Research Article Comet-FISH with rDNA Probes for the Analysis of Mutagen-Induced DNA Damage in Plant Cells Jolanta Kwasniewska, * Marta Grabowska, Miroslaw Kwasniewski, and Bozena Kolano Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydra- zide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treat- ment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleo- lus formation and differences in chromatin structure between the two loci may explain the different sus- ceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment. Environ. Mol. Mutagen. 00:000–000, 2012. V V C 2012 Wiley Periodicals, Inc. Key words: comet assay; Crepis capillaris; fluorescence in situ hybridization; maleic hydrazide INTRODUCTION The comet assay is a method used for analyzing genomic DNA damage and repair by measuring the level of single-, double-strand DNA breaks, and alkali-sensitive sites in individual cells [Collins et al., 2008]. The assay is based on embedding cells/nuclei in agarose on microscopic slides after exposure to a genotoxic agent and the applica- tion of an electrical field to cause the migration of frag- mented nuclear DNA and loops from inside the nucleus (the head) to outside the nucleus (the tail). The comet assay has been widely accepted as a reliable marker for DNA damage [Collins, 2004]. It is becoming an important tool in the environmental monitoring of atmosphere, water and soil contamination [Fairbairn et al., 1994; Poli et al., 1999; Restivo et al., 2002] as well as in genotoxic risk assessment [Cotelle and Ferard, 1999]. Percent DNA in the tail (TD, %), tail length (TL, lm) and tail moment (TM) are the most common parameters utilized to describe the level of DNA damage [Collins et al., 2008]. Most studies involving the comet assay have used ani- mal and human cells. In contrast to animal and human cells, the plant cell wall is a barrier that makes it impossi- ble to embed whole cells into agarose and therefore iso- lated nuclei are used. Protocols for comet assays in many plant species (e.g., Vicia faba, Nicotiana tabacum, Allium cepa) have been developed [Koppen and Verschaeve, 1996; Gichner and Plewa, 1998; Gichner et al., 2002, 2003] and applied to the measurement of DNA damage in various plant tissues including: seeds [Cerda et al., 1997], roots [Gichner et al., 2000a], and leaves [Gichner and Plewa, 1998]. Fluorescence in situ hybridization (FISH) applied on microgels with embedded nuclei (comet-FISH) was introduced few years ago but relatively few studies have utilized it. Centromere, telomere and ribosomal DNA repeats are the most widely used probes for comet- FISH. Chromosome arm- or whole-chromosome painting probes, as well as gene-specific probes have also been applied [Glei et al., 2009]. This technique increases the resolution of the comet assay by analyzing the *Correspondence to: Dr. Jolanta Kwasniewska, Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. E-mail: juchimiu@us.edu.pl Grant sponsor: National Science Centre, Poland; Grant Number 3178/B/ P01/2011/40. Received 21 December 2011; provisionally accepted 3 April 2012; and in final form 5 April 2012 DOI 10.1002/em.21699 Published online in Wiley Online Library (wileyonlinelibrary.com). V V C 2012 Wiley Periodicals, Inc. Environmental and Molecular Mutagenesis 00:000^000 (2012)