Effects Of Elicitors On Adventitious Roots Of Panax Ginseng And Establishment Of A Simple Bioreactor Culture System

Ginseng（Panax ginseng C.A.Mey.）,a perennial herb belonging to the Panax genus under the Araliaceae family,is a valuable Chinese herb known for its ability to tonify vital energy,nourish the spleen and lungs,nourish the blood,support righteous energy,and dispel evil.Ginsenoside,the main secondary metabolite of ginseng,is the primary active ingredient responsible for the herb’s medicinal properties.It has been shown to effectively regulate human immune function,suppress inflammation and inhibit cancer cells.However,the problems of the long growth cycle,unstable yield,multiple pests and diseases,and the reuse of ginseng-cultivated land have limited the scale and development speed of the ginseng industry.Consequently,increasing the production of secondary metabolites by adding elicitors to the tissue culture has emerged as a common and effective method for increasing the content of active ingredients in medicinal plants.As a new resource food,the ginseng adventitious root has been applied to the industrial production of ginsenoside due to its advantages of high growth rate,stable production capacity of secondary metabolites.Therefore,to increase the yield of secondary metabolites in ginseng adventitious root by adding elicitors in its culture has great research significance and further application value.In this study,the four-year-old fresh ginseng main root was used as an explant to induce adventitious root,and its culture system was expanded in B5 liquid medium.In this culture system,the effects of three kinds of elicitors including Aspergillus niger,folic acid（FA）,and calcium ion(Ca²⁺)on ginseng adventitious root were compared which taking the biomass accumulation and ginsenoside content of ginseng adventitious root as indicators.Therefore,the suitable elicited concentration,suitable elicited duration time,and co-elicited effect of these three elicitors were studied,and the FA elicitor was selected out as the best elicitor in the growth of ginseng adventitious root and the production of ginsenoside.Then,by studying the effect of the best elicitor,folic acid,on the gene expression of key catalytic enzymes in the process of ginsenoside biosynthesis,we preliminarily explored the induction mechanism of folic acid elicitor on the growth of ginseng adventitious root and the accumulation of ginsenoside content.Finally,the suspension culture system of ginseng adventitious root elicited by folic acid was expanded to a 5 L bioreactor to further improve the production efficiency of ginsenoside.The result lay a theoretical foundation for the large-scale production of adventitious root of ginseng.The main findings of study were as follows:（1）As an elicit,the Aspergillus niger homogenate can significantly promote the synthesis of ginsenoside in ginseng adventitious root,and the suitable elicited concentration is 80 mg·L^-1,the suitable elicited duration time is 72 hours.The content of 12 kinds of mono-ginsenoside detected in ginseng adventitious root were all changed significantly with the elicited of Aspergillus niger homogenate.The content of majority mono-ginsenoside reached their accumulation peak during the first 24-72 hours of elicited.Specifically,the PPD-type ginsenosides response to the Aspergillus niger homogenate elicited faster within the first 24 hours,and the accumulation of PPT-type ginsenoside increased with longer elicited duration time.However,the Aspergillus niger homogenate elicitor can significantly inhibit the growth of adventitious root.（2）As an elicit,the folic acid（FA）can significantly promote the biomass accumulation and the biosynthesis of ginsenoside in ginseng adventitious root,and the suitable elicited concentration is 50μmol·L^-1,the suitable elicited duration time is 144 hours.When applying 50μmol·L^-1 folic acid as the elicitor,the fresh and dry weights of ginseng adventitious roots were 1.797 and 1.678 times higher than the control,respectively.Additionally,the total ginsenoside content was 1.982 times higher than the control.The content of 12 kinds of mono-ginsenoside detected in ginseng adventitious root were all changed significantly with the elicited of FA.The content of majority mono-ginsenoside reached their accumulation peak during the 72-168 hours of elicited.Specifically,the accumulation speed of PPD-type ginsenosides content was faster than the PPT-type ginsenoside.（3）As an elicit,the Ca²⁺can significantly promote the biomass accumulation and the biosynthesis of ginsenoside in ginseng adventitious root,and the suitable elicited concentration is 100 mmol·L^-1,the suitable elicited duration time is 144 hours.When applying 100 mmol·L^-1Ca²⁺as the elicitor,the fresh and dry weights of ginseng adventitious roots were 1.741 and 1.627 times higher than the control,respectively.Additionally,the total ginsenoside content was 1.740 times higher than the control.The content of 12 kinds of mono-ginsenoside detected in ginseng adventitious root were all changed significantly with the elicited of Ca²⁺.The content of majority mono-ginsenoside reached their accumulation peak during the 72-168 hours of elicited.Specifically,the accumulation speed of PPD-type ginsenosides content was faster than the PPT-type ginsenoside.（4）The elicited of three elicitors above will lead to the increase of catalytic enzyme content or activity which related to the growth and secondary metabolic synthesis in ginseng growth,including malondialdehyde content（MDA）,catalase activity（CAT）,peroxidase activity（POD）and superoxide dismutase activity（SOD）.Among them,the elicited effect of Ca²⁺and FA can significantly increase the content and activity of the above four enzymes,while the SOD activity under the elicited effect of Aspergillus niger did not show significant changes.It is speculated that the elicited mechanism of Ca²⁺and FA elicitors in ginseng adventitious root may be more similar,while that of Aspergillus niger as elicitors is different from the former two.（5）The above three elicitors were combined into co-elicitors respectively.The co-elicitors could promote the biomass accumulation and ginsenoside biosynthesis of ginseng adventitious root to varying degrees,except the co-elicitor composed of Aspergillus niger homogenate and folic acid which had no significant effect on the growth of adventitious root of ginseng.The co-elicitor composed of Ca²⁺and 50μmol·L^-1 folic acid had the best elicited effect,with a total ginsenoside content of 1.900 times higher than the control.However,this result still slightly lower than the effect of folic acid elicitor alone.（6）The optimal endogenous reference genes in ginseng adventitious roots treated with folic acid were identified by q RT-PCR and multiple software.The gene of CYP was selected as the most suitable reference gene.Based on this result,the relative expression levels of three key catalytic enzyme genes in the ginsenoside biosynthesis pathway,which including UGTPg1,Pg CYP716A47,and Pg CYP716A53v2,were detected,and the correlation analysis of expression levels of the above genes and the corresponding ginsenoside content were further conducted.The results showed that the expression of these three key enzyme genes could significantly respond to the folic acid elicitor,and their expression changes were significantly or extremely significantly correlated with different mono-ginsenoside contents.This result preliminary indicates that the folic acid has an elicited mechanism to promote ginsenoside biosynthesis by increasing the expression of key catalytic enzyme genes.（7）Based on the above results,the folic acid elicitor was used to optimize the 5 L suspension culture system of ginseng adventitious root.Firstly,using the evaporation weight of liquid culture medium in a 5 L bioreactor suspension culture system as an indicator,0.5 L·min^-1 is determined as the optimal ventilation rate.Then add the folic acid elicitor in the 5 L suspension culture system of ginseng adventitious root for optimization.The results showed that the total ginsenoside content of ginseng adventitious root optimized with folic acid elicitor in a 5 L bioreactor was 1.42 times higher than that of the control group without folic acid elicitor,and 16.386%higher than that of the 250 m L shake flask group with folic acid elicitor.The above results indicate that it is effective to increase the content of ginsenosides in ginseng adventitious root by expanding the culture scale and adding appropriate elicitors,which lays a theoretical foundation for further improving the production efficiency of secondary metabolites in ginseng adventitious root.