越橘离体腋芽小滴玻璃化超低温保存  

Cryopreservation of Axillary Buds of Vaccinium in vitro by Droplet Vitrification

作　　者：李娴 张志东[1,2] 刘海广 陈丽[1,2] 李亚东 孙海悦[1,2] LI Xian;ZHANG Zhidong;LIU Haiguang;CHEN Li;LI Yadong;SUN Haiyue(College of Horticultrure,Jilin Agricultural University,Changchun 130118,China;Jilin Provincial Engineering Center of Genetic Breeding and Innovative Utilization of Small Fruits,Changchun130118,China)

机构地区：[1]吉林农业大学园艺学院,长春130118 [2]吉林省小浆果遗传育种与创新利用工程中心,长春130118

出　　处：《吉林农业大学学报》2024年第4期589-599,共11页Journal of Jilin Agricultural University

基　　金：吉林省科技发展计划项目(20180201076NY,20200402080NC)。

摘　　要：以越橘试管苗“密斯梯”的腋芽为材料,筛选了小滴玻璃化超低温保存过程中的各项技术参数;用8种66份越橘基因型验证该方法的广谱性;用10个ISSR引物检测超低温保存后5个品种再生株系遗传稳定性。结果表明:从30 d苗龄试管苗切取长1~1.5 mm单腋芽茎段分别在继代培养基(WPM+0.3 mg/L ZT+7 g/L琼脂+30 g/L蔗糖,SCM)和预培养基(WPM+7 g/L琼脂+0.3 mol/L蔗糖,PCM)上各培养1 d[(25±2)℃黑暗];腋芽用加载液(WPM+2.0 mol/L甘油+1.0 mol/L蔗糖,LS)处理30 min(室温),然后浸入植物玻璃化保护溶液Ⅱ(WPM+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,PVS2)中40 min(0℃);在铝箔纸上制成小滴(1腋芽/小滴)之后投入液氮中保存;取出的材料立即用卸载液(WPM+1.2 mol/L蔗糖,ULS)处理20 min(室温);在SCM上腋芽黑暗培养1 d后转至正常条件下再生培养。广谱性验证结果为平均成活率81.3%和再生率47%。5个越橘品种的分子标记检测结果显示未出现特异性条带。The technical parameters were selected during the cryopreservation by droplet vitrification with axillary buds of Vaccinium“Misty”in vitro.The wide spectrum of this method was tested with to⁃tal 66 genotypes of Vaccinium including 8 spieces.The genetic stability of strains regenerated from 5 cultivars after cryopreservation was tested by ISSR with 10 primers.The results showed that the single axillary bud(length 1-1.5 mm)cut from 30-day-old shoots in vitro was cultured on subculture me⁃dium(WPM+0.3 mg/L ZT+7 g/L agar+30 g/L sucrose,SCM)and on preculture medium(WPM+7 g/L agar+0.3 mol/L sucrose,PCM)for 1 day,respectively,at(25±2)℃in the dark.The axillary buds were treated in loading solution(WPM+2.0 mol/L glycerol+1.0 mol/L sucrose,LS)for 30 min(room temperature)and immersed into Plant Vitrification SolutionⅡ(WPM+300 g/L glycerol+150 g/L ethylene glycol+150 g/L DMSO+0.4 mol/L sucrose,PVS2)for 40 min(0℃).The droplets(one bud per droplet)were formed on aluminum foil paper and quickly put into liquid nitrogen(LN).The material taken out from LN was treated immediately in unloading solution(WPM+1.2 mol/L su⁃crose,ULS)for 20 min(room temperature).The axillary buds were cutltured on SCM medium for regen⁃eration under the normal culture condition after 1 day in the dark(25±2)℃.The testing results of wide spectrum showed that the average survival rate and regeneration rate were 81.3%and 47%,respec⁃tively.The molecular marker detection results of 5 bilberry varieties showed no ploymorphic band.

关 键 词：越橘 腋芽 小滴玻璃化 超低温保存 遗传稳定性 

分 类 号：S663.9[农业科学—果树学]