摘要
本研究以秦巴山区三种濒危植物秦岭冷杉（Abies chensiensis）、连香树（Cercidiphyllum japonicum）、中国红豆杉（Taxus chinensis）的种子胚、定芽或种子为试验材料，采用单因素试验和正交试验研究了冷冻保护剂类型、冷冻保护剂处理方式、冷冻方式、保存温度和解冻方式以及保存时间等因素对三种试验材料超低温保存后细胞活力的影响，进而确立了三种濒危植物种质资源的最佳超低温保存技术条件；采用氯化三苯基四氮唑（TTC）还原法和细胞微结构分析法对保存效果进行了评价，分析了超低温保存对细胞超微结构的影响及其与细胞活力的关系，初步揭示了超低温冷冻保存对植物组织细胞活力的影响机理。
研究结果表明：
（1）秦岭冷杉种子胚最佳超低温保存条件是：秦岭冷杉种子胚消毒后用PVS（435%甘油+20%乙二醇+0.6mol/L蔗糖的脱氨MS液体培养基）玻璃化保护剂经250W超声冰浴处理30min，在﹣20℃条件下预冻2h，投入液氮中保存7d，37℃恒温水浴解冻30min，秦岭冷杉种子胚冻存后细胞存活率最高，达到91.73%。
（2）连香树定芽细胞最优超低温保存条件是：连香树定芽消毒后，用含35%甘油、20%单乙烯乙二醇、15.0%二甲基亚砜（DMSO）和1%甘氨酸的3%蔗糖脱氨MS液体培养基作为冷冻保护剂装载于冷冻管中，于400mmHg真空常温下处理30min，再经﹣20℃预冻6h，投入液氮保存60d后，40℃恒温水浴解冻30min，连香树定芽细胞相对存活率最高达95%以上。
（3）中国红豆杉种子最优超低温保存条件是：经机械去壳的中国红豆杉种子加入8%DMSO，0℃超声（250W）浸泡处理10min，于﹣20℃预冻1h后投入液氮中冷冻保存72h，37℃恒温水浴中快速解冻1h，红豆杉种子的超低温保存活力最高，TTC还原量可达为2.4697±0.0221mg/g；而PVS4更有利于红豆杉种子的超低温长期保存。
（4）影响超低温保存后细胞活力的主要因素：冷冻保护剂类型、冷冻方式和保存温度，都直接影响着细胞内冰晶的形成状态，从而引起对细胞超微结构的损伤。在不同的超低温冷冻保存条件下，细胞的活力和细胞超微结构的损伤程度不同，细胞超微结构损伤程度越大，细胞活力越弱，影响细胞活力主要由冷冻过程内细胞中形成的冰晶体数目、体积和形状。
Three kinds of endangered plant species in the Qinba Mountains: Abies chensiensis,Cercidiphyllum japonicum and Taxus chinensis were used as experimental material using theseed, embryo or bud to cryopreserve by single factor and orthogonal experiment design.Theeffects of the cryopreservative type, mode of cryopreservative treatment, freezing, storage andthawing temperature as well as storage time on the cell viability after cryopreservation wereinvestigated to establish the best cryopreservation conditions for the three endangered plant inthis experiment. After the cryopreservation the cell relative survival rate was evaluated byTTC reduction and mic-structure analysis method. The relationship between cell ultrastructureof cryopreserved plant tissues and the cell viability was analysised, the influence mechanismof the preservation process for cryopreservation on cell viability of the plant tissue wasinitially revealed
The results were shown below：
(1) The best cryopreservation condition of Abies chensiensis embryo is that the Abieschensiensis embryo was removed and then dehydrated with a highly concentrate vitrificationsolution(PVS4) after pretreatment, the compositon of PVS4is35%Gly+20%EG+0.6mol/Lsuc of MS﹣NH4+, then was treated for30min with250W ultrasonic wave in ice-water bath,and was pre-frozen for2h at﹣20℃and plunged directly into the liquid nitrogen saving for7d,rapidly thawn at37℃, the highest relative cell survival rate was obtained to91.73%.
(2) The optimal cryopreservation conduction for Cercidiphyllum japonicum buds is thatthe buds were pre-treated, followed by cryoprotective treatment in de-ammonia MS liquidmedium containing35%glycerine,20%monomer ethylene glycol(MEG),15%dimethylsulfoxide(DMSO),1%Glycine and3%sucrose, loading into refrigerated tubes, then processedfor30min at room temperature under400mmHg vacuum condition and cooled to﹣20℃,held for6h and then immersed in liquid nitrogen, saved for60d before quickly thawing at40℃water bath reached to the best relative cell survival rate(more than95%).
(3) The best suitable cryopreservation condition for Taxus chinensis seeds is that theseeds were mechanical shelled and then loaded into tubes by the protective agent of thede-ammonia MS liquid medium with3%sucrose and8%DMSO and were treated for10minin0℃ice-water bath with250W ultrasonic, then were cooled at﹣20℃for1h, the tubes was put into liquid nitrogen immediately and saved for72h and defrost at37℃, the TTCreduction amount of past-thawing Taxus chinensis seeds successfully reached the highest to2.4697±0.0221mg/g. Furthermore, under the same conditions, use PVS4as cryoprotectantcan be more conducive for long term conservation of Taxus chinensis seeds.
(4) The transmission electron microscope test result indicated that the main factors whichaffect the cell viability after cryopreservation is cryoprotectant type, freezing and preservationtemperature and so on, also they had a direct impact on the changes of cell ultrastructure.Under the different cryopreservation conditions, the viability of the post-thaw cell and theextent of damagment in cell ultrastructure are different. The greater the extent damage of thecell ultrastructure, the cell viability beacome weaker. This is mainly because that cell viabilityis driven by the ice crystals generated in the ultrastructure of cells, such as its number, sizeand shape.
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