烟草染色体制备体系的建立与优化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0915

生物技术通报 ›› 2025, Vol. 41 ›› Issue (3): 44-50.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0915

烟草染色体制备体系的建立与优化

韩梦荞¹(), 吴疆², 李丽华³, 王昭懿⁴, 邓习¹, 韦凤杰⁵, 任民¹, 孙洋洋¹(), 李富欣⁵()

^1.中国农业科学院烟草研究所，青岛 266101
^2.河南省烟草公司南阳市公司，南阳 473000
^3.河南省烟草公司洛阳市公司，洛阳 471000
^4.宁夏大学农学院，银川 750021
^5.河南省烟草公司，郑州 450018

收稿日期:2024-09-20 出版日期:2025-03-26 发布日期:2025-03-20
通讯作者: 李富欣，男，博士，高级农艺师，研究方向：烟叶生产管理；E-mail: lfx4413@163.com
孙洋洋，男，博士，助理研究员，研究方向：烟草种质资源和染色体工程；E-mail: sunyangyang@caas.cn
作者简介:韩梦荞，女，硕士研究生，研究方向：作物科学；E-mail: hanmengqiao0502@163.com
第一联系人：吴疆为共同第一作者
基金资助:
中国烟草总公司重大科技项目（110202201002（JY-02）），中国烟草总公司河南省公司创新项目(2024410000240025);中央级公益性科研院所基本科研业务费专项(1610232023022)

Establishment and Optimization of a Tobacco Chromosome Preparation System

HAN Meng-qiao¹(), WU Jiang², LI Li-hua³, WANG Zhao-yi⁴, DENG Xi¹, WEI Feng-jie⁵, REN Min¹, SUN Yang-yang¹(), LI Fu-xin⁵()

^1.Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101
^2.Nanyang Branch of Henan Provincial Tobacco Company, Nanyang 473000
^3.Luoyang Branch of Henan Provincial Tobacco Company, Luoyang 471000
^4.School of Agriculture, Ningxia University, Yinchuan 750021
^5.Henan Provincial Tobacco Company, Zhengzhou 450018

Received:2024-09-20 Published:2025-03-26 Online:2025-03-20

摘要/Abstract

摘要：

目的开发一套适合烟草的染色体制备体系，为推进烟草染色体工程以及远缘杂交研究的系统开展奠定基础。方法以普通烟草为供试材料，利用酶解法进行染色体制备体系的构建。试验针对根尖组织8个时间段进行采样；利用4种方法进行预处理，包括对照（前期无任何处理）、20%风油精室温处理2 h、冰水混合物0℃处理48 h以及N₂O处理，其中，N₂O处理又分为20‒70 min每间隔10 min取一次样；根尖样品分别在35、45和55 min不同时间下进行酶解。对每种处理方法或时长均进行了染色体制备并观察，根据染色体长度、清晰度及分散程度鉴定烟草染色体制备最佳方法。整合优化后的处理方法和时长，形成烟草染色体制备方法，并通过制备不同类型烟草品种的染色体样品进行体系的适用性验证。结果 10∶30‒12∶30为烟草根尖取样的理想时间，N₂O预处理30‒40 min，酶解45 min较为适宜，并利用该优化体系应用于普通烟草祖先种、普通烟草、远缘杂交衍生后代3个不同种质资源，均获得高质量染色体样品。结论建立了适用于烟草染色体的制备体系。利用该体系可在3 h内完成从烟草根尖取样到制备出合格的染色体样品。

关键词: 烟草, 染色体制备, 酶解法, 种质资源, 染色体工程

Abstract:

Objective To develop a specialized chromosome preparation system for tobacco, laying the groundwork for advancements in tobacco chromosome engineering and the systematic exploration of distant hybridization research. Method The chromosome preparation system was established using the enzyme digestion method, with common tobacco as the test material. The experiment was designed to sample the root tip tissues at eight time intervals. Pre-treatment included four methods: A control group (without any pre-treatment), treatment with 20% wind medicated oil at room temperature for 2 h, exposure to an ice-water mixture at 0℃ for 48 h, and N₂O treatment, in which the N₂O treatment was further divided into samples collected at 10-min intervals from 20 to 70 min; the root tip samples were enzymatically digest for different durations, including 35, 45, and 55 min. Chromosome preparation was performed and observed for each treatment and duration, with the optimal method for tobacco chromosome preparation identified based on chromosome length, clarity, and dispersion. Merging improved processing methods and durations to establish a tobacco chromosome preparation system. The validation applicable to the method was further confirmed by preparing chromosome samples from various tobacco varieties. Result It is the optimal time between 10‍‍∶‍30 and 12‍‍∶‍‍30 for tobacco root tip sampling, 30‒40 min for N₂O pretreatment and 45 min for enzymatic digestion were suitable for preparing chromosomes. High-quality chromosome samples were successfully obtained by applying this optimization system to three different germplasm resources: the ancestral species for common tobacco, common tobacco, and progeny derived from distant hybridization. Conclusion A preparation system for tobacco chromosomes is established, capable of preparing high-quality chromosome samples from tobacco root tips within 3 h. This system provides crucial technical support for research in distant hybridization and chromosome engineering.

