Objective  After cryopreservation, pollen viability shows various changes, studies show that ROS is one of the main reasons for the changes of pollen viability after cryopreservation. Therefore, this study investigated the relationship between pollen viability and ROS induced oxidative stress and programmed cell death after cryopreservation, to further reveal the role of ROS in pollen viability changes after cryopreservation.

  Method  The pollen of P. lactiflora ‘Fen Yu Nu’ was used as material, the changes of pollen viability, ROS production, oxidative stress and programmed cell death after cryopreservation for different lengths of time were compared and analyzed.

  Result  During the cryopreservation of pollen, the O₂•− content of ROS components was relatively high after 1 and 3 months of cryopreservation, and the H₂O₂ content significantly increased with the extension of storage time, while •OH content began to decrease after 3 months, both H₂O₂ and •OH contents were significantly correlated with pollen viability. Secondly, the content of malondialdehyde (MDA), an index of oxidative damage, was significantly increased after 3 months of liquid nitrogen (LN) storage, which was significantly correlated with pollen viability, H₂O₂ and •OH contents. The activity of caspase-3-like protease, an indicator of programmed cell death (PCD), was higher than that of the fresh pollen after 1 and 3 months of liquid nitrogen freezing, and the apoptosis rate was significantly increased after 5 and 8 months of LN stored, and the apoptosis rate was significantly correlated with pollen viability, H₂O₂ and •OH contents. In addition, appropriate concentration of exogenous oxidative damage inhibitors (AsA, GSH) and apoptosis-like inhibitors (caspase-3 inhibitors) significantly improved pollen viability after 8 months of cryopreservation.

  Conclusion  During the cryopreservation of P. lactiflora ‘Fen Yu Nu’ pollen, ROS components have an important effect on pollen viability after cryopreservation, the effects of H₂O₂ and •OH are particularly prominent. The oxidative stress response and programmed cell death induced by H₂O₂ and •OH are important reasons for the changes of pollen viability after liquid nitrogen cryopreservation.