Abstract: Using embryogenic calli of Dioscorea bulbifera L. as materials, its dark culture time after cryopreservation of embryogenic calli was studied, the microstructure of embryogenic calli cultured in dark after cryopreservation and its regenerated plants were observed with semithin sections in this paper. The results showed that: After cryopreservation by encapsulation-vitrification, dark culturing for 1-5 d, the survival rate of D. bulbifera embryogenic calli increased with dark culture time prolonged, when dark culture time was over 5 d, the survival rate of D. bulbifera embryogenic calli decreased significantly. Semithin section observation results showed that after being cultured directly in the photoperiod after freezing, the cells of the calli arranged loosely and some parts would appear larger gaps. When cultured in the photoperiod with suitable dark culture after freezing, the cells of the calli arranged more closely and the cell gaps became smaller. Root, stem and leaf structure of plantlets regenerated from embryogenic calli of D. bulbifera cultured in darkness after cryopreservation had no significant difference compared with plantlets regenerated subcultured normally, and their average number of chloroplast of mesophyll cells also had no significant difference. Therefore, dark culture after cryopreservation could ensure the cellular structural integrity of embryo callus tissue to a certain extent and no morphological variation was observed in regenerated plants.

Key words: Dioscorea bulbifera L., embryogenic callus, cryopreservation, dark culture after freezing, regenerated plantlet, semithin section observation