Optimization Of Eucommia Ulmoides In Vitro Culture And Regulatory Characteristics Of Chlorogenic Acid Synthesis In Induced Tissues

Eucommia ulmoides Oliver(EUO)is rich in a variety of medicinal ingredients,with high economic value and broad application prospects.Due to the long reproductive cycle of EUO,culture in vitro is the best method to obtain plants quickly.Therefore,in this study,leaves,terminal buds,stems,and stems with axillary buds of EUO were used as explant to optimize the conditions of disinfection and hormone concentration of culture in vitro.The optimal induction tissue for obtaining high medicinal active substances was screened.To reveal the regulatory mechanism of chlorogenic acid synthesis in vitro.This study provides a good experimental basis for the establishment of culture in vitro and rapid propagation system of EUO and the sustainable use of medicinal resources.The optimal disinfection conditions for EUO are 75 % alcohol for 30 s follow by 4 %sodium hypochlorite for 90 s and botany probiotics 2.50 m L/L.Morphological observation shows that the stems are easily induced into callus,and the terminal buds and stems with axillary buds are easily induced into clumps,which are divided into clumps I and II according to their morphological differences.According to the results of induction rate,the optimal ratio of hormone concentration is 0.50 mg/L 6-BA + 0.01 mg/L NAA for induction of cluster seedlings.the optimal ratio of hormone concentration is 0.50 mg/L 6-BA + 0.09 mg/L NAA for induction of callus.Principal component analysis shows that 0.50 mg/L 6-BA + 0.01 mg/L NAA is the optimal hormone ratio for the culture of different explants.Among the different induced tissue cultures of Eucommia ulmoides,the callus shows the highest growth rate of flavonoids content.The highest growth rate of lignin content is observed in cluster seedlings I.The contents of phenylpropanoids,phenols and iridoids in cluster II show the highest growth rate.High performance liquid chromatography-mass spectrometry analysis shows that the compounds in clumps are similar to those in seedlings,and the contents of caffeic acid,cinnamic acid,L-phenylalanine and p-hydroxycinnamic acid in clumps are relatively higher.With the extension of culture time,the content of chlorogenic acid increase,while the contents of cinnamic acid and p-hydroxycinnamic acid decrease firstly and then increase.According to q RT-PCR analysis shows that the expression of 4CL,an upstream gene of chlorogenic acid synthesis pathway,is significantly up-regulated at 0-3 h of culture(P<0.05).The expression of PAL is first down-regulated and then up-regulated,and the expression of the downstream gene HCT is first up-regulated and then down-regulated.Correlation analysis shows that the contents of caffeic acid,cinnamic acid and p-hydroxycinnamic acid are significantly positively correlated with the content of chlorogenic acid(P<0.01).The expression of PAL gene is positively correlated with the contents of chlorogenic acid,cinnamic acid and p-hydroxycinnamic acid(P<0.05).These results indicate that the regulation of PAL gene expression is beneficial to promote the synthesis of chlorogenic acid in the of culture in vitro for cluster seedlings.In summary,we optimized the culture in vitro conditions of EUO and screened the most suitable induction tissues.The induced clumps can rapidly accumulate the active components of medicinal plants.This study provides a reference for further study on the regulation of secondary metabolism in vitro culture of EUO.To provide theoretical basis and technical support for the development of EUO factory seedlings and medicinal resources.