玻璃化冷藏条件对连香树定芽细胞存活率及微结构的影响

选择6种保护剂、2种玻璃化方式和2种低温处理方式对连香树定芽的冷藏条件进行筛选,用TTC还原法测定冷藏定芽的细胞存活率,并用电子显微镜观察定芽细胞冷冻时的受损情况,以优选连香树定芽的冷藏技术条件。结果表明:(1)保护剂、玻璃化方式、冷藏温度均对莲香树定芽的细胞存活率具有显著影响,不同冷藏处理的定芽组织细胞内冰晶形状、数量不同,细胞破裂数也不同。(2)连香树定芽的最佳冷藏条件为:用含甘油35.0mL、乙烯乙二醇20.0mL、DMSO 15.0mL、甘氨酸1.0mg和3%蔗糖脱氨MS液体培养基组成的100mL保护剂于真空下浸泡30min,再经-20℃预冻处理6h后,于液氮中玻璃化和冷藏60d,其定芽细胞的存活率最高,相对存活率达95%。(3)细胞微结构观察显示,在该优选条件下明显抑制了连香树定芽细胞内冰晶体的形成,消除了冰晶对细胞的涨破和刺伤,其细胞结构完整无损伤。

Influence of Vitrifying Cryopreservation Conditions on Cell Viability and Ultrastructure of Cercidiphyllum japonicum Normal Buds

Cryopreservation conditions of normal buds of Cercidiphyllum japonicum were selected by different treatments combined with six protecting reagents,two vitrifying approaches and two low temperatures.The cells viability of the cryopreserved buds were investigated by TCC reduction test.The injuries of the cell ultrastructure of the cryopreserved buds were observed and analyzed by electron microscopy.The optimal cryopreservation technological conditions of the buds were confirmed by the experiments.The results showed that:(1)the protecting reagents,vitrifying methods and cryopreserving temperatures are all given significant impact on the cell viability of the buds.The different treatments of vitrifying cryopreservation formed different shapes and quantities of ice crystalloid within cells of the buds,resulted in the different amounts of the cell rupture.(2)The optimal cryopreservation condition of buds is that the buds can be marinated for 30 min in vacuum with the protecting reagents which consist of 35.0 mL glycerin,20.0 mL ethylene glycol,15.0 mL DMSO,1.0 mg glycine and 3% sugar deamination MS liquid in 100.0 mL volume,prefrozen for 6 h at-20℃,vitrified and cryopreserved in liquid nitrogen for 60 d,and lead to the highest cell relative viability of 95%.(3)the cell ultrastructure show that the elected optimal condition restrains obviously the formation of ice crystalloid inside cell and eliminates the swelling break of the cells effectively.The cell structures of cryopreserved buds are integrated and no injury.