The Mechanism Of Stress Response To Vitrification-Cryopreservation Treatment In Rice Embryo

Cryopreservation,which keeps the materials be preserved in liquid or vapor phase of liguid nitrogen(LN),is considered as the most advanced methods for the long-term storage of plant germplasm.During the cryopreservation procedure,materials will suffer from mechanical injury,osmotic stress,toxic injury result from cryoprotectants,high dehydration,extreme hypothermia and ROS stress,etc.These stresses may cause damage to cells,involving abnormal physiological metabolism,abnormal cytological changes,and injuries of cell membrane system,etc,which affect the survival and regrowth of materials after exposure to LN.In the present study,the changes of cell ultrastructure and physiological metabolism in rice "9311"(Oryza sativa,Indica)germinated embryos during the cryopreservation procedure were investigated,which may facilitate the research of response mechanism of cells to stresses during cryopreservation.The main results were as follows:1)The vitrification cryopreservation procedures of rice 48h-germinated embryo was optimized using the orthogonal experiment design methods:the embryos were precultured for 36h in preculture solution which supplemented with 0.5mol/L sucrose,loaded for 10min,dehydrated in PVS2 solution for 10min in the ice bath,and then exposed to LN.When the embryos were rewarmed and recovered,the survival and regrowth percentages were 96.8%and 82.9%,respectively.Besides,our results also revealed that the significant correlation between the germination duration of rice germinated embryos and survival and regrowth after cryopreservation when the germination duration of embryos were from 48h to 72h.Importantly,the investigations of significantly changes of viability and stress response in 24h-,48h-,72h-germinated embryos revealed the tissue-temporal and spatial pattern during cryopreservation procedures.Embryo germination with 48h-72h can be used as the materials to study the stress mechanism of cryopreservation.2)The ultrastructual changes of cells in meristems and their surrouding region of 24h-,48h-and 72h-germinated embryos during cryopreservation procedures of embryo germination for,i.e.preculture,loading,dehydration,and LN exposure,were observed using TEM methods.The results showed that the water contents and their compositions of cells may significantly affect the survival and regrowth after cryopreservation.Remarkably,the dehydration treatment during cryopreservation procedures improved the survival and regrowth percentages of germinated rice embryos after cryopreservation,meanwhile lead to the stresses or injuries,e.g.cell structure deformation,mitochondrial swelling,heterochromatin increase and so on.3)Results of the investigations of the cell viability,ROS generation,MDA content,REL etc,showed that when the embryos were treated with preculture,loading,and PVS2 dehydration,ROS accumulation and significant oxidative stress in the embryos were observed,which lead to serious damage of lipid peroxidation and membrane integrity.Meanwhile,it was found that antioxidant systems were triggered to reponses of these stress by investigation of antioxidant contents(ASA,DHA,GSH and GSSG),antioxidant enzyme activities and their expression(SOD,CAT,APX,MDHAR,DHAR and GR).Besides,the supplementation with 6 mol/L exogenous ASA significantly improved the survival and regrowth after cryopreservation.4)Results of the changes compositions and unsaturation degree of lipid components,especially the phospholipids(GP),showed that dehydration treatments reduced the lipid metabolism in terms of the signal transduction,lipid synthesis and membrane fusion;however,these treatments have no fatal effects on the bimolecular structure,elasticity and fusion of the membrane system.In addition,the unsaturation degree of lipids components decreased,and it was found that the serious oxidative stress in terms of lipid peroxidation during cryopreservation procedures.Remarkably,PG(18:1(9Z)/18:3(9Z,12Z,15Z))was considered as the potential indicator for oxidant stress in terms of the acceleration of lipid peroxidation.When the materials were exposed to LN,in the surviving cells,it was observed that the fluidity,elasticity,fusion and integrity of the membrane systems were kept,and the regulation in the signal transduction,lipid synthesis,development and function were activated.Moreover,18:2-glc-campesterol and integral-sitosterol 3-o-popali-d-galactopyranoside were selected as the potential indicators for survival.In conclusion,many factors influenced the survival and regrowth percentages.The serious damages in cell ultrastructural were observed and the fresh embryos were dead when exposing to LN without any treatments.Dehydration treatment was found to promote to maintain the ultrastructure in meristem cells,activate the antioxidant systems,keep the integrity of membrane system and regulation in the signal transduction,lipid synthesis,development and function,which may be necessary to survive and regrowth after cryopreservation.However,these treatments also caused ROS accumulation and severe oxidative stress,weakened the ability to participate in the signal transduction,membrane fusion and lipid synthesis,as well as the significant changes in the cell ultrastructure,i.e.deformation,mitochondrial swelling,increased heterochromatin.Exogenous antioxidants can reduce oxidative stress and facilitate the survive and regrowth after cryopreservation.