Cryopreservation of Embryogenic Callus of Dioscorea bulbifera by Vitrification

Cryopreservation of callus of Dioscorea bulbifera by vitrification was optimized. Calli of Dioscorea bulbifera were pretreated in liquid Murashige and Skoog (MS) medium supplemented with 2 mg L−1 kinetin (KT), 0.5 mg L−1 NAA, 0.5 mg L−1 2,4-D and 0.2 M sucrose for 5 d under continuous light (36 μM m−2 s−1) at 25±1°C. The material was then loaded with 60% vitrification solution (PVS2) for 20 min at room temperature and dehydrated with 100% PVS2 for 30 min at 0°C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen and conserved for 1-360 d. After rapid thawing in a water-bath at 35°C, the calli were washed three times with liquid MS medium supplemented with 2 mg L−1 KT, 0.5 mg L−1 NAA, 0.5 mg L−1 2, 4-D and 1.2 M sucrose and then transferred onto solid MS medium supplemented with KT 2 mg L−1, NAA 0.5 mg L−1, 0.09 M sucrose and 0.75% (w/v) agar. The cultures were kept in the dark for 2 days prior to exposure to the light (12 h light/dark cycle). The TTC test showed that 80-90% of the calli survived this cryoprocedure and there was a 60-70% regeneration of plantlets from the calli. The regenerated material did not exhibit any morphological variations.