The Study Of The Culture Conditions And Influencing Factors Of Atractylodes Lancea Tissue And Suspension Cell Cultures

Atractylodes lancea is a famous genuine Chinese medicine used for thousands of years.Because of over exploitation and habitat destruction,the storage amount and distribution area of A.lancea reduce sharply in recent years.Therefore,A.lancea has been one of the four endangered medicinal plants of Jiangsu Province.Most studies of A.lancea focused on the pharmacological composition,function,preparation,and interaction mechanism between bacteria and host,but there are few studies comparing the differences between essential oil yields in A.lancea obtained from biotechnology system and wild ones,and how to determine the optimum growth and production condition.Firstly,we studied the impacts of different macroelements and their different concentrations in Murashige-Skoog(MS)rooting medium on the growth and volatile oil accumulation in A.lancea.Based on the results of single factor experiments,we used response surface method to run an optimization experiment of different macroelements and their different concentrations.It was found that lack of CaCl2 and MgSO4 affect essential oil yield severely,and the optimum conditions obtained after concentration optimization of CaCl2,KNO3,NH4NO3,KH2PO4 and MgSO4 were 1.5 times,1.13 times and 0.5 times,1.06 times,139 times of original MS medium,respectively.These results obtained an optimal condition:Obtained value obtained 54.94%higher than the control.Subsequently,we analyzed the effects of endophytes,saprophytes,and pathogens on essential oil accumulation in A.lancea.We selected endophytes Acremonium strictum AL16 and Pseudomonas fluorescens ALEB7B,saprophytic fungi Aspergillus niger(HQM),saprophytic bacteria Escherichia coli(E.Coil),and beneficial fungi Rhodotorola mucilaginosa(JHJM),pathogenic bacteria Erwin ia carotovora subsp.carotovora ECC.3,and pathogenic fungi Athelia rolfsii SY4 to inoculate sterile tissue of A.lancea,then monitored the accumulations of biomass and essential oil in A.in 20 days which inoculated with non-pathogens and in 5 days Which inoculated with pathogens.According to the results,we analyzed and compared the different effects of microorganisms on the growth and volatile oil production in plantlet.It showed that application of the pathogenic bacteria ECC.3 on A.lancea will die with the changes of biomass and contents of some principal components in volatile oil produced in large amounts.The endophytic bacteria ALEB7B can promote the volatile oil accumulation,while AL16 is not dominant in the indoor experiment.The results of pot experiment showed the growth advantage of plantlets which inoculated with AL16.Saprophytic fungi A.niger can affect plant volatile oil accumulation in a certain extent,while saprophytic bacteria Escherichia coli has little effect.R.mucilaginosa showed a significant inhibition of plant growth and volatile oil production.Finally,we have established the suspension cell line of A.lancea,and then used it as material to optimize the conditions for culture by the experiments designed with response surface methodology based on the five variable and five levels.The five kinds of variables are the content of hormone 1-Naphthylacetic acid(NAA),2,4-dichlorophenoxy acetic acid(2,4-D)and the exogenous elicitor D-manose,inoculum volume,and rotation speed.Then we determined an optimal fermentation conditions for biomass accumulation of A.lancea which are NAA 0.43 mg L-1;2,4-D 0.48 mg L-1;inoculation amount of 50%;shaking speed of 125.44 rpm;D-mannose 60 mg L-1.Meanwhile have getted an optimum conditions for the accumulation of essential oil were NAA 0.25 mg L-1,2,4-D0.75 mg L-1,the amount of 37.52%inoculation,speed of 131.81 rpm,D-mannose 60 mg L-1.Eventually,shaking verification showed that its accumulation of biomass is higher than that of the control by 14330%and 18.51%,and its volatile oil content is higher than that of the control by 57.72%and 106.80%.