Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability

During biotic and abiotic stress in plants, reactive oxygen species (ROS) may play two very different roles: high ROS concentrations can exacerbate damage, whereas low concentrations can activate defense responses. The aim of this study was to investigate the relationship between ROS generation and pollen viability after cryopreservation. ROS generation was detected from 'Siberia' (Lilium × siberia) pollen using flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate as a fluorescent probe. Pollen viability was determined by 2,3,5-triphenyltetrazolium chloride staining. ROS generation was slightly increased by rapid cooling (26.13 ± 4.74 vs. 15.80 ± 2.30 for fresh pollen) and significantly increased by vitrification (49.74 ± 1.43; P < 0.01). Pollen viabilities after rapid cooling and vitrification were significantly increased (58.88 ± 3.76% and 70.35 ± 2.90%, respectively) over that of fresh pollen (46.65 ± 1.61%; P < 0.01). No significant differences in ROS generation were associated with cold acclimation at different temperatures before rapid cooling. However, sharp decreases in viability were observed with cold acclimation at 4°C and -20°C relative to rapid cooling without acclimation (P < 0.01). We observed nonsignificant decreases in ROS generation among vitrification treatments that omitted different steps and a significant decrease when the unloading step was omitted (P < 0.05). Pollen viabilities were significantly reduced when the loading or dehydration steps were omitted (P < 0.01). No significant differences were observed in ROS generation or pollen viability among the treatments when 200 U/ml catalase was added to different solutions used in the vitrification process. Comprehensive analysis of all data indicated a positive correlation between ROS generation and pollen viability (r = 0.651, P < 0.001). Therefore, increasing ROS generation during cryopreservation may improve the viability of 'Siberia' pollen. © 2014 The Society for In Vitro Biology.