Exogenous Catalase and Pyruvate Dehydrogenase Improve Survival and Regeneration and Affect Oxidative Stress in Cryopreserved Dendrobium nobile Protocorm-like Bodies.

BACKGROUND: Reactive oxygen species (ROS)-induced oxidative damage is responsible for viability loss in plant tissues following cryopreservation. Antioxidants may improve viability by preventing or repairing the injury. OBJECTIVE: This work aimed at studying the effect of catalase (CAT) and pyruvate dehydrogenase (PDH), which are involved in ROS metabolism and are differentially expressed during pollen cryopreservation, for cryopreservation of Dendrobium nobile Lindl. 'Hamana Lake Dream' protocorm-like bodies (PLBs). MATERIALS AND METHODS: Different concentrations of exogenous CAT or PDH were added at the loading, PVS2 treatment, unloading steps during vitrification-cryopreservation of PLBs. Their survival and regeneration were evaluated and correlated with physiological oxidative indexes. RESULTS: PLB survival increased significantly when CAT and PDH were added separately to the unloading solution at a suitable concentration. CAT at 400 U·ml-1 increased PLB survival and regeneration by 33.5 and 14.6 percent respectively. It had no impact on the production of superoxide anion radical (·O2-) and on superoxide dismutase (SOD) activity, but it reduced the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and enhanced ascorbic acid (AsA) and endogenous CAT levels compared to PLBs cryopreserved using the standard vitrification protocol (CK1). PDH at 0.1 U·ml-1 significantly improved PLB survival (by 2.5 percent), but it had no marked effect on regeneration compared to the CK1 group. It induced the same variations in ·O2-, AsA and endogenous CAT levels that were observed following CAT addition. However, PDH did not affect the H2O2 and MDA content but significantly increased SOD activity. CONCLUSION: These results indicate that the addition of 400 U·ml-1 CAT and 0.1 U·ml-1 PDH at the unloading step increased survival of cryopreserved PLBs and that this improvement was associated with scavenging of H2O2 and the repair of oxidative damage. Exogenous CAT also significantly improved PLB regeneration after cryopreservation, while PDH had no obvious effect. The effect of exogenous CAT on PLB survival and regeneration was stronger than that of PDH, which may be due to the increased SOD activity by PDH addition.