基于流式细胞仪分析的花魔芋倍性检测条件筛选

doi:10.14088/j.cnki.issn0439-8114.2026.01.014

湖北农业科学 ›› 2026, Vol. 65 ›› Issue (1): 78-83.doi: 10.14088/j.cnki.issn0439-8114.2026.01.014

基于流式细胞仪分析的花魔芋倍性检测条件筛选

吕恋心^1,2, 焦盈盈^1,2, 张诗婉^1,2, 李晓倩^1,2, 刘淼^1,2, 何斐^1,2

1.富硒魔芋陕西省高校工程研究中心,陕西安康 725000;
2.安康学院现代农业与生物科技学院,陕西安康 725000

收稿日期:2025-07-24 出版日期:2026-01-25 发布日期:2026-02-10
通讯作者: 何斐(1988-),女,陕西南郑人,副教授,博士,主要从事魔芋资源保护与利用研究工作,(电话)19891560830(电子信箱)hefei6000@163.com。
作者简介:吕恋心(2003-),女,陕西渭南人,主要从事魔芋资源保护与利用研究工作,(电话)15719254667(电子信箱)1677800412@qq.com。
基金资助:
陕西省科技厅项目(2025-JC-QN-1597; 2025NC-YBXM-029); 国家级大学生创新创业重点项目(202411397001)

Screening of ploidy detection conditions in Amorphophallus konjac based on flow cytometric analysis

LYU Lian-xin^1,2, JIAO Ying-ying^1,2, ZHANG Shi-wan^1,2, LI Xiao-qian^1,2, LIU Miao^1,2, HE Fei^1,2

1. Engineering Research Center of Selenium-rich Konjac, Universities of Shaanxi Province, Ankang 725000, Shaanxi, China;
2. School of Modern Agriculture & Biotechnology, Ankang University, Ankang 725000, Shaanxi, China

Received:2025-07-24 Published:2026-01-25 Online:2026-02-10

摘要/Abstract

摘要： 以花魔芋(Amorphophallus konjac K. Koch)新鲜愈伤组织为材料,比较解离缓冲液、荧光染色剂、过滤次数和离心处理对倍性检测结果的影响。结果表明,MG^b解离缓冲液处理的花魔芋细胞核数量明显高于MgSO₄和Tris-MgCl₂处理组,且主峰明显,碎片峰较少,CV为4.47%。荧光染色剂DAPI与PI相比,染色效果无明显差异,但DAPI无需加入RNAase即可减少荧光噪音产生的背景干扰,节省检测时间。1次过滤法和2次过滤法都可以有效去除黏连细胞和细胞碎片,2次过滤法收集到的细胞核数量更多,效果更好。在制备细胞核悬浮液时,过滤后直接避光染色上机的检测效果最佳。研究初步建立的花魔芋流式细胞术倍性分析方法为取0.25 g花魔芋新鲜愈伤组织,加入1 mL MG^b解离缓冲液,混合切碎,过滤后加入50 μL DAPI染色剂避光染色30 min即可上机检测。

关键词: 花魔芋(Amorphophallus konjac K. Koch), 流式细胞仪, 倍性检测

Abstract: Taking the fresh callus tissue of Amorphophallus konjac K. Koch as materials, the influences of dissociation buffers, fluorescent nucleic acid stains, filtering times, and centrifugation protocols on ploidy detection were compared. The results showed that, MG^b lysis buffer yielded significantly higher counts of A. konjac nucleus compared to MgSO₄ and Tris-MgCl₂ buffers, with a prominent peak and minimal fragment peaks observed. The coefficient of variation (CV) was calculated at 4.47%. Fluorescent staining with DAPI demonstrated comparable efficacy to PI, with the added advantage of reducing background fluorescence noise without RNAase treatment, thereby conserving analysis time. Both the primary filtration and the secondary filtration methods could effectively remove adhered cells and cell debris. The secondary filtration method collected more cell nuclei and had a better effect. Optimal nuclear suspension preparation involved direct light avoidance and in situ staining post-filtration. The proposed flow cytometric protocol for A. konjac ploidy analysis involved: homogenizing 0.25 g of fresh callus tissue in 1 mL MG^b lysis buffer, filtering, staining with 50 μL DAPI in darkness for 30 minutes, followed by immediate analysis.

Key words: Amorphophallus konjac K. Koch, flow cytometry, ploidy detection

中图分类号:

S632.2

吕恋心, 焦盈盈, 张诗婉, 李晓倩, 刘淼, 何斐. 基于流式细胞仪分析的花魔芋倍性检测条件筛选[J]. 湖北农业科学, 2026, 65(1): 78-83.

LYU Lian-xin, JIAO Ying-ying, ZHANG Shi-wan, LI Xiao-qian, LIU Miao, HE Fei. Screening of ploidy detection conditions in Amorphophallus konjac based on flow cytometric analysis[J]. HUBEI AGRICULTURAL SCIENCES, 2026, 65(1): 78-83.

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基于流式细胞仪分析的花魔芋倍性检测条件筛选

Screening of ploidy detection conditions in Amorphophallus konjac based on flow cytometric analysis

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