After cryopreservation, the occurrence of apoptosis-like programmed cell death events induced by the accumulation of ROS reduces pollen viability.

Cryopreservation, as a biotechnological means for long-term preservation of pollen, has been applied to many species. However, after cryopreservation, the viability of pollen significantly decreases via a mechanism that is not completely clear. In this study, the pollen of Paeonia lactiflora ‘Zi Feng Chao Yang’, which exhibits significantly reduced viability after liquid nitrogen (LN₂) storage, was used to study the relationship among pollen viability, programmed cell death (PCD) and reactive oxygen species (ROS). The apoptosis rate was increased significantly in pollen with decreased viability after cryopreservation, and the changes in ROS generation and hydrogen peroxide (H₂O₂) were consistent with the apoptosis rate. Correlation analysis results showed that the apoptosis rate is positively correlated with ROS generation and H₂O₂ content. In addition, ascorbic acid (AsA), glutathione (GSH) and ascorbic acid reductase (APX) levels were significantly correlated with ROS and H₂O₂. After LN₂ preservation for 8 months, the exogenous antioxidants AsA and GSH at appropriate concentrations significantly decreased H₂O₂ content, inhibited PCD indicator levels, and increased cryopreserved pollen viability. These observations suggest that PCD occurred in pollen during LN₂ preservation for 1–8 months and was induced by the accumulation of ROS in pollen after cryopreservation, thus explaining the main reasons for the reduction in pollen viability after cryopreservation in LN₂.