Cryopreservation technique of Dioscorea opposita germplasm by vitrification / 中草药

ABSTRACT

Objective To study the cryopreservation technique of Dioscorea opposita germplasm resources by vitrification. Methods Stems with buds of B# D. opposita germplasm were used as materials for cryopreservation by vitrification and the survival rates were examined by culture method in order to optimize a cryopresevation by vitrification technical system for D. opposita germplasm. Results A best procedure of cryopresevation by vitrification was as below At first, the aseptic plantlets of stems with buds of B# D. opposita subcultured in vitro for 60 d were treated at 4 ℃ for 7 d. The stems with buds of D opposita about 1-1.5 cm in length were cut and precultured at 4 ℃ for 2 d in 5% sucrose and 3% mannose media, they were dehydrated with 60% vitrification solution (PVS_1 22% glycerol+13% glycol+13% polyethyleneglycol+10% dimethyl sulfoxide) at 0 ℃ for 60 min and then dehydrated with 100% vitrification solution at 0 ℃ for 60 min. At last, the stems were immersed immediately into liquid nitrogen directly and conserved for 24 h. After rapidly thawing in a water bath at 37 ℃, the stems were washed four times with MS medium supplemented with 7% sucrose and 10 min each time, then transferred and re-cultured on the MS medium supplemented with KT 2 mg/L and NAA 0.02 mg/L. The survival rate was up to 75%. The regenerated plantlets of D. opposita showed no more difference than the normal plantlets in morphology. Conclusion This test sets up successfully a cryopresevation by vitrification system technique for D. opposita germplasm, which provides an effective way for long-lasting conservation in vitro of D. opposita germplasm resources.