High-Efficiency Encapsulation-vitrification Protocols for Cryopreservation of Embryogenic Calli of the Oriental Medicinal Plant Anemarrhena asphodeloides Bunge

Embryogenic calli from in vitro grown tillers of Anemarrhena asphodeloides Bunge were successfully cryopreserved by the encapsulation–vitrification technique. Excised embryogenic calli were precultured for 4 days in liquid MS medium supplemented with 2 mg L^-1 kinetin (KIN), 0.1 mg L^-1 α -naphthalene acetic acid (NAA) and 0.75 M sucrose, then encapsulated in calcium alginate beads and loaded with a mixture of 2 M glycerol + 0.4 M sucrose for 60 min at 25±1°C. Calli were then dehydrated with the PVS2 solution for 80 min at 0°C. After changing the solution with fresh PVS2, calli were directly immersed in liquid nitrogen (LN). After rapid rewarming in a water-bath at 35°C for 5 min, calli were washed three times with liquid MS medium supplemented with 2 mg L^-1 KIN, 0.1 mg L^-1 NAA and 1.2 M sucrose, then transferred on solid MS medium supplemented with 2 mg L^-1 KIN, 0.1 mg L^-1 NAA, 3% (w/v) sucrose and 0.75% (w/v) agar. Cryopreserved cultures were kept in the dark for 5 days prior to exposure to a 14 h light/10 h dark photoperiod with a light intensity of 36 μ mol m^-2 s^-1 provided by white cool fluorescent tubes at 25 ±1°C. Survival of cryopreserved embryogenic calli reached 80%, including after storage for c. 1 year. No significant difference was observed in the morphological development of plants coming from control and cryopreserved embryogenic calli. This encapsulation-vitrification method appears promising for the cryopreservation of A. asphodeloides Bunge germplasm.