Optimization Of Ginseng Adventitious Root Suspension Culture System And Establishment Of 5L Culture System

Ginseng is an expensive traditional Chinese medicine,and ginseng resources are close to exhaustion.In order to protect ginseng resources and to meet the market demand,in this study,Ginseng explants from four different sources were choosed,the best clones with good growth potential were selected by comparing the morphological differences of adventitious roots.And on this basis,we built a shake flask culture system,simple bioreactor cultivation system,and optimized the related training parameters,and finally examined the bioreactor saponins accumulation and related gene expression of key enzymes of ginseng adventitious roots induced by methyl jasmonate in bioreactor cultivation,which provided theoretical and technical basis for mass production of ginsenoside from ginseng adventitious roots.The main results were as follows:(1)Callus tissue could be induced by different explants of ginseng,and callus was more easily induced by the leaves of ginseng aseptic seeding and more difficult to be induced by the petiole of ginseng aseptic seeding.(2)The number of adventitious roots redifferentiated in callus tissue of four ginseng explants was different,and the number of adventitious roots redifferentiated in petiole of aseptic seeding was the highest and adventitious roots grew better.(3)Adventitious roots induced by callus of aseptic seeding petiole in raw materials,we compared influence of the combination of 2 kinds of inorganic element concentration(1.0 times,0.5 times)in B5 medium on ginseng adventitious root growth,selected three most significant factors using software and experimental design,which respectively were:MgSO4·7H2O?NH4NO3 and MnSO4·4H2O,and got the best cultivation conditions of ginseng adventitious roots by response surface method,which wasMgSO4·7H2O:167.29mg/L,NH4NO3:84.20mg/L,MnSO4·4H2O:8.28 mg/L,under this condition,the fresh weight of ginseng adventitious root reached 11.57g.(4)Optimization of 250 mL suspension culture conditions:by single factor experiments,we studied the influence of hormone concentration,inoculum size and shaking speed on ginseng adventitious roots growth,and got the best training parameters as the IBA concentration of 4.18 mg/L,quantity of 0.509 g,shaking bed of 128.99 RPM through Box-Behnken experiment design and response surface optimization.Under this condition,saponin production in the ginseng adventitious roots suspension culture was 2.44 mg.(5)Optimization of 500 mL suspension culture conditions:four different carbon sources and three different concentrations were studied.It was found that sucrose added at a concentration of 30 g/L was more suitable for the culture of Panax ginseng adventitious roots.(6)In order to optimize training system of cultivation of adventitious roots and saponins accumulation in 5L bioreactors,we studied the influence of inoculation amount,fluid volume,ventilation,sucrose concentration on ginseng adventitious roots and the saponins accumulation.The results showed that under the condition of 5 L bioreactor with 3 L fluid volume ventilation 0.15 VVM,10 g of ginseng adventitious roots,cultivated in the optimized B5 medium(4 mg/L IBA+30 g/L sucrose,pH:5.8)had the largest saponin production,as follows:52.52 mg.(7)By comparing the contents of saponin in adventitious roots cultured by four years of life cultivation in bioreactor,it was found that the contents of saponin in ginseng roots were 6.93 times than in adventitious roots.The content of Rd saponins in adventitious roots(0.51 mg/g)in bioreactor culture was close to that of four-year plants(0.86 mg/g).(8)The treatment with methyl jasmonate inhibited the biomass accumulation of adventitious roots and increased the content of darmacane ginsenoside,in which the change rate of Rd was the highest at 48 h,and the total ginsenoside was the highest at 48 h.(9)Induction of methyl jasmonate increased expression of PgFPS,PgUGT and PgDDS genes in the pathway of ginsenoside synthesis.