Optimization Of Agapanthus Embryogenic Callus Cryopreservation Based On The Arabidopsis Antioxidant Mechanism

This research was conducted to study the ROS and antioxidant mechanism of different Arabidopsis seedlings during cryopreservation, for optimizing the Agapanthus embryogenic callus(EC) cryopreservation system using exogenous substances. The regulating effect of optimal exogenous substances to ROS metabolism and antioxidant system of Agapanthus EC cryopreservation was investigated in physiology and gene levels, which further verified the result of Arabidopsis antioxidant mechanism. The results were as follows:1 、 The antioxidant mechanism of cryopreservation of Arabidopsis seedlings germinated for 48 h ~ 72h(1) The regrowth rates of different Arabidopsis seedlings had an negatively relationship to the lipid peroxidation level. The levels of lipid peroxidation of different Arabidopsis seedlings were increased during cryopreservation, and the dehydration and rapid-warming were the main steps for oxidative stress, which induced by H2O2 generated from shoot tip and foot. While the excessive production of OH· was detrimental to the72 h seedlings.(2) CAT and As A were scavengers of H2O2 as the main components of antioxidant system in Arabidopsis.(3) Recovery for 24 h was the main phase in Arabidopsis seedlings to maintain redox state. MSD(AT3G56350) was the important genes restored growth state after cryopreservation. POD3(AT1G05260) was the main regulater to the antioxidant activity of cryopreservation. The regrowth ratesof antioxidant mutants were reduced generally, especially the 75.6%reduced rate of Catalase-2 mutant, which proved the significance of antioxidant system to cryopreservation.2 、 Optimization of Agapanthus embryogenic callus cryopreservation based on the Arabidopsis antioxidant mechanism(1) Exogenous antioxidants with different concentrations had positive effects on the relative survival rates of Agapanthus EC after cryopreservation. The addition of 0.08 mmol/L GSH could highly enhance the relative survival rate of Agapanthus EC. The following were 0.05μmol/L MEL, 1 mmol/LAs A, 1 μ M ABA and 10 mmol/L GB.(2) Establishment of optimized Agapanthus EC cryopreservation was to added 0.08mmol/L GSH to the vitrification solution.(3) Lipid peroxidation of the Agapanthus EC were increased gradually during cryopreservation, and reached the maximum after rapid warming and dilution. However, lipid peroxidation level in optimized system was relatively low.(4) H2O2 was also the main ROS lead to the oxidative stress as the verification to the Arabidopsis results. Exogenous GSH improved the antioxidant ability significantly, especially the POD activity, CAT activity and As A contents. They worked in dehydration, rapid-warming and dilution phases.(5) The expression of CSD was consistent to the SOD activity, which was further validated the response to oxidative stress of SOD during cryopreservation on gene level. Elevated gene expression of APX,MDHAR, GPX and GR in the recovery stage indicated that the promotion effect of GSH on the Agapanthus EC recovery.