The chromatin structure is significantly influenced by some epigenetic modifications including DNA methylation. The nuclear organization plays an essential role in the cell response to external stresses including mutagens. We present an analysis of the correlation between epigenetic modifications and the instability of the Brachypodium distachyon genome, which are observed as micronuclei, following maleic hydrazide (MH) and nitroso-N-methylurea (MNU) treatments. We compared the level of DNA methylation in the control (untreated) and mutagen-treated B. distachyon nuclei. An immunostaining method using specific antibodies against modified DNA anti-5-methylcytosine was used for the evaluation of DNA methylation in a single nucleus and micronucleus. Interestingly, we showed an alteration of DNA methylation in cells after mutagenic treatments. The results indicate that DNA methylation might be involved in the response of the B. distachyon genome to mutagenic treatments. This demonstrates that analyses of the epigenetic modifications should be integrated into current plant genetic toxicology in order to explain the mechanisms of DNA damage and repair in plants.

Cytogenetic approaches play an essential role as a quick evaluation of the first genetic effects after mutagenic treatment. Although labor-intensive and time-consuming, they are essential for the analyses of cytotoxic and genotoxic effects in mutagenesis and environmental monitoring. Over the years, conventional cytogenetic analyses were a part of routine laboratory testing in plant genotoxicity. Among the methods that are used to study genotoxicity in plants, the micronucleus test particularly represents a significant force. Currently, cytogenetic techniques go beyond the simple detection of chromosome aberrations. The intensive development of molecular biology and the significantly improved microscopic visualization and evaluation methods constituted significant support to traditional cytogenetics. Over the past years, distinct approaches have allowed an understanding the mechanisms of formation, structure, and genetic activity of the micronuclei. Although there are many studies on this topic in humans and animals, knowledge in plants is significantly limited. This article provides a comprehensive overview of the current knowledge on micronuclei characteristics in plants. We pay particular attention to how the recent contemporary achievements have influenced the understanding of micronuclei in plant cells. Together with the current progress, we present the latest applications of the micronucleus test in mutagenesis and assess the state of the environment.

This study is an example of using 5-ethynyl-2′-deoxyuridine (EdU) for detecting sister chromatid exchanges (SCEs) at chromosomal level. Here we report a detailed protocol for differential labeling sister chromatids in barley (Hordeum vulgare, 2n = 14) cells that is based on the incorporation and simple detection of EdU. The perfect distinguishing of sister chromatids enabled an analysis of the effects of two model agents-maleic acid hydrazide (MH) and gamma rays-on the formation of SCEs. Using this method, we demonstrated the high sensitivity of barley cells to maleic hydrazide, which is expressed as an increased level of SCEs. A gamma ray induced only slightly more SCEs than in the control cells. The possible mechanisms of MH and gamma ray action in respect to distinguishing chromatids using EdU are discussed. Recommendation for SCEs visualization using EdU as an easy and quick method that can be successfully adapted to other plant species and potentially for human genotoxicity studies is presented.

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