桤叶唐棣茎尖小滴玻璃化超低温保存体系优化  

Optimization of Cryopreservation System of Shoot Tips in Vitro of Saskatoon(Amelanchier alnifolia) by Droplet Vitrification

作　　者：苏原慧 张志东 刘海广 李亚东 李金英 王学山 SU Yuanhui;ZHANG Zhidong;LIU Haiguang;LI Yadong;LI Jinying;WANG Xueshan(College of Horticultrure,Jilin Agricultural University,Jilin Provincial Engineering Center of Genetic Breeding and Innovative Utilization of Small Fruits,Changchun 130118,China;Modern Ag?ricultural College,Changchun Polytechnic,Changchun 130033,China)

机构地区：[1]吉林农业大学园艺学院,吉林省小浆果遗传育种与创新利用工程中心,长春130118 [2]长春职业技术学院现代农学院,长春130033

出　　处：《吉林农业大学学报》2022年第3期275-285,共11页Journal of Jilin Agricultural University

基　　金：吉林省科技发展计划项目(20180201076NY,20200402080NC);吉林省“三区”人才支持计划项目(202022309)。

摘　　要：以桤叶唐棣(Amelanchier alnifolia)组培苗为材料,对其茎尖小滴玻璃化超低温保存方法的影响因素进行研究,并用11份桤叶唐棣种质资源验证了所建立的超低温保存规程的广谱性。结果表明:从苗龄30 d的组培苗上剪取带顶芽的茎段(长约0.5 cm),用继代培养基(MS+1.0 mg/L 6-BA+35 g/L蔗糖+7 g/L琼脂,SSM)低温炼苗14 d(4℃,黑暗),将剥取的茎尖(长1.5~2 mm)依次在预培养基(MS+0.3 mol/L蔗糖+7 g/L琼脂)预培养1 d[(25±2)℃,黑暗],在室温下用加载液(MS+2.0 mol/L甘油+0.4 mol/L蔗糖)处理20 min,在0℃用植物玻璃化溶液PVS2(MS+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)处理40 min,转至铝箔条上由PVS2溶液制成的4.5μL小滴(0℃)中,并迅速浸入液氮10 min,装入冷冻管后在液氮中保存1 h。从液氮中取出的材料立即在卸载液(MS+1.2 mol/L蔗糖)中解冻和卸载20 min(室温),茎尖转至SSM上暗培养3 d[(25±2)℃,黑暗],之后在正常温光条件下再生培养,获得最高的存活率和再生率分别为71.67%和68.33%,11份种质资源的平均再生率为50.92%。该研究首次报道了桤叶唐棣种质资源的小滴玻璃化超低温保存规程。The shoot tips from terminal buds of saskatoon( Amelanchier alnifolia) in vitro were successfully cryopreserved by droplet vitrification.The influencing factors of cryopreservation method were studied,and 11 germplasm resources of saskatoon were used to verify the wide-spectrum of the established cryopreservation protocol.The results showed that the nodal segments(about 0.5 cm in length) including terminal buds from 30-day-old shoots were cold-hardened in the dark at 4 ℃ for2 weeks with subculture medium(MS + 1.0 mg/L 6-BA + 35 g/L sucrose + 7 g/L agar,SSM).The shoot tips(1.5~2 mm in length) excised from the terminal buds were precultured in solid medium(MS+ 0.3 mol/L sucrose + 7 g/L agar) for 24 h at( 25±2) ℃ in the dark,treated with loading solution(MS + 2.0 mol/L glycerol + 0.4 mol/L sucrose) for 20 min at room temperature,and exposed to PVS2(MS + 300 g/L glycerol + 150 g/L ethylene glycol + 150 g/L dimethyl sulfoxide + 0.4 mol/L sucrose)for 40 min at 0 ℃.The shoot tips were then transferred into 4.5 μL PVS2 droplets on aluminum foil strips at 0 ℃,and immersed into liquid nitrogen(LN) for 1 h.The materials taken out from LN were thawed and unloaded immediately in MS medium solution containing 1.2 mol/L sucrose for 20 min at room temperature.The shoot tips were transferred onto SSM at(25±2) ℃ in the dark for 3 days and then cultured under normal temperature and light conditions for regrowth.The highest survival rate and regeneration rate were 71.67% and 68.33%,respectively.The average regeneration rate of 11germplasm resources was 50.92%.To our knowledge,this is the first report on saskatoon cryopreservation protocol by droplet vitrification.

关 键 词：桤叶唐棣 超低温保存 小滴玻璃化 离体茎尖 

分 类 号：S663.9[农业科学—果树学]