Tissue Culture And Hairy Root Inducing Of Echinacea Angustifolia And Studies On The Accumulation Of Echinacoside And Chlorogenic Acid

Echinacea angustifolia DC (Asteraceae) ,also named narrow-leafed purple coneflower, is native to North American and it is a traditional American indian's herb. The main active compounds of E. spp. are alkamides, polysaccharides,polyphenolic , caffeic acid derivatives and so on. Since they have the effect of immunostimulation, echinacea preparations have been widely used in Europe and North America. Echinacoside is the main caffeic acid derivatives of E. angustifolia. According to the results of recent pharmacopoeial articles, it has many effects such as antimicrobial, cell apoptosis regulation and so on. In recent years this species have been successfully introduced into some areas of China such as Beijing ,Shenyang and Shangdong. Because of dormancy, the seed germination percentage of E.angustifolia is very low, which highly limited the fast regenaretion of this plant. Influenced by climate and soil factors, the content of the active components fluctuates and it is difficult to control the quality of the related medicine. So, biotechniques is an essential way to solve these problems especially to improve the production of bioactive secondary metabolites.In this research , a technical system for rapid propagation of E. angustifolia was established by Orthogonal Test , using mature seeds of the wild Canada E.angustifolia. The hairy roots inducing system of E.angustifolia was also established by two agropine strains. A method of High Performance Liquid Chromatography(HPLC) was developed for simultaneous determination of chlorogenic acid and echinacoside in the tissue cultures and hairy roots of E.angustifolia. The main research aspects are as follows:1. Tissue culture and plants regenaretion of E. angustifolia: Efficient plant regeneration was achieved via organogenesis from leaf and stem explant of E. angustifolia. According to the results of Orthogonal Test L9(3~4) Murashige and Skoog(MS)medium supplemented with 6-benzylaminopurine (6-BA)0.5 mg/L and naphthaleneacetic acid (NAA)1.0 mg/L was most effective for callus inducing, while MS medium supplemented with 6-BA 1.0 mg/L and NAA 0.5 mg/L was best for shoots genisis. Plantlets were rooted on 1 / 2MS medium supplemented with different concentrations of indole- 3-butyric acid(IBA) high rooting and survival was achieved when the IBA concentration was 0.5 mg/L. The contents of chlorogenic acid and echinacoside in the callus, roots and upper ground parts of E. angustfolia were analyzed by HPLC respectively .The contents of the two secondary metabolites were both high in the roots ,while in the callus both of them were relatively low, especially echinacoside was hardly detectable .2. Establishment of hairy root inducing system of E.angustifolia: Axenically grown E.angustifolia plantlets were inoculated with two Agrobacterium rhizogenes strains A4 and R1000. Hairy root lines were established 20 days after inoculation with the two agropine strains. Polymerase chain reaction with primers for the gene rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A.rhizogenes to the genome of hairy roots. The content of echinacoside and chlorogenic acid in four hairy root strains ,A4-1,A4-2,R1000-1and R1000-2 were analyzed by HPLC.The results indicated there are differences in the content of secondary metabolites among the four strains.The content of echinacoside (0.16 mg/g DW)and chlorogenic acid(0.82mg/g DW) in A4-2 are more than the amount in nature roots (echinacoside 0.15 mg/g DW, chlorogenic acid 0.68mg/g DW). The amount of chlorogenic acid in R1000-2 was 0.94 mg/g DW 1.4 times of the content in nature roots(0.68mg/g DW).3. Establishment of root suspension system for E.angustifolia: The root suspension system was established by supplementing 1 / 2MS liqid medium with different plant hormone. According to the results , adding IBA 0.5 mg/L in the medium was more profitable for root growth than adding 2,-4-D and NAA ,because 2,-4-D and NAA will lead to callus genesis of the suspension root.By adding phenylalanine to the medium for root suspension, we found that 5mg/L phenylalanine benefit for the growing of roots and when the phenylalanine concentration was 20mg/L the highest content of secondary metabolites were detected.