Cryopreservation of Chinese Taxus chinensis seeds | 中国红豆杉种子超低温保存技术研究

Chinese. 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。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0.6 MPa抽真空和冰浴250 W超声波处理，处理时间分别为0，5，10和15 min；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。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PVS-4，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1 h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。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12 h，脱去种子内皮，在无菌条件下用体积分数75%酒精消毒30 s，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10 min，于-20 ℃预冻1 h后置于液氮中冷冻保存72 h，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PVS-4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。

English. 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. 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 standing 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 bath, thawing in water flow, standing at 5 ℃ and standing at 25 ℃). The relationship between preserving time and the viability of Chinese T. chinensis seeds under the three superior cryoprotectans condition was discussed. TTC (2, 3, 5-triphenyl tetrazolium chloride) seed viability quantitative testing method was used to get the seeds viability. The result shows that cryoprotectants, cryoprotectant treating methods, cryoprotectant treating times, freezing methods, defreezing methods and the cryopreservation times all significantly influenced the viability of the cryopreserved Chinese T. chinensis seeds. The cryoprotectants which can maintain cells in a high activity after cryopreservation were: 8% DMSO, 5% EG and PVS-4. The TTC reduction amount of cryopreserved seeds were (2.679 7±0.255 6), (2.290 4±0.040 7) and (2.055 5±0.009 9) mg/g, separately; 250 W ultrasonic wave treatment in ice bath for 10 min was the best, TTC reduction amount was (2.143 1±0.098 6) mg/g; staying at -20 ℃ for 1 h then put into LN2, the cell activity was relatively high, TTC reduction amount was (2.454 4±0.069 5) mg/g; thawing in 37 ℃ water bath (rapid thawing) had higher cell viability after, TTC reduction amount was (2.469 7±0.022 1) mg/g. 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 PVS-4 cryoprotectant is more conducive for long-term (more than 1 months) ultra- low-temperature preservation of Chinese T. chinensis seeds.