Comet Assay: A Strong Tool for Evaluating DNA Damage and Comprehensive Guidelines for Plant Cells

Abstract

<jats:p>Heavy metals affect plant system in various toxic ways including morphophysiological alterations and genotoxic damages inside a plant cell. The extent of DNA damage under any genotoxic agents can be effectively measured in single cells applying comet assay approach. Comet assay primarily measures DNA strand breakage in single cells and its use has increased in different areas: clinical applications, human monitoring, radiation biology and genetic ecotoxicology. This paper is a review of the detailed protocol and precautions to be taken while performing comet assay and may have been slightly modified from other original protocols according to the plant, organ, cell type, etc. In conclusion, the study reviewed in this paper demonstrate that the comet assay application in plants provides a reliable, sensitive and rapid system for the study of environmental genotoxicity caused by heavy metals.</jats:p>

Comet Assay: Multifaceted Options for Studies of Plant Stress Response

“…A Comet assay protocol includes the following main steps: the preparation of a suspension of cells or nuclei; the preparation of slides covered with agarose (gel slides); the resuspension of cells or nuclei in low melting point agarose; the application of the agarized suspension to gel slides; lysis; alkaline denaturation or preincubation in neutral solutions, depending on the protocol; the electrophoresis at alkaline pH or at neutral pH conditions, depending on the protocol; fixation (combined with neutralization step if electrophoresis was performed in alkaline conditions); staining; and microscopic analysis [14]. In recent decades, a number of highly informative and comprehensive reviews devoted to the peculiarities of Comet procedures in algae and higher plants have been published [10][11][12][13]96]. Recommendations and even guidelines pinpoint the most reliable laboratory practices to promote the generation of reproducible data, comparable between individual laboratories and research groups [16,94].…”

“…All operations should be performed in a dark room under very dim light (0.1-1 lux) and at ice-cold temperature conditions, to prevent the destruction of nuclei and any artificial DNA damage during the assay. This is required because after the digestion of chromatin proteins, DNA is considered to be sensitive to light, especially to ultraviolet waves [13,16]. The duration of the different steps strongly depends on the type of plant cells under investigation [14].…”

“…Remarkably, the freezing of plant cells or nuclei for later analysis, including their submersion in liquid N 2 and subsequent storage (at −80 • C), has been reported only rarely [4,13]. Usually, fresh plant material is cooled on ice and disintegrated with a razor or scalpel blade.…”

“…From this experiment, it became clear that the Comet assay is well suited for applications in plants, and that the sensitivity of the Comet test is comparable to, or even higher than, the traditional chromosome aberration test or micronucleus test [5,6], as reviewed in [7]. By now, Comet assays have been applied to plants exposed to different adverse conditions, and described in a number of reviews and protocols [8][9][10][11][12][13][14]. The adaptation of the method to plants has led to a burst in research in the field of ecotoxicology, while highly valuable results were obtained also in phytopathology, embryology, and plant cell biology.…”