利用流式细胞仪检测草莓倍性方法的优化

Optimization of strawberry ploidy identification method using flow cytometry

【目的】建立一套利用流式细胞仪快速鉴定草莓倍性的方法,为草莓新品种选育提供技术支持。【方法】以二倍体森林草莓、四倍体东方草莓、六倍体智利草莓和八倍体凤梨草莓为试材,通过改良提取液、增加离心和过滤次数等,应用流式细胞仪对大田生长的草莓植株根尖、茎尖和幼叶进行倍性检定。【结果】不同部位材料检测峰值有所不同,其中匍匐茎茎尖和根尖的细胞检测效果优于幼叶;在提取液中加入巯基乙醇,可使荧光通道值峰形更饱满;增加离心次数和上机前再过滤,可使峰值更清晰。【结论】利用流式细胞仪检测草莓倍性的优化条件为:采用匍匐茎茎尖或根尖的细胞为检测试材,在常规提取液中添加巯基乙醇来制备DNA悬浮液,DNA悬浮液离心漂洗3次后经300目尼龙网过滤再上机检测。

[ Objective ]In this research, a rapid and accurate method was developed for the identification of strawber- ry ploidy by flow cytometry in order to provide improving techniques for breeding new strawberry cuhivar. [Method]The root apex, stolon apex, and young leaf from 4 types of strawberry ploidies viz., diploid Senling strawberry, tetraploid Ori- ent strawberry, hexaploid Chile strawberry, and oetaploid bromeliad strawberry planted in field were collected to identify their ploidy with different protocols; each protocol was modified depending on different extraction buffer ingredients and relevant corresponding concentrations, the various times of centrifugations, and different amounts of filtration. [Result]The research results showed different identification peaks presented in different tissues, and stolon and root apex were relative- ly better than that of fresh leaves, therefore stolon and root apex were then selected as the test materials. With the addition of mercaptoethanol into the extract, a full-peaked shape can be observed at the fluorescence channel ; increasing centrifu- gation time and re-filtering suspension before testing could make peak value more clear. [Conclusion ]The optimization conditions on identifying strawberry ploidy by flow cytometry were： taking stolon apex or root apex as materials, adding mercaptoethanol into the conventional extract to prepare DNA suspension, and DNA suspension centrifuging 3 times and re-filtering through 300-mesh nylon net before testing on machine.