  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.
