**3. Current Methods and Functional Applied Strategies**

#### *3.1. Standard Techniques*

From an operational point of view, different approaches can be utilized to pinpoint the genotoxic effects of ENMs on plant DNA [7]. All these approaches are able to assess ENM genotoxicity from different points of view, showing potential advantages and disadvantages in terms of sensitivity and resolution, respectively. Methods described and relevant examples are schematized in Figure 1.

**Figure 1.** Schematic representation of the methodologies utilized to highlight plant ENM genotoxicity: (**a**) microscopic techniques to highlight chromosomal aberrations, (**b**) electrophoresis-based methods (e.g., comet assay) to highlight genomic DNA (gDNA) damage, (**c**) molecular markers (e.g., RAPD) to show mutational events and (**d**) Real time PCR based methods to highlight copy number variation (stoichiometric or sub-stoichiometric shift) in plastid (ptDNA) or mitochondrial (mtDNA) genomes. These techniques can be utilized as Alternative Testing Strategies (ATS), in assessing and/or characterizing the risk associated with ENMs exposure/effects, not only in experimental controlled conditions, but also in monitoring of realistic scenarios, at early exposure stages.

Among the major effects observed from the exogenous genotoxic effects on plant genomes, the chromosomal aberrations, which are the result of structural and numerical chromosome changes, preferentially within heterochromatic regions, are composed mainly of repetitive DNA sequences [21]. Optical, fluorescence and confocal laser scanning microscopy techniques are able to highlight aberrations at the level of the chromosome

structure, including chromosomal breaks, sticky, multipolar, and laggard chromosomes, as well as micronucleus formation [22–24].

Chromosomal aberrations have been observed by Pakrashi et al. [25], studying the effect of titania nanoparticles (TiO2 NPs) on *Allium cepa* L. root tips, in the range 0–100 mg L−1. Optical and fluorescence microscopic analyses showed a dose-dependent frequency of the aberration appearance, which includes chromosomal breaks, chromosome stickiness during metaphase, multiple micronucleus formation, as well as the occurrence of binucleate cells. Confocal microscopic images highlighted the formation of chromosomal bridges, in addition to a distorted and notched nucleus.

Similarly, Panda et al. [26] observed micronucleus mitotic aberrations formations in *Allium cepa* L. cells exposed to 0–80 mg L−<sup>1</sup> of different forms of silver ionic colloidal nanoparticles (Ag NPs). Additionally, in this case, the percentage of increase in aberrations was concentration dependent.

Silva and Monteiro [27] investigated the genotoxic and phytotoxic impacts of silicabased nanomaterials (SiO2 NPs, in a range between 0.54–1.82 g L−1) using root tip cells of *Allium cepa* L., highlighting chromosomal aberrations and delays in mitosis due to disturbed metaphase. Sun et al. [28] studied the cytotoxic and genotoxic effects of ZnO NPs (5–50 mg L<sup>−</sup>1) in root meristems of *Allium cepa* L. cells by cell membrane integrity, metabolic activity, reactive oxygen species (ROS) accumulation, DNA damage and chromosomal aberration, highlighting how ZnO NP accumulation within cell nucleuses affected cell mitosis, inducing chromosome breaks, bridges, stickiness, and micronuclei formation. As often reported, the utilization of *Allium cepa* L. is considered an efficient bioindicator in genotoxicity testing, due to its reduced number of chromosomes and rapid root growth rate [29]. Abdelsalam et al. [30] investigated the effects of foliar application of (nitrogenphosphorus-potassium) NPK nanoparticles (2.5 to 5 kg ha−1) for two harvest seasons on *Triticum aestivum* L. as an alternative to conventional fertilizers, assessing yield and genotoxic effects. Although fertilization with NPK nanoparticles produced an increase in yield, root-tip cells showed various types of chromosomal aberrations such as multinuclei, micronuclei, chromosome deletion, lagging chromosome and cell membrane damage, and the NPK nanoparticles treatment at 5 kg ha−<sup>1</sup> produced 35.7–38.9% of abnormal cells. With a similar approach, Abdelsalam et al. [31] tested on *Triticum aestivum* L. seeds the utilization of (amino-zinc) AZ nanoparticles (50–150 mg L<sup>−</sup>1) on in vitro medium for 8, 16, or 24 h. Genotoxicity was evaluated in root meristems, revealing mitotic activity variations, chromosomal aberrations, and micronuclei formation and a growth inhibit of the normal cellular function.
