The Effect Of Different Species Of Physicochemical Factors And Endophytes On The Chlorogenic Acid Content And The Growth Of Cell In Suspension Cultures Of Lonicera Macranthoides Hand.-Mazz. ‘Yulei1’

Lonicera macranthoides Hand.-Mazz. ‘Yulei1’belonged to Lonicera japonica, was approved as a new variety of honeysuckle and had an important medicamentosus value.Compared with traditional honeysuckle, this variety has high yield and stress resistance,and most importantly high content of chlorogenic acid.The level of chlorogenic acid is the main evaluation index of quality of honeysuckle.However, traditional extraction of chlorogenic acid depended on the raw materials of honeysuckle foliag.This study was designed to optimize cell suspension culture system and investigate the production of chlorogenic acid in the suspension cultures of L. macranthoides Hand.-Mazz.In addition, this study also weighed the biomass of the ‘Yulei1’ cells and analyzed the content of chlrogenic acid by the addition of different kinds of endophytes in cell suspension cultures of ‘Yulei1’.（1） Optimization of physicochemical culture conditions: The appropriate callus was inducued by explants of ‘Yulei1’ and the appropriate suspension cultures was established. The study investigates effect of different species of physicochemical factorson cell in suspension cultures of L. macranthoides Hand.-Mazz. ‘Yulei1’. The result showed the optimized conditions for biomass accumulation and chlorogenic acid production were 15 days after culture growth, initial medium p H of 5.5,30-40 g.L-1 sucrose,50 g·L-1 of inoculum on fresh weight basis and full strength B₅ medium.For the study of single homones, maximum accumulation of chlorogenic acidand biomass were observed when the culture medium was supplemented with2.0 mg·L-16-BA.For the study of combination of homones, optimal accumulation of chlorogenic acid was observed using combination of homones with 6-BA 2.0mg.L-1+2,4-D 0.5 mg.L-1, while optimal accumulation of biomass was observed with6-BA 2.0 mg.L-1+2,4-D 2.0 mg.L-1. In addition, phenylalanine also contributed to the synthesis of chlorogenic acid at a concentration > 50mg·L-1.Different species of inorganic salt on the chlorogenic acid content and the growth of cell in suspension cultures of L. macranthoides Hand.-Mazz. ‘Yulei1’ were tested. The result showed the suspension culture would be benefit to the accumulation of chlorogenic acid and growth of cell when NO3-/NH4+（nitrate nitrogen/ ammonium nitrogen）ratio was 13:1.The biomass of callus reached the maximum（19.26 g·L-1） in 3.5 g·L-1KNO3.However,while KNO3 concentration was at relatively low levels（0.5 g·L-1and1.5 g·L-1）,high chlorogenic acid production was tested.when the highest growth of biomass and the high chlorogenic acid production were tested, Na H2PO4·2H2O concentration was 0.10g·L-1. In addition, when the highest growth of biomass and the high chlorogenic acid production were tested, Ca Cl2 concentration was 0.20 g·L-1.So far as Mg2+was concerned, a low Mg SO4·7H2O concentration could promote the growth of cell, while excessive Mg SO4·7H2O concentration would promote the accumulation of chlorogenic acid. Taking the biomass growth and chlorogenic acid production into consideration, the optimal concentration of Mg was moderate.（2） Effects of Endophytes of L. macranthoides Cultivar ‘Yulei1’ on the Biomass Accumulation and Chlorogenic Acid Production: We isolated 17 kinds of endophytes from L. macranthoides cultivar ‘Yulei1’. Besides, the 6kinds of endophytes were identified through the 16 S r DNA or 18 S r DNA sequences homologous analysis,andmorphological observation.The 6kinds of endophytes included Rhizobium sp.（cc2）, Pseudomonas sp.（cc4）, Coniothyrium minitans（cc5）, Bacillus altitudinis（cc13）, Bacillus stratosphericus（cc14）, and Bacillus pumilus（cc15）. In addition,the biomass accumulation and chlorogenic acid production of L.macranthoides ‘Yulei1’were influenced by 6 kinds of endophytes. As for biomass accumulation, the maximum accumulation of biomass was observed while 6 kinds of endophytes concentration was 25 mg.L-1. In terms of chlorogenic acid production, the highest chlorogenic acid production of L.macranthoides ‘Yulei1’ was tested while 4 kinds of endophytes（Uncultured alpha proteobacterium-Rhizobium sp., Pseudomonas sp.,Coniothyrium minitans and Bacillus stratosphericus） concentration was 25 mg.L-1.However, the highest chlorogenic acid production of L. macranthoides Hand.-Mazz‘Yulei1’ was tested while 2 kinds of endophytes（Bacillus altitudinis and Bacillus pumilus） concentration was 12.5 mg.L-1.In summary, the physicochemical culture conditions were optimized in suspensio n cultures of L.macranthoides Hand.-Mazz. ‘Yulei1’. And relationship between endop hytes and chlorogenic acid in suspension cultures of L. macranthoides Hand.-Mazz. ‘Yulei1’ were studied.This study also had practical significance to large-scale productions of chlorogenic acid.