The influences of culture conditions on the callus induction,tissue culture and regulation of secondary metabolism of Eucommia ulmoides Oliver

Studies on the callus induction,tissue culture and secondary metabolism regulation of Eucommia ulmoides were carried out in a controlled illuminating incubator.The main results obtained were as fol- lows:( 1 ) the callus induction rate( CIR) was significantly influenced by the explantcharacters.CIR of ex- plant derived from the leaves of the coarse- bark cultivar is92 .61 %± 1 0 .0 2 % ,significantly higher than 30 .93%± 1 0 .1 6% of the smooth- bark one. CIR of the explantderived from 1 0～ 1 5 d- old leaves in the up- permost part of current growing shoot is 71 .45 %± 1 0 .44% ,significantly higher than 8.77%± 1 2 .1 3%from 2 0～ 2 5 d- old leaves and 0 .0 % from 30 d- over leaves.Explantfrom the middle to proximal partgave 60 .70 %± 5 .78% of CIR while the explant from the apical partin same leaf only gave 2 1 .87%± 1 3.72 % . Explantfrom vein- free leaf produced higher CIR.In addition,CIR was also significantly influenced by ex- plantsize and installing method;( 2 ) Media type,hormone concentration and illuminating conditions had significantinfluence on CIR.Callus were successfully obtained in Harada Medium supplemented with2 ,4- D( 0 .5 mg·L-1) and BA( 0 .6mg·L-1) ,and in Bourgin Nistch Medium supplemented with2 ,4- D( 0 . 5 mg· L-1) and BA ( 0 .6mg· L-1) with 2 4 h dark initiated culture plus light- dark- alternative culture and replacement of medium within 1 2 d after inoculation.1 2 h dark initiated culture is a prerequisite to a suc- cessful callus induction;( 3) Progressively increasing concentration of pyruvic acid added into MS Medium with the supplement of0 .7mg·L-1KT,0 .8mg· L-1NAA and1 0 mg glucosan resulted in the changes of the secondary metabolite aucubin production.There was a significant relationship between the yield of au- cubin in the callus( Y) and the concentration of pyruvic acid added into the medium( P) .A quadratic co- re- lation equation was Y( % ) =- 0 .0 0 1 2 P2 + 0 .0 2 83P+ 0 .5 1 5 under present conditions;( 4 ) The growth rate of callus and the production of chlorogenic acid in the callus was significantly influenced by culture conditions including temperature,medium type,carbon source,hormone concentration and so on.Condi- tions which favorable to callus growth was differentfrom thatsuitable for chlorogenic acid accumulation in the callus.White Medium supplemented with0 .6mg·L-1BA,0 .5 mg·L-1NAA,3% sucrose as carbon source,5 mg·L-1phenylalanine and2 8℃ was favorable to the production of chlorogenic acid of callus, which greatly varied under different culture condition.