Research On Cryopreservation Technique Of Tissue Cells Of Three Species Endangered Plants In Qinba Mountainous Area

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 revealedThe 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.