培养条件对杜仲愈伤组织形成及次生代谢过程的影响

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

对杜仲愈伤组织诱导研究表明 ,诱导出愈率在不同外植体类型、外植体年龄与部位、外植体接种方式间存在显著差异 ,5 mm× 5 m m大小、叶龄为 15 d左右的糙皮杜仲叶片的中下部外植体在采用叶片背面贴置于培养基的培养方式出愈率最高 ;培养基类型、激素水平和光照条件对出愈效果均有显著的影响 ,在附加 2 ,4 - D(0 .5 m g·L- 1 )和 BA(0 .6 m g· L- 1 )的 Harada培养基和附加 2 ,4 - D(0 .5 mg· L- 1 )、BA(0 .6 m g· L- 1 )和 BA(0 .6 m g· L- 1 )或附加 NAA(0 .5 m g· L- 1 )的 Bourgin & Nistch培养基上 ,以 2 4 h的黑暗启动培养加以光暗交替培养 (光照 14 h+黑暗 10 h) ,并在接种后 12 d内更换培养基 ,均能诱导出杜仲愈伤组织 ,以叶片作外植体时 ,BA的浓度建议不高于 1.0 mg· L- 1 ,KT的存在不利于愈伤组织的诱导 ;在附加了 KT(0 .7mg· L- 1 )、NAA(0 .8m g· L- 1 )和 10 mg· L- 1 葡聚糖的 MS培养基上 ,通过向培养基中添加递进浓度的丙酮酸研究表明 ,杜仲愈伤组织中的次生代谢产物桃叶珊瑚甙的产量 ;通过继代培养的 4因素 3水平正交试验发现 ,培养温度、碳源、培养基类型及激素水平对杜仲叶片愈伤组织生长速率及愈伤组织中的氯原酸含量均有显著的影响 ,适宜愈伤组织生长的培养条件有别于适?

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.