雨生红球藻FACHB-712营养细胞和厚壁孢子低温保藏效果及优化

以高产虾青素的雨生红球藻（Haematococcus pluvialis Flotow）FACHB-712藻株为材料，研究2种细胞形态（营养细胞和厚壁孢子）在低温保藏下的复苏率及其差异原因。结果显示，采用两步法（先预冻降温后再投入液氮中）冻存其营养细胞，在不同冻存条件下，其存活率均低于5%，以10%甘油作为保护剂、冻存速率为0.5℃/min、预冻温度为-40℃、保留30 min，然后再投入液氮罐（-196℃）中保藏，其存活率可达到13.3%。采用两步法冻存厚壁孢子，其复苏存活率高达66.13%，复苏萌发后细胞的生长特性、虾青素含量与液氮保藏前无明显差异（P > 0.05）。对液氮保藏前后藻细胞形态和超微结构观察结果表明，超低温保藏后，营养细胞的结构受到较大损伤，而厚壁孢子受到的损伤相对较小。当添加不同保护剂后，直接将厚壁孢子分别冻存在-20℃、-80℃低温及液氮中，发现-80℃低温冻存处理组的复苏存活率相对较高，可达27%。研究表明采用两步法先预冻降温后再投入液氮中冻存厚壁孢子，是长期保藏雨生红球藻FACHB-712的最佳方法，也可采用一步法将厚壁孢子冻存于-80℃冰箱中。

Effect and optimization of cryopreservation on vegetative cells and akinete of Haematococcus pluvialis Flotow FACHB-712

The survival rate of cryopreservation based on vegetative cells and akinetes of Haematococcus pluvialis Flotow FACHB-712 were compared. Results showed that in most cases the survival rate of the vegetative cells was lower than 5% in liquid nitrogen with two-step cryopreservation, but the rate increased to 13.3% if the protocol was modified as follows:cell suspensions were added with glycerol as a cryoprotectant (10%), cooled at a rate of 0.5℃/min until -40℃, and then maintained for 30 min before being placed to liquid nitrogen. The survival rate of the akinetes reached 66.13% in liquid nitrogen with the two-step protocol. There were no obvious differences (P > 0.05) in the growth characteristics and the astaxanthin content of akinetes before and after preservation in liquid nitrogen. Comparatively, the ultrastructure of the vegetative cells was seriously damaged, whereas that of the akinetes was much less damaged after two-step cryopreservation treatment. If directly placed in -20℃, -80℃, or liquid nitrogen, respectively, the highest survival rate of the samples was about 27% in the treatment under -80℃. In conclusion, the two-step cryopreservation protocol in liquid nitrogen using akinete-stage cells was preferable for the long-term preservation of H. pluvialis FACHB-712, with the one-step protocol at -80℃ also applicable as an alternative approach.