红豆越橘组培苗茎尖小滴玻璃化超低温保存  

Cryopreservation of Shoot Tips of Lingonberry in vitro Culture by Droplet-Vitrification

作　　者：杨晶文 毛宇金 李娴 张志东[1,2] 孙海悦 李亚东[1,2] 刘海广 YANG Jingwen;MAO Yujin;LI Xian;ZHANG Zhidong;SUN Haiyue;LI Yadong;LIU Haiguang(College of Horticulture,Jilin Agricultural University,Changchun 130118,China;Jilin Provin-cial Engineering Center of Genetic Breeding and Innovative Utilization of Small Fruits,Changchun 130118,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3期439-445,共7页Journal of Jilin Agricultural University

基　　金：吉林省科技发展计划项目(20170414023GH,20180201076NY)。

摘　　要：研究了红豆越橘组培苗茎尖的小滴玻璃化超低温保存方法。剪取苗龄30 d的组培苗带顶芽茎段,在WPM(Woody plant medium)+0.5 mg/L ZT(Zeatin玉米素)+30 g/L蔗糖+7 g/L琼脂培养基(SSM)上,于4℃黑暗条件下炼苗25 d;将剥取的长约2.0 mm茎尖在黑暗和(25±2)℃条件下用预培养基(WPM+0.3 mol/L蔗糖+7 g/L琼脂)培养24 h;室温下茎尖在加载液(WPM+2.0 mol/L甘油+1.0 mol/L蔗糖)中处理30 min;0℃下用植物玻璃化溶液Ⅱ(PVS2)处理茎尖40 min;0℃下在铝箔条上将茎尖与PVS2制成小滴(2.5μL),并迅速投入液氮(-196℃)中保存1 h;从液氮中取出带茎尖的铝箔条迅速转入卸载液(WPM+1.2 mol/L蔗糖)中,室温下解冻和卸载20 min;将卸载的茎尖在SSM培养基上黑暗培养(25±2)℃1 d;之后在正常光照条件下再生培养。结果表明:再生培养30 d后,试验的4份种质资源的平均再生率为76%,这为红豆越橘种质资源保存提供了一种新的途径。A droplet-vitrification for cryopreservation of lingonberry shoot tips in vitro was studied.Nodal segments with a terminal bud,excising from 30 day-old stock shoots in vitro,were cultured on WPM(woody plant medium)+0.5 mg/L ZT(zeatin zein)+30 g/L sucrose+7 g/L agar(SSM)in the dark at 4℃for 25 d.Shoot tips excised in length of about 2.0 mm were precultured on WPM+0.3 mol/L sucrose+7 g/L agar in the dark at(25±2)℃for 24 h.Shoot tips precultured were treated with a loading solution(WPM+2 mol/L glycerol+1.0 mol/L sucrose)for 30 min at room temperature.Then they were exposed to plant vitrification solution 2(PVS2)at 0℃for 40 min.On aluminum foil strips,each of the shoot tips was transferred into a droplet containing 2.5μL PVS2 at 0℃,followed by a direct immersion in liquid nitrogen(−196℃)for 1 h.Foils with shoot tips removed out from liquid nitrogen were immediately put into an unloading solution(WPM+1.2 mol/L sucrose)at room temperature for 20 min.The unloaded shoot tips were cultured on SSM in the dark at(25±2)℃for 1 d,and then cultured at normal light and temperature condition for recovery.The result showed that after 30 days of regeneration cultivation,the average regrowth rate of the four germplasm resources tested was 76%,providing a new approach for the preservation of lingonberry germplasm resources.

关 键 词：红豆越橘 超低温保存 小滴玻璃化 组培苗茎尖 

分 类 号：S663[农业科学—果树学]