Abstract
Cryopreservation is an alternative, safe, and cost-effective method for long-term plant genetic resource conservation. This study was conducted to optimize the conditions for cryopreserving the protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid with the PVS3 vitrification method. Five parameters were assessed in this study: PLB size, sucrose concentration, preculture duration, PVS3 duration, and unloading duration. The viability of the cryopreserved PLBs was determined using the triphenytetrazolium chloride assay and growth recovery assessments. The optimum condition for the cryopreservation of the PLBs of Brassidium Shooting Star orchid is based on the size range between 3 and 4mm precultured with half-strength semi-solid MS media supplemented with 0.25M sucrose for 24h, followed by treatment with loading solution mixture of 2M glycerol and 0.4M sucrose supplemented with half-strength liquid MS media at 25°C for 20min. The PLBs were then dehydrated with PVS3 at 0°C for 20min prior to immersion in liquid nitrogen; finally, the PLBs were immersed with half-strength liquid MS media supplemented with 1.2M sucrose for 30min. Histological analyses displayed denser cytoplasm and voluminous nucleus in the cryopreserved PLBs of Brassidium Shooting Star orchid.
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