黄独胚性愈伤组织冻后黑暗培养及其再生植株半薄切片观察

Dark culture of embryogenic calli after cryopreservation and semithin section observation of its regenerated plantlet of Dioscorea bulbifera L.

以黄独胚性愈伤组织为材料,对黄独胚性愈伤组织冻后黑暗培养的时间进行探讨,并对冻后黑暗培养胚性愈伤组织的显微结构及其再生植株进行半薄切片观察。结果表明,包埋玻璃化法超低温保存后,黑暗培养1~5 d,黄独胚性愈伤组织的冻后成活率随着时间的延长而增加,超过5 d,成活率显著下降。半薄切片观察结果表明,冻后黄独胚性愈伤组织直接置于光周期下培养,会造成细胞排列疏松,局部出现较大的细胞空隙;而经黑暗培养后再置于光周期下培养,细胞排列较为紧密,细胞空隙较小。经半薄切片观察,黄独冻后黑暗培养胚性愈伤组织再生植株与常温继代再生植株相比较,根、茎、叶结构无显著差异,叶肉细胞的平均叶绿体数量也无显著差异。因此,冻后黑暗培养一定程度上保证了胚性愈伤组织细胞结构的完整性,且其再生植株无形态学变异。

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.