Establishment and Optimization of a Tobacco Chromosome Preparation System

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

Abstract

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

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.

Figures/Tables 5

References 32

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