW.In case of callus,the highest contents of polysaccharide,total saponin,and total flavonoids were achieved at the 40^th,40^th,and 16^thday respectively,while in case of the cell suspension culture,the maximum yields of polysaccharide,total saponin,and flavonoids were achieved at the 40th day and were 1259.25,398.42 and 474.26 mg.L^-1respectively which was thought mainly due to the homogenization of the cell groups in the suspension culture.A.senticosus callus suspension culture process,the pH of the medium or after the first drop in the entire training cycle and the pH value of biomass showed a strong correlation;carbon, nitrogen and phosphorus in training 36 days.After the basic consumption completely,callus suspension of good nutrition metabolism.Our data also showed that the concentration of sucrose had significant effects on the synthesis and accumulation of polysaccharide and total saponins except for total flavonoids and biomass. The effective component in the callus of A.senticosus was enhanced via a treatment of five fungal elicitors separately Colletorichum lagnarinm,Botrytis cinerea,2020,2006,and 2014.Among these five elicitors,Botrytis cinerea and 2014 had the highest inducing efficiency of total saponins and 2020 had the highest inducing efficiency of polysaccharide,but non of them manifests any noticeable effects on the cell growth.The total saponins content of callus treated with Botrytis cinerea and 2014 were separately 32.8 mg.g^-1DW and 34.8 mg.g^-1DW,and were higher than the control(3.14 mg.g^-1DW).The polysaccharide content of callus treated with 2020 was 77.25 mg.g^-1DW,with 20.7%higher than the control(64.74 mg.g(-1)DW).