Key words: tobacco, chromosome preparation, enzymatic hydrolysis, germplasm resources, chromosome engineering

韩梦荞, 吴疆, 李丽华, 王昭懿, 邓习, 韦凤杰, 任民, 孙洋洋, 李富欣. 烟草染色体制备体系的建立与优化[J]. 生物技术通报, 2025, 41(3): 44-50.

HAN Meng-qiao, WU Jiang, LI Li-hua, WANG Zhao-yi, DENG Xi, WEI Feng-jie, REN Min, SUN Yang-yang, LI Fu-xin. Establishment and Optimization of a Tobacco Chromosome Preparation System[J]. Biotechnology Bulletin, 2025, 41(3): 44-50.

图/表 5

表1 不同取样时间下根尖细胞观察情况

Table 1 Observations on root tip cells at different sampling times

取样时间

Sampling time

根尖细胞观察情况

Observations on the chromosomes of root apical cells

8∶30‒9∶30

分裂中期细胞数量最少，约占1.2%

Minimum number of cells in mid-division， about 1.2%

9∶30‒10∶30

分裂中期细胞数量上升，约占18.8%

The number of cells rises in mid-division， about 18.8%

10∶30‒11∶30

分裂中期细胞数量最多，约占35.2%

Maximum number of cells in mid-division， about 35.2%

11∶30‒12∶30

分裂中期细胞数量下降，约占27.8%

Decrease in cell number during mid-division， about 27.8%

12∶30‒13∶30

分裂中期细胞数量较少，约占1.6%

Smaller number of cells in mid-division， about 1.6%

13∶30‒14∶30

分裂中期细胞数量较少，约占10.8%

Smaller number of cells in mid-division， about 10.8%

14∶30‒15∶30

分裂中期细胞数量较少，约占6.4%

Smaller number of cells in mid-division， about 6.4%

15∶30‒16∶30

分裂中期细胞数量上升，约占19.2%

The number of cells rises in mid-division， about 19.2%

图1 不同处理方式制备的烟草根尖染色体形态A：对照；B：20%风油精处理2 h；C：冰水混合物0℃处理48 h；D：N2O处理70 min；E：N2O处理60 min；F：N2O处理50 min；G：N2O处理40 min；H：N2O处理30 min；I：N2O处理20 min。bars=50 μm

Fig. 1 Different chromosomes morphologies of root tip cells in different treatmentsA: CK; B: 20% essential balm treatment for 2 h; C: ice-water mixture 0℃ treatment for 48 h; D: N2O treatment for 70 min; E: N2O treatment for 60 min; F: N2O treatment for 50 min; G: N2O treatment for 40 min; H: N2O treatment for 30 min; I: N2O treatment for 20 min. Scale bar=50 μm

图2 不同酶解时间根尖细胞不同状态A：酶解35 min；B：酶解45 min；C：酶解55 min。bars=50 μm

Fig. 2 Different states of root tip cells at different enzymolysis timesA: Enzymolysis 35 min. B: Enzymolysis 45 min. C: Enzymolysis 55 min. Scale bar=50 μm

图3 烟草染色体制备流程

Fig. 3 Preparation process of tobacco chromosome

图4 酶解法制备的不同烟草种质资源染色体样品A：绒毛状烟草；B：中烟100；C：L8；D：绒毛状烟草DAPI图像；E：中烟100 DAPI图像；F：L8 DAPI图像（A‒C：bars=50 μm；D‒F：bars=5 μm）

Fig. 4 Chromosome samples from various tobacco germplasm resources prepared using an enzymatic digestion methodA: N. tomentosiformis; B: ZY100; C: L8; D: N. tomentosiformis DAPI image; E: ZY100 DAPI image; F: L8 DAPI image (A‒C: scale bar = 50 μm; D‒F: scale bar=5 μm)

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烟草染色体制备体系的建立与优化

Establishment and Optimization of a Tobacco Chromosome Preparation System

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