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Estimation of nuclear DNA content in plants using flow cytometry
Jaroslav Dolezel1, Johann Greilhuber, Jan Suda
1Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, Olomouc, Czech Republic. dolezel@ueb.cas.cz
Nature Protocols
|September 15, 2007
Summary
Flow cytometry (FCM) is the leading method for measuring plant nuclear DNA content, offering efficient genome size estimation. This study details FCM protocols for plant sample preparation and data analysis, addressing common challenges.
Area of Science:
- Plant Biology
- Cytometry
- Genetics
Background:
- Flow cytometry (FCM) is the predominant technique for quantifying nuclear DNA content in plants.
- It surpasses methods like Feulgen densitometry for estimating genome size, polyploidy, and endopolyploidy due to its efficiency and throughput.
Purpose of the Study:
- To present optimized protocols for plant cell nucleus isolation for FCM analysis.
- To guide the measurement, data analysis, and interpretation of nuclear DNA content in plants using FCM.
Main Methods:
- Developed four distinct protocols for preparing suspensions of intact plant cell nuclei.
- Utilized DNA-selective fluorochromes for FCM analysis of nuclear DNA amounts.
Main Results:
- Detailed procedures for sample preparation, chemical and equipment requirements, and the FCM measurement process.
- Addressed common issues in plant FCM, including secondary metabolite interference and standardization requirements.
Conclusions:
- FCM provides a robust and efficient method for plant genome size and ploidy analysis.
- Standardization and correct terminology are crucial for accurate DNA content and genome size determination in plants.
