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Journal of Botany
Volume 2010 (2010), Article ID 320609, 7 pages
http://dx.doi.org/10.1155/2010/320609
Research Article

Improved and Reproducible Flow Cytometry Methodology for Nuclei Isolation from Single Root Meristem

Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil

Received 29 January 2010; Revised 30 March 2010; Accepted 6 May 2010

Academic Editor: João Loureiro

Copyright © 2010 Thaís Cristina Ribeiro Silva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Root meristems have increasingly been target of cell cycle studies by flow cytometric DNA content quantification. Moreover, roots can be an alternative source of nuclear suspension when leaves become unfeasible and for chromosome analysis and sorting. In the present paper, a protocol for intact nuclei isolation from a single root meristem was developed. This proceeding was based on excision of the meristematic region using a prototypical slide, followed by short enzymatic digestion and mechanical isolation of nuclei during homogenization with a hand mixer. Such parameters were optimized for reaching better results. Satisfactory nuclei amounts were extracted and analyzed by flow cytometry, producing histograms with reduced background noise and CVs between 3.2 and 4.1%. This improved and reproducible technique was shown to be rapid, inexpensive, and simple for nuclear extraction from a single root tip, and can be adapted for other plants and purposes.