Influence Mechanism Of ROS Induced Oxidative Stress And Apoptosis On Cell Viability Of Agapanthus Praecox Callus During Cryopreservation

This research was conducted to study the action mechanism of ROS induced oxidative stress and PCD of Agapanthus EC and NEC during Cryopreservation, for optimizing the Agapanthus cryopreservation system using exogenous substances. The regulating effect of optimal exogenous substances to ROS metabolism, antioxidant system and PCD of Agapanthus NEC cryopreservation was investigated in physiology levels, which further verified the result of the antioxidant mechanism and PCD action mechanism of Agapanthus. The results were as follows: 1 、 The antioxidant mechanism and PCD action mechanism of Agapanthus EC and NEC during Cryopreservation.(1) The cell relative viability of Agapanthus EC and NEC had an negatively relationship to the lipid peroxidation level. The levels of lipid peroxidation of Agapanthus were increased during cryopreservation, and the dehydration and dilution were the main steps for oxidative stress, which induced by H2O2 generated..(2) CAT 、POD and SOD were scavengers of H2O2 as the main components of antioxidant system in Agapanthus EC and NEC cryopreservation. Quantitative analys is of differentially expressed genes related to antioxidant system also confirmed the importance of the antioxidant system of Agapanthus EC during cryopreservation.(3) The cell relative death rate had the same variation tendency to PCD in Agapanthus EC and NEC cryopreservation, and PCD is the important factor to result in the low relative viability. The whole process of cryopreservation is accompanied by PCD, especially in the dehydration and dilution steps. And quantitative analysis of differentially expressed genes related to PCD also confirmed the occurrence of PCD during Agapanthus EC cryopreservation. 2、Optimization of Agapanthus NEC cryopreservation system.(1) Exogenous antioxidants and apoptosis inhibitors with different concentrations had positive effects on the relative survival rates of Agapanthus EC and NEC after cryopreservation. The addition of 1.0 μmol/L D-CHO could highly enhanced the relative survival rates of Agapanthus EC and NEC. The following were 0.06mmol/L CinnamtanninB-1.(2) Establishment of optimized Agapanthus NEC cryopreservation was to added 1.0 μmol/L D-CHO and 0.06mmol/LCinnamtannin B-1 to the vitrification solution.(3) Lipid peroxidation of the Agapanthus NEC were increased gradually during cryopreservation. However, lipid peroxidation level in optimized system was relatively low. Exogenous Cinnamtannin B-1 significantly reduced the content of H2O2 and improved the antioxidant ability significantly, especially the POD activity and CAT activity.(4) Exogenous D-CHO obviously alleviated the occurrence of PCD in Agapanthus NEC cryopreservation, and significantly improve the cell survival rate after frozen. And successfully optimized the cryopreservation system of Agapanthus NEC.