花烛胚性悬浮细胞玻璃化超低温保存条件研究

Study on vitrification cryopreservation condition of Anthurium andraeanum embryonic suspension cells

为建立适宜的花烛（Anthurium andraeanum Lind.）胚性悬浮细胞玻璃化超低温保存技术,采用单因素实验方法对影响玻璃化超低温保存后细胞相对存活率的主要因素进行了研究。结果表明,经玻璃化超低温保存后花烛悬浮细胞的相对存活率与悬浮细胞的继代培养时间、渗透调节剂的种类和浓度及预培养时间、装载液种类和预处理时间、PVS2脱水时间以及超低温保存后的化冻温度均有一定的关系。继代培养3和5 d,细胞的相对存活率较高（约20%）;分别以0.3、0.5、0.7 mol·L^-1山梨醇和60、80、100、120 g·L^-1蔗糖为渗透调节剂预培养0-4 d,以0.5 mol·L^-1山梨醇预培养2 d的效果最好,细胞的相对存活率为26.2%;用体积分数25%PVS2预处理15 min,细胞的相对存活率最高（29.0%）;分别用体积分数100%PVS2脱水0、5、10、15、20、25和30 min,其中脱水10 min的悬浮细胞相对存活率最高（32.1%）;分别在10℃、20℃、30℃、40℃、50℃和60℃水浴条件下进行化冻处理,其中用40℃水浴化冻的悬浮细胞相对存活率最高（32.1%）。花烛胚性悬浮细胞玻璃化超低温保存和化冻的适宜流程为：将继代培养3-5 d的胚性悬浮细胞团（直径2 mm）在含0.5 mol·L^-1山梨醇的1/2MS液体培养基中预培养2 d后,于4℃条件下处理24 h,然后先用体积分数25%PVS2室温预处理15 min,再用体积分数100%PVS2在0℃条件下脱水10 min,最后迅速投入液氮中冷冻保存;将经过冷冻保存的细胞置于40℃水浴中化冻3 min,用含1.2mol·L^-1蔗糖的1/2MS液体培养基洗涤3次（每次10 min）,之后即可进行恢复培养。

In order to establish suitable vitrification cryopreservation technology of Anthurium andraeanum Lind.embryonic suspension cells,the main factors effected relative survival rate of the cells after vitrification cryopreservation were studied by the single factor experiment.The results show that the relative survival rate of the embryonic suspension cells after vitrification cryopreservation has a certain relationship with subculture time,type and concentration of osmotic regulating agent and pre-culture time,loading solution type and pre-treatment time,dehydration time of PVS2 and thawing temperature.Relative survival rate of the suspension cells subcultured for 3 and 5 d is higher with a rate of about 20%.Among using 0.3,0.5,0.7 mol·L^-1 sorbitol and 60,80,100,120 g·L^-1sucrose as osmotic regulating agents and pre-culturing for 0-4 d,the effect with 0.5 mol·L^-1 sorbitol and pre-culturing for 2 d is the best with a relative survival rate of 26.2%.Relative survival rate of the cells is the highest with a value of 29.0% after pre-treating for 15 min with 25% PVS2.Dehydrating with 100% PVS2 for 0,5,10,15,20,25 and 30 min,respectively,the dehydrating for 10 min has the highest relative survival rate of the cells with a value of 32.1%.Among using 10 ℃,20 ℃,30 ℃,40 ℃,50 ℃ and 60 ℃ water baths to thaw,relative survival rate of the suspension cells is the highest（32.1%） at 40 ℃ thawing condition.The suitable technology process of vitrification cryopreservation of A.andraeanum embryonic suspension cells is as follow： embryonic suspension cell mass（diameter 2 mm） subcultured for 3-5 d is pre-cultured in 1/2MS liquid medium containing 0.5 mol·L^-1 sorbitol for 2 d,then treated for 24 h in 4 ℃,and pre-treated with 25% PVS2 for 15 min in room temperature,dehydrated with 100% PVS2 for 10 min in 0 ℃,at last,rapidly put the cell mass into liquid nitrogen for cryopreservation.Put the cryopreservation cells in 40 ℃ water bath to thaw for 3 min,then wash with 1/2MS liquid medium containing 1.2 mol·L^-1 sucrose for three times（each time for 10 min）,afterward carry on renewal culture.