摘要
【目的】探索中国红豆杉种质资源的超低温保存技术,为红豆杉种质资源的开发利用提供支持。【方法】采用多重单因素试验法,将中国红豆杉种子前处理后,采用9种不同的冷冻保护剂处理,之后分别进行0℃冷浴静置、25℃室温0.6MPa抽真空和冰浴250W超声波处理,处理时间分别为0,5,10和15min;然后按照4种不同的冷冻方式投入液氮中冷冻保存,保存结束后分别采用37℃水浴快速、20℃流动自来水、5℃慢速和25℃室温静置进行解冻,最后探讨了在较优的3种冷冻保护剂条件下保存时间对红豆杉种子活力的影响。以氯化三苯基四氮唑(TTC)还原法测定冷冻保存后种子的活力。【结果】冷冻保护剂、保护剂处理方式和处理时间、冷冻方式、解冻方式、保存时间均对超低温冷冻保存后的红豆杉种子活力具有明显影响。9种冷冻保护剂中较优的是体积分数8%二甲基亚砜(DMSO)、体积分数5%乙二醇和PVS4,冻存后中国红豆杉种子TTC还原量分别为(2.679 7±0.255 6)、(2.290 4±0.040 7)和(2.055 5±0.009 9)mg/g;冰浴250 W超声波处理10 min的效果最好,TTC还原量为(2.143 1±0.098 6)mg/g;4种冷冻方式中-20℃预冻1h后投入液氮保存红豆杉细胞活性相对较高,TTC还原量为(2.454 4±0.069 5)mg/g。37℃水浴快速解冻后细胞活力更高,TTC还原量为(2.469 7±0.022 1)mg/g。【结论】红豆杉种子的最佳超低温保存技术条件为:采收后的新鲜种子干燥后于5℃条件下放置9个月打破其休眠,然后机械去掉种子硬壳,用蒸馏水浸泡12h,脱去种子内皮,在无菌条件下用体积分数75%酒精消毒30s,无菌水冲洗2～3次,沥干,移入冷冻管中,加入体积分数8%DMSO冷冻保护剂,封口,于冰浴250 W超声波条件下浸泡处理10min,于-20℃预冻1h后置于液氮中冷冻保存72h,然后在37℃水浴中快速解冻。在此条件下,中国红豆杉种子活力最高,TTC还原量可达(2.469 7±0.022 1)mg/g,而且同等条件下,选用PVS4冷冻保护剂更有利于红豆杉种子的超低温长期(1个月以上)保存。
[Objective] The study was to explore ultra-low-temperature preservation technology of Chinese Taxus chinensis germplasm resource and to provide support for its development and utilization. [Method] Multiple single factor test method was used in the study. After pre-treatment, the Chinese T. chinensis seeds were added into 9 different cryoprotectants;The cryoprotective methods included stand- ing in ice bath,0.6 MPa the vacuum treatment at 25℃ and 250 W ultrasonic wave treatment in ice bath; The time was set to be 0,5,10 and 15 min. The seeds were put into liquid nitrogen for cryopreservation by 4 different freezing methods. Then the seeds were thawed by 4 defreezing methods（thawing in 37℃ water ℃ water bath （rapid thawing） had higher cell viability after, TTC reduction amount was （2. 469 7± 0. 022 1） mg/g. [Conclusion] The optimum cryopreservation technology conditions are as follows. The dormancy of new seeds are broken by putting them at 5℃ for 9 months, then the seeds are peeled and soaked for 12 h in distilled water,the endodermis removed,disinfected by 75% alcohol for 30 s under aseptic condition,washed 2--3 times by aseptic water, drained the surface water, then the seeds put were into freezing tubes,added the 8% DMSO cryoprotectant,sealed and soaked the tubes in water for 10 min under ultrasonic wave condition,pre-frozen at -20℃ for 1 h, put into liquid nitrogen for cryopreservation and then thawed in 37℃ water bath. Under this condition,the seeds would obtain higher cell viability,TTC reduction amount is （2. 469 7±0. 022 1） mg/g. Moreover, under the same conditions,the PVS4 cryoprotectant is more conducive for long-term （more than 1 months ）ultra-low-temperature preservation of Chinese T. chinensis seeds.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2012年第2期71-78,共8页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家林业局林业公益性行业科研专项(200704009)
关键词
中国红豆杉种子
超低温保存
种子活力
定量TTC法
Chinese Taxus chinensis seeds
cryopreservation
seed viability
TTC seed viability quanti- tative testing
作者简介
程利红（1985-），女，陕西宝鸡人，在读硕士，主要从事食品科学及食品工程新技术研究。
[通信作者]孙宝胜（1958-），男，陕西米脂人，副研究员，主要从事濒危植物保护研究。E-mail：sunbs1958@sohu．com
