Optimization Of Callus Induction And The Effect Of MeJA On The Accumulation Of Terpenoids In Callus Of Isodon Amethystoides

Isodon amethystoides?Benth.?H.Hara is a perennial erect herb of the family Lamiaceae,which can be used as a whole herb in many parts of the country and belongs to traditional chinese medicine.The Isodon amethystoides cells contain a variety of compounds with extremely high medicinal properties,such as the wangzaozin A of the diterpenoids and the oleanolic acid and ursolic acid of the triterpenoids.In this paper,the induction conditions of callus were optimized by different media types and with or without light.The hormonal conditions of loose callus proliferation and browning factors controlling callus were explored.Callus was treated with different concentrations of exogenous elicitors to investigate the effects of callus on its terpenoids content,growth,proliferation,browning and related physiological and biochemical characteristics.It provides experimental basis for tissue culture of Isodon amethystoides and reference for large-scale production of medicinal components by using plant cells.The main results of this paper are as follows:?1?The seeds of Isodon amethystoides collected in the same year and the sterile seedlings were cultured for 15 days in a sterile environment.The young leaves of healthy sterile seedlings of the same growth state were used as explants,and the single factor experiment was used to study effects of MS,1/2MS and B₅ medium on callus induction and callus induction under light or shading culture conditions.The results showed that the callus induction time,induction rate and growth state of Isodon amethystoides were the best in B₅ medium,followed by 1/2MS medium,and the induction effect of MS was a little worse than that of the first two.A large amount of chlorophyll is produced in callus under light condition,and the texture of callus is hard.The callus in the condition of light-shading culture was light yellow and the texture was soft.Therefore,B₅ medium was selected as the basic medium and shading culture medium for callus induction.?2?B₅+NAA 0.5 mg·L^-1+6-BA 1.0 mg·L^-11 was used as the basic medium for callus induction of Isodon amethystoides and healthy callus of it was used as culture material.L₉?3²?orthogonal design was used to study the effects of different concentrations of 2,4-D and KT on the formation of loose callus.The results showed that at different concentrations of 2,4-D and KT,the callus could grow at different levels,of which 2,4-D had a significant effect on the effect and KT was not significant.Based on the results of analysis of variance and range analysis,the optimum medium for the proliferation of Isodon amethystoides was B₅+2,4-D 1.5mg·L^-1+KT 0.5 mg·L^-1.?3?The effects of different concentrations of anti-browning agents?activated carbon,citric acid and polyvinyl pyrrolidone?on callus browning were studied in healthy callus of Isodon amethystoides.The results showed that the appropriate concentration of anti-browning agent can inhibit the browning degree and promote the growth of callus.The proliferation rate,browning status and related physiological and biochemical characteristics of callus were comprehensively analyzed.Adding 0.5g·L^-11 polyvinyl pyrrolidone to the medium to control the browning degree of callus was the best,followed by adding 50 mg·L^-11 citric acid.The total anti-browning effect of the activated carbon is not ideal.?4?The effects of exogenous addition of different concentrations of methyl jasmonate?MeJA?on the contents of ursolic acid and oleanolic acid were studied in healthy callus of Isodon amethystoides.At the same time,the effects of different treatments on the growth,proliferation,browning and physiological and biochemical characteristics of callus were analyzed.The results showed that the contents of ursolic acid and oleanolic acid increased with the addition of MeJA concentration and reached the maximum when the concentration of MeJA was 300?mol·L^-1.The proliferation rate and browning rate generally showed an upward trend which the activity of related protective enzymes?such as peroxidase and catalase?,the content of protective components?such as soluble sugar and soluble protein?increased and the content of MDA increased basically.In the callus of Isodon amethystoides,the components of with oridonin as reference substance were not detected,which may be considered to be due to the variety and lack of characteristic components of diterpenoids to make it impossible to detect.