Cryopreservation of Dendrobium draconis Rchb. f. protocorms using V cryo-plate method

Cryopreservation of Dendrobium draconis Rchb. f. protocorms, a beautiful Thai orchid was successful using V cryo-plate method. Protocorms were selected from seeds after sowing for 30 d and then precultured on modified VW agar medium supplemented with 0.4 M sucrose at room temperature (29±2°C) for 1, 2, and 3 d. Precultured protocorms were placed in wells on the aluminum cryo-plate containing 12 wells and then embedded with 3% sodium alginate gel. Osmoprotection was performed using cryo-plates with protocorms immersed in loading solution (LS) containing 2 M glycerol and different concentrations of sucrose (0.4, 0.6, and 0.8 M sucrose) for 15 min at 29±2°C and then dehydrated with PVS2 solution for 0-40 min at 29±2°C. Cooling was performed by transferring the cryo-plates with protocorms into cryotubes and then directly plunged into liquid nitrogen for 40 min. Cryo-plates with protocorms were rapidly warmed in 1 M sucrose for 15 min at 29±2°C. Non-cryopreserved and cryopreserved were cultured on modified VW agar medium supplemented with 100 g L^‑1 banana, 150 mL L^‑1 coconut water, 50 g L^‑1 potato at pH 5.2. The results showed that survival of cryopreserved protocorms increased with increasing time for preculture. The highest survival of cryopreserved protocorms was 75% when precultured with 0.4 M sucrose for 3 d, treated with 0.6 M sucrose in LS and dehydrated with PVS2 for 10 min. No survival of cryopreserved protocorms was observed for 1 d of preculture, treated with 0.4 and 0.6 M sucrose in LS. Cryopreserved protocorms developed into planlets when cultured on modified VW agar medium for 6 months. No significant difference was observed in morphology of non-cryopreserved and cryopreserved plantlets developed from protocorms.