仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。

Cryopreservation of Cyclamen persicum Mill. Callus

In this paper we studied cryopreservation of Cyclamen persicum Mill. callus to avoid variations produced by sub-culture. The callus in the logarithmic phase after sub-culture were used for experiments. Firstly, the callus were pre-cultured in culture-medium containing 4%, 6% or 8% sucrose for different time periods, transferred to different cryoprotectants to directly cryopreserve or incubated for 2 hours at -20°C, then submersed in liquid nitrogen, lastly thawed rapidly in a waterbath at 37°C, and washed with liquid culture-medium containing the corresponding concentration of sucrose. Cell survival rate was computed after stained by Neutral Red, and SPSS 13.0 software was used for statistical analysis. The results showed that sucrose concentration, pre-culture time, cryoprotectants had various impacts on cell survival rate. We have developed a simple but effective protocol for the cryopreservation of callus of C. persicum. Of the different protocols tested, 4%sucrose, pre-culturing for 3 days, No. 9 cryoprotectant and freezing directly after 30 minutes at 0°C results in the highest cell survival rate.