Cryopreservation Of Paeonia Emodi Seeds And The Mechanism Of Seed Water Content Affecting The Viability

Cryopreservation is one of biological germplasm preservation techniques,and it has the potential to preserve germplasm permanently.Seed water content is a key factor affecting seed viability and cryopreservation effect.Only the seeds with suitable water content have higher viability.However,the mechanism of seeds with different water content respond to cryopreservation is currently not completely clear.Based on the research on the cryopreservation technology procedures of wild plant Paeonia emodi seeds,this paper studied the occurrence of oxidative stress and programmed cell death（PCD）before and after cryopreservation of seeds with different water contents;Added H₂O₂ and Ca²⁺regulators to the source to study the effects of changes in endogenous Ca²⁺and H₂O₂ on oxidative stress and PCD,explored the mechanism of differences in viability of seeds with different water content after cryopreservation.The main results are as follows:（1）The seed viability of the naturally harvested P.emodi was directly proportional to the water content.There is no significant difference between the seed viability when the water content was21.36%～8.77%,which was 98.89%～97.74%;but when the seed water content was 7.07%～At 3.72%,it droped to 82.22%～0.00%.The critical value of safe water content of P.emodi seeds at room temperature was 8.77%.（2）The seed of P.emodi with 8.77%water content could be successfully cryopreserved with 97.80%seed viability.The seed were thrown into liquid nitrogen directly or slowly and thawing in tap water or water bath after liquid nitrogen storage.The seed viability was 95.48%and 97.82%,respectively,but no obvious difference.After 90 days of cryopreservation,the seed viability was 96.67%by this technology procedure,which was not significantly different from the that of naturally harvested seeds.（3）When the seed water content of P.emodi decreased to 7.07%,the ROS and the MDA content in the seeds increased significantly,and the seed viability decreased to 82.22%significantly.But at the same time,the PCD related indicators did not significant increase.That indicates that the main reason of seed vitality decline caused by the reduced seed water content is cell membrane lipid peroxidation at room temperature.（4）After cryopreservation,the MDA content and PCD related indicators from the seeds with 8.77%water content did not change significantly.And the seeds ultrastructure also did not change significantly.However,when the seed water content was higher（21.36%,18.33%）,all ROS components increased,which was consistent with the MDA content increased.The O₂^-concentration and the MDA content showed a very significant positive correlation.The seed ultrastructure showed the typical PCD characteristics:the cytoplasmic wall was clearly separated;the cell membrane ruptured;the cytoplasmic material leaks,and the nucleus shrunk;and the nucleus-to-cytoplasm ratio significantly reduced.At the same time,the apoptotic enzymes activity was significantly increased,and the mitochondrial membrane potential dropped significantly.It showed that the O₂^-plays a major role by damaging the oxide film and together with the PCD pathway to reduce,the viability in the cryopreservation of the seed with higher water content.（5）Adding exogenous H₂O₂（50 mmol/L）increased the endogenous H₂O₂ content of cryopreserved seeds with 7.07%-21.36%water content,deepened the degree of cell membranes lipid peroxidation;promoted the occurrence of PCD,resulting in a significant decrease in seed viability by 37.07%-50.70%.The exogenous antioxidant As A（7.5μmol/L）reduced the endogenous H₂O₂ content of cryopreserved seeds,alleviated the degree of peroxidation of seed membrane lipids,significantly reduced Caspase-3 like activity,inhibited the occurrence of PCD in certain extent,and improved seeds viability.It indicated that H₂O₂can reduce the seed viability by mediating the damage of the oxide film and inducing the occurrence of PCD.（6）The external addition of Ca Cl₂（100 mmol/L）could increase the endogenous Ca²⁺content of cryopreserved seeds,promoted the PCD occurrence,and significantly improve the·OH production capacity.The exogenous Ca²⁺chelating agent EGTA（50μmol/L）reduced the endogenous Ca²⁺content of the seeds,inhibited the PCD occurrence,significantly reduced the MDA content of high-water content seeds（18.33%and 21.36%）,and alleviated the lipid peroxidation degree of the cell membrane and the seed viability increased by 50.03%and 30.08%respectively.It shows that seed water content affects PCD and the degree of membranes lipid peroxidation by changing the Ca²⁺concentration and ROS content,thereby affecting seed viability.This study determines the technical procedures for seed cryopreservation of P.emodi.It clarifies that the change of seed water content of P.emodi affects seed vitality by affecting cell membrane lipid peroxidation at room temperature,but by controlling cell membrane lipid peroxidation and PCD during cryopreservation,which H₂O₂ and Ca²⁺played a key role in the two ways.