红锥组培高效无菌系建立及丛芽诱导研究

Sterilization and Primary Culture of Seeds from Castanopsis hystrix

红锥(Castanopsis hystrix)为壳斗科栲属常绿乔木,种皮坚硬、渗透性强,这是阻碍以种子为外植体启动组织培养的主要原因之一。为成功建立红锥组培快繁体系,系统研究了红锥种子灭菌方案、种子萌发与丛生芽诱导培养基。结果表明,红锥种子最佳的灭菌方案为:先将带种皮种子用75%酒精浸泡40 s,用无菌水冲洗1次,然后用0.1%升汞浸泡12 min,无菌水冲洗5～6次,接着剥除种皮,再用0.1%升汞浸泡6 min,无菌水冲洗5～6次,污染率最低,为26.7%,发芽率最高,为73.3%;较好的萌发培养基为改良MS+GA3 3.0 mg·L^(-1),接种后第2天种子即开始萌动,萌发率93.3%,生长旺盛;较好的丛芽诱导培养基为改良MS+6-BA2.0～3.0 mg·L^(-1)+NAA 0.2 mg·L^(-1)+GA3 3.0 mg·L^(-1),丛芽数量平均6.01个。外植体诱导效果与灭菌方案、基本培养基、培养基中激素种类和浓度等有显著的关系。较低盐浓度的改良MS培养基、较高水平的6-BA有利于丛芽的诱导。

Castanopsis hystrix is a genus of superior,valuable evergreen trees.Its seed coat were of strong permeability but very hard,which leads to difficulty in primary culture of seeds.Disinfection procedure,germination and shoot-inducing medium were systematically studied.The results showed that the best disinfection procedure for C.hystrix seed was that,the seeds with the outer seed coat and subsequent tissue were disinfected with 75%(v/v)ethanol for 40 s followed by one rinse(1 min)with sterile distilled water,and then surface disinfected with an aqueous solution of 0.1%of mercuric chloride(w/v)for 12 min followed by five or six rinses(1 min each)with sterile distilled water.Subsequently,the outer seed coat and subsequent tissue were removed to expose intact embryos,and then the embryos were disinfected with an aqueous solution of 0.1%of mercuric chloride(w/v)for 6 min followed by five or six rinses(1 min each)with sterile distilled water,yielding 73.3%contamination-free germinated buds with 26.7%contamination rate.Modified-MS supplemented with 3.0 mg·L^-1gibberellic acid(GA3)was found to be best for seed germination,seed began to germinate the next day after being put into the medium,and the germination frequency reached up to 93.3%.Modified-MS containing 2.0-3.0 mg·L^-1 6-benzyladenine(6-BA),0.2 mg·L^-1 1-napthyl acetic acid(NAA),3.0 mg·L^-1 GA3 was proved to be optimal for adventitious shoot induction with average 6.01 buds per explant.The induction effect of the explant was significantly related to the sterilization protocol,the basic medium,and the hormone type and concentration in the medium.The improved MS medium with lower salt concentration and higher level of 6-BA were beneficial to the induction of the shoot.