*3.2. Gel Electrophoresis-Based Methods*

DNA damage in individual plant cells can be highlighted by gel electrophoresis-based methods [32]: cells embedded in agarose on a microscope slide are lysed with detergent and high salt concentrations to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix; subsequent electrophoresis conducted at high pH produces structures resembling comets, which can be observed by fluorescence microscopy. The intensity of the "comet tail" reflects the breaks in DNA sequences. Comet assay is able to detect DNA single-strand breaks, DNA double-strand breaks, and the formation of apoptotic nuclei [33]. This assay is often utilized as a confirmation method for microscopic evidence [26,34,35].

Several examples can be found in recent literature related to ENM genotoxicity in plants: Panda et al. [26], through comet assay, observed a significant DNA damage rate determined by dose-dependent Ag NPs exposure and correlated to ROS formation. Faisal et al. [36] utilized the comet assay to assess the genotoxic effects in *Solanum lycopersicum* L. seedlings exposed to NiO NPs (0–2 g L−1). Analyses showed a significant increase in genomic DNA damage, and an increase in the number of apoptotic (21.8%) and necrotic (24.0%) cells. Ci ˘gerci et al. [34] studied Indium tin oxide (ITO, In2O3/SnO2, ration 90/10%) particles (1–100 mg L−1), observing a significant increase in DNA damages in *A. cepa* root meristematic cells, highlighting potential alterations in the cell cycle, as demonstrated

by the higher number of cells able to enter into mitosis, as compared with the untreated controls. Thiruvengadam et al. [37] studied physiological, metabolic, and transcriptional effects of Ag NPs (1–10 mg L−1) *Brassica rapa* spp. observing a dose dependent DNA damage effects in turnip cells. Sun et al. [28] confirmed by comet assay the chromosomal aberration generated in *A. cepa*, highlighting a significant increase in DNA fragmentation after ZnO NPs exposure.

#### *3.3. Molecular Markers and Biomarker Assays*

Not only electrophoresis-based methods and chromosomal aberration analyses are utilized to detect potential genotoxic effects. Molecular markers can be also implemented as tools to detect the ENMs effect on genetic materials [38]. Molecular markers are defined as fragments or amplicons of DNA associated with a certain location within the genome. Molecular markers can be used as a biotechnological tool to identify and characterize a particular sequence of DNA when there is a limited knowledge of the sequence itself. This is the case, for example, of Random Amplified Polymorphic DNA (RAPD), markers based on PCR amplification of DNA fragments from random segments of genomic DNA, with a single primer of an arbitrary nucleotide sequence [39]. RAPDs do not require specific knowledge of the DNA sequence of the target organism. The occurrence of mutation at the level of DNA, particularly at the site that was previously complementary to the primer, will not allow amplicon production, resulting in a different pattern of amplified DNA fragments, which results in a molecular marker that is mainly dominant [40]. Since the early 1990s, several molecular marker tools have been developed in order to increase the detail of the physical genomic mapping and QTL analysis, with pros and cons related to the intrinsic properties of each molecular marker, respectively [41].

Molecular markers can be also utilized as tools to determine potential mutations at the level of the DNA sequence [42], which can support or validate data previously obtained, but also to isolate potential targets functional to biomarker characterization and development [43,44].

Hosseinpour et al. [45] studied the effects of the application of ZnO NPs (0–40 mg L<sup>−</sup>1) and plant growth promoting bacteria on *S. lycopersicum* L. under salt stress, with particular regard to DNA damage and cytosine methylation changes. RAPD analysis has been performed to determine the effects of co-exposure to bacteria and ZnO NPs on tomato genomic DNA. The rate of polymorphism observed in case of salinity stress treatment (42.2%) was a decrease in case of exposure to ZnO NPs and/or plant growth promoting bacteria from 32.4% to 25.3%, respectively. The results obtained through the application of different bacteria and ZnO NPs concentrations suggest the inverse relationship between the level of cytosine methylation and salinity stress tolerance. Mosa et al. [46] studied the genotoxic effects and genomic alterations in *Cucumis sativus* L. of copper-based nanoparticles (Cu NPs) using the RAPD technique. Cu NPs (0–200 mg L−1) showed a concentration-dependent increase rate of polymorphism occurrence, highlighting the Cu NPs genotoxic effect. Kokina et al. [47] studied the impact of iron oxide nanoparticles (Fe3O4 NPs, 0–4 mg L<sup>−</sup>1) on *Medicago falcata* L. The utilization, in this case, of the RAPD technique highlighted the genotoxic effect of Fe3O4 NPs, which induced genomic DNA modifications. This type of PCR-based molecular marker for its randomic amplification nature may be subject to experimental or technical variability, and thus requires procedures of validation [38,39]. Several other type of molecular markers and biomarkers can be utilized as more reliable tools to assess genomic variations, either at the level of genomic DNA (gDNA) and plastid and mitochondrial DNA (ptDNA, mtDNA). Pagano et al. [44] highlighted a modulation of the organellar functionality in *Arabidopsis thaliana* L. Heynh in direct comparison to a modulated organelle genome replication level, upon exposure to CeO2 NPs, FeOx NPs, ZnS QDs, CdS QDs (80–500 mg L−1). In this case, multiple target genes at the level of ptDNA and mtDNA were utilized as structural markers to assess the potential variations at the level of DNA replication by real time qPCR. In particular, CdS QD exposure triggered potential variations at the sub-stoichiometric level of the two organellar genomes, while

nanoscale FeOx NPs and ZnS QDs exposure triggered an increase in organellar DNA copy numbers. These findings suggested how modification in organellar genomes stoichiometry may result from a potential morpho-functional adaptive response to ENM exposure, which led to decreased rates of photosynthesis and cellular respiration.
