Hydrogen peroxide participated in programmed cell death by mediating ethylene during the cryopreservation of Dendrobium nobile protocorm-like bodies

Hydrogen peroxide (H 2 O 2 ) is a signaling molecule that is directly or indirectly involved in the occurrence of programmed cell death (PCD). Although H 2 O 2 was clearly involved in the occurrence of PCD in plant cryopreservation studies, the pathway has not been studied. In this study, the protocorm-like bodies (PLBs) of Dendrobium nobile Lindl. ‘Hamana Lake Dream’ were used as materials, and exogenous H 2 O 2 regulators were added to investigate the changes in the endogenous ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) content and the activities of ACC oxidase (ACO) and ACC synthase (ACS) during the cryopreservation of PLBs. By adding exogenous ethylene regulators, we compared the changes in caspase-3-like protease and PCD regulatory genes during PLB cryopreservation to explore the possible pathway of H 2 O 2 in the occurrence of PCD during PLB cryopreservation. The results showed that exogenous H 2 O 2 significantly reduced the cell survival rate, endogenous ACC content, and ACO and ACS enzyme activities of PLBs after cryopreservation, while the addition of the H 2 O 2 scavenger catalase (CAT) had the opposite effect. The addition of the ethylene donor ethephon (ETH) significantly decreased the activity of caspase-3-like protease, downregulated the expression of pro-PCD genes, and upregulated the expression of anti-PCD genes and signal transduction-related genes, while the addition of the ethylene synthesis inhibitor cobalt (II) nitrate (Co(NO 3 ) 2 , Co) had the opposite effect. These results indicated that H 2 O 2 was involved in the occurrence of PCD during the vitrification cryopreservation of Dendrobium nobile PLBs by affecting the endogenous ethylene synthetic pathway and thus affecting the survival rate of cells after freezing with liquid nitrogen.