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Cryopreservation of seeds of blue waterlily (Nymphaea caerulea) using glutathione adding plant vitrification solution, PVS+

Nymphaea caerulea is a valuable freshwater aquatic plant, not only because of its ornamental value but also its extractions for chemical and medical uses. It is necessary to store its seeds as backup resources. Cryopreservation is the reliable, cost-effective method for long-term preservation of botanical genetic banks, especially for recalcitrant plants. In this study, we demonstrated that due to unable to tolerate desiccation and low-temperature, N. caerulea is recalcitrant. Since viability was lost before 6 months storage, N. caerulea seeds were not appropriate to long-term store by traditional storage method. The only way to long-term store N. caerulea seeds is cryopreservation. However, Plant Vitrification Solution 3 (PVS3; 50% w/v sucrose, 50% w/v glycerol), a commonly used plant vitrification solution, was ineffective on N. caerulea seeds cryopreservation. The maximum survival rate of cryopreserved seeds treated by PVS3 was only 23%. Oxidative stress induced by reactive oxygen species (ROS) accumulation within seeds was the reason to cause inefficiency of PVS3 treated seeds cryopreservation. We developed a new plant vitrification solution (PVS+), by adding glutathione (GSH) into PVS3. PVS+ rescued the inefficiency by decreasing ROS accumulation and elevated survival rate of cryopreserved seeds to 97%. Our results showed some inefficiencies of plant tissue cryopreservation may be caused by ROS-induced oxidative stress. Antioxidants can reduce ROS-induced oxidative stress and improve seeds survival after cryopreservation. In conclusion, N. caerulea seeds were identified as recalcitrant and successfully cryopreserved. Suppressing ROS accumulation during cryopreservation may be a potential strategy to improve survival rate of recalcitrant seeds after cryopreservation.