黄独胚性悬浮细胞的小滴玻璃化法超低温保存  

Cryopreservation of Embryogenic Suspension Cells of Dioscorea bulbifera L.by Droplet-vitrification

作　　者：尹明华[1,2,3,4] 李诚欣 李祥媛 刘凯盈 封昀 卢霞 柯维忠 YIN Ming-hua;LI Cheng-xin;LI Xiang-yuan;LIU Kai-ying;FENG Yun;LU Xia;KE Wei-zhong(College of Life Sciences,Shangrao Normal University,Jiangxi Shangrao 334001,China;Key Laboratory of Protection and Utilization of Medicinal and Edible Plant Resources in Shangrao City,Jiangxi Shangrao 334001,China;Shangrao Key Laboratory of Germplasm Conservation and Utilization of Potato and Taro Crops,Jiangxi Shangrao 334001,China;Shangrao Agricultural Technology Innovation Research Institute,Jiangxi Shangrao 334001,China)

机构地区：[1]上饶师范学院生命科学学院,江西上饶334001 [2]上饶市药食同源植物资源保护与利用重点实验室,江西上饶334001 [3]上饶市薯芋类作物种质保存与利用重点实验室,江西上饶334001 [4]上饶农业技术创新研究院,江西上饶334001

出　　处：《西南农业学报》2021年第11期2332-2339,共8页Southwest China Journal of Agricultural Sciences

基　　金：2019年度江西省教育厅科学技术研究一般项目(GJJ190877);国家自然科学基金资助项目(31360072)。

摘　　要：【目的】建立和优化黄独胚性悬浮细胞小滴玻璃化法超低温保存的技术体系。【方法】通过单因子试验探究各种条件对黄独胚性悬浮细胞小滴玻璃化法超低温保存效果的影响,并通过RAPD分子标记检测其冻后再生苗的遗传稳定性。【结果】黄独胚性悬浮细胞小滴玻璃化法超低温保存较佳的技术体系如下:黄独胚性悬浮细胞(25±1)℃下在MS+2 mg/L KT+0.5 mg/L NAA+0.5 mg/L 2,4-D+0.8 mol/L蔗糖的液体培养基中预培养4 d;(25±1)℃下在装载液(MS+2 mol/L甘油+0.4 mol/L蔗糖,pH 5.8)中装载70 min;0℃下在PVS2(300 g/L甘油+150 g/L乙二醇+150 g/L二甲基亚砜+0.4 mol/L蔗糖,pH 5.8)中脱水100 min;冻后铝箔条浸入37℃恒温预热过的培养液(MS+KT 2 mg/L+NAA 0.5 mg/L+2,4-D 0.5 mg/L+30 g/L蔗糖,pH 5.8)中化冻;(25±1)℃下用洗涤液(MS+KT 2 mg/L+NAA 0.5 mg/L+2,4-D 0.5 mg/L+1.2 mol/L蔗糖,pH 5.8)进行洗涤,洗涤3次,每次洗涤时间为10 min;洗涤后先黑暗培养5 d再置于光周期下培养。黄独胚性悬浮细胞冻后相对存活率为90.5%±4.4%。液氮保存时间对黄独胚性悬浮细胞冻后存活率无显著影响。RAPD检测结果表明黄独胚性悬浮细胞小滴玻璃化法超低温保存再生植株无变异,未发现遗传稳定性发生变化。【结论】本试验建立的黄独胚性悬浮细胞小滴玻璃化法超低温保存技术程序较为可靠,可为黄独胚性悬浮细胞的长期保存提供技术参考。【Objective】The present paper aimed to establish and optimize the cryopreservation technology system of embryogenic of Dioscorea bulbifera L.by droplet-vitrification.【Method】The effects of different conditions on cryopreservation of embryogenic suspension cells of Dioscorea bulbifera L.by droplet-vitrification were studied by single factor test,and the genetic stability of regenerated plantlets after freezing was detected by RAPD molecular markers.【Result】The best cryopreservation technology system of embryogenic suspension cells of Dioscorea bulbifera L.by droplet-vitrification was as follows:Embryogenic suspension cells of Dioscorea bulbifera L.was pre cultured in MS+2 mg/L KT+0.5 mg/L NAA+0.5 mg/L 2,4-D+0.8 mol/L sucrose at(25±1)℃for 4 days,loaded in the loading solution(MS+2 mol/Lglycerol+0.4 mol/L sucrose,pH 5.8)at(25±1)℃,dehydrated in PVS2(300 g/L glycerol+150 g/Lglycol+150 g/LDMSO+0.4 mol/Lsucrose,pH 5.8)at 0℃,thawed by the aluminum foil being immersed in the culture solution(MS+KT 2 mg/L+NAA 0.5 mg/L+2,4-D 0.5 mg/L+1.2 mol/L sucrose,pH 5.8)preheated at 37℃after freezing,washed with washing solution(MS+KT 2 mg/L+NAA 0.5 mg/L+2,4-D 0.5 mg/L+1.2 mol/L sucrose,pH 5.8)for three times(each washing time was 10 min)at(25±1)℃,cultured in darkness for five days and then cultured in light cycle after washing.The relative survival rate of embryogenic suspension cells of Dioscorea bulbifera L.by droplet-vitrification cryopreservation was 90.5%±4.4%.There was no significant effect of liquid nitrogen storage time on the survival rate of embryo suspension cells of Dioscorea bulbifera L.after cryopreservation by droplet-vitrification.The detection result of RAPD showed that there was no variation in the plantlets regenerated from embryogenic suspension cells of Dioscorea bulbifera L.after cryopreservation by droplet-vitrification,which could ensure the genetic stability.【Conclusion】The cryopreservation procedure of embryogenic suspension cells of Dioscorea bulbifera L.by droplet-vitrification

关 键 词：黄独 胚性悬浮细胞 小滴玻璃化法 超低温保存 

分 类 号：R282[医药卫生—中药学]