Cryopreservation of embryogenic cultures of Dioscorea bulbifera l. by encapsulation- dehydration.

Embryogenic cultures of Dioscorea bulbifera were cryopreserved using an encapsulation- dehydration procedure with subsequent plant regeneration. Embryogenesis was induced by culturing in vitro grown axillary bud meristems on MS medium supplemented with 2.0 mg per liter 2,4-D. After cryopreservation, recovery growth of embryogenic culture up to 53.3 percent was recorded when excised proliferating embryogenic cultures of 1.5-2.0 mm in diameter were: encapsulated in 3 percent calcium alginate containing 0.15 M sucrose followed by preculturing with 0.5 M sucrose for 3 d; dehydrated in the laminar air flow for 4 h, thereby reducing the bead moisture content to 19.4 percent ( fresh weight basis); plunged into liquid nitrogen; thawed at 40 degree C; and cultured on recovery growth medium, i.e. MS supplemented with 2.0 mg per liter 2,4-D and 0.3 mg per liter BAP. However, preculturing for an extended period of 7 d increased the recovery growth further to 67.8 percent. During recovery growth the embryogenic tissue protruded out of the beads without loss of structural integrity of the cryopreserved embryos. Subculturing of these cultures on to embryo conversion medium, i.e. MS medium with 0.5 mg per liter zeatin and 400 mg per liter glutamine, resulted in production of plantlets through embryo conversion. The regenerated plantlets exhibited the same morphology as that of originally maintained in vitro plantlets and were established in vivo, in a net house with 80 percent success.

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration.

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.