**1. ENM Genotoxicity in Plant: The Current State**

The global market for nanotechnology might grow from USD 5.2 bln in 2021 to USD 23.6 bln by 2026, with annual growth rate (CAGR) of 35.5%, respectively for the years 2021–2026. The North American market for nanotechnology is estimated to grow from USD 1.6 bln in 2021 to USD 7.2 bln by 2026, at a CAGR of 34.5% for the period 2021–2026, while the Asia–Pacific market for nanotechnology is estimated to grow from USD 1.2 to USD 6.0 bln, at a CAGR of 37.6%, respectively, for the same time period, as reported by Nanotechnology Services Global Market Report 2022.

Nanotechnology has captured the attention of a wide range of industries in many sectors, gaining in a short period large attraction and significant public investments in research and development, in addition to increasing private-sector investments. Many governments are implementing the application of nanotechnology notwithstanding the associated risks and uncertainties [1]. Nanotechnology allows the development and improvement of completely new products, processes, and services [2].

However, engineered nanomaterials (ENMs) are in the process of being dispersed into the environment, coming into contact with non-mammal organisms and plants [3,4]. So far, scientists have just started to investigate the impact of nanomaterials on plants, which has contrasting outcomes depending on the type of nanomaterial and on the plant species [5]. The field of nanotoxicology has been extended from microorganisms to plants and animals, even if the idea of ENM genotoxicology for plants is not so widespread. In fact, a search in Scopus [6] for publications with the word "Nanotoxicology" since 2013 produced 625 results. Research in the same timeframe, from 2013 to 2022, using the word "nanomaterial genotoxicology" produced only four outcomes. A more extensive database research has been conducted by Ghosh et al. [7], who found that there are few papers dealing with

the genotoxicity of the nanoparticles in respect to other effects that nanoparticles exert on plants.

In the field of toxicology, the term genotoxicity generally refers to any kind of damage to the genetic material, the genome, as cytotoxicity indicates injury to the cell instead. Toxic effects to the genetic material have attracted great attention for many reasons, including in particular that the genome of germ cells, the reproductive cells, determine all heritable characteristics of organisms [8]. Investigation of injury to the genome has led to the definition of a specific kind of toxicity, genotoxicity, and to the development of the subspecialty of genetic toxicology [9].

Several plant species have the intrinsic capability of being used as multiple genetic assay systems. These plant genetic systems have played important roles in detecting new mutagens and developing techniques later used in other systems for advancing mutagenesis knowledge. Some of the mainly used higher plant species are: *Allium cepa* L. (2n = 16), *Arabidopsis thaliana* L. (2n = 10), *Crepis capsularis* (L.) Wallr (2n = 6), *Glycine max* L. (2n = 40), *Hordeum vulgare* L. (2n = 14), *Solanum lycopersocum* L. (2n = 16), *Nicotiana tabacum* L. (2n = 48), *Pisum sativum* L. (2n = 14), *Tradescantia* Ruppius ex L. (2n = 24), *Vicia faba* L. (2n = 12) and *Zea mays* L. (2n = 20) [10].

#### **2. Mechanisms of ENