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

This study successfully developed a method for the long-term cryopreservation of recalcitrant Blue Waterlily (Nymphaea caerulea) seeds by creating an enhanced cryoprotectant solution.

Summary:

The research first confirmed that Nymphaea caerulea seeds are recalcitrant, meaning they cannot tolerate desiccation (drying) or freezing temperatures. Traditional seed banking methods (drying and storing at -20°C) were ineffective, with seeds losing viability within 6 months, and direct exposure to -20°C or liquid nitrogen (LN) killing them outright.

The standard cryoprotectant, Plant Vitrification Solution 3 (PVS3), was also insufficient, yielding a maximum survival rate of only 23% after cryopreservation in LN. The study identified oxidative stress from the accumulation of Reactive Oxygen Species (ROS), particularly hydrogen peroxide (H₂O₂), as the primary cause of this failure. The cryopreservation process itself induced a massive surge in H₂O₂ levels within the seeds.

To address this, the researchers developed a modified solution called PVS+ by adding the antioxidant glutathione (GSH) to PVS3. The optimal concentration was found to be 0.08 mM GSH. PVS+ dramatically reduced ROS accumulation during cryopreservation, keeping H₂O₂ levels near normal. This intervention rescued seed viability, increasing the survival rate after cryopreservation in LN to an impressive 97%.

Key Highlights:

1. Recalcitrant Seeds: Nymphaea caerulea seeds were definitively classified as recalcitrant, being highly sensitive to drying and cold, making traditional storage methods unsuitable for long-term conservation.
2. PVS3 Inefficacy: The commonly used cryoprotectant PVS3 failed to adequately protect the seeds, achieving only a 23% survival rate after cryopreservation.
3. ROS as the Culprit: The primary reason for PVS3’s failure was identified as oxidative stress caused by a significant buildup of ROS (specifically H₂O₂) during the cryopreservation process.
4. PVS+ Solution: A novel vitrification solution, PVS+, was created by adding 0.08 mM glutathione (GSH), a potent antioxidant, to PVS3.
5. High Success Rate: PVS+ effectively mitigated oxidative stress by scavenging ROS, resulting in a dramatically improved survival rate of 97% for cryopreserved seeds.
6. New Strategy: The study demonstrates that suppressing ROS-induced oxidative stress using antioxidants like glutathione is a highly effective and promising strategy for improving the cryopreservation of recalcitrant seeds.

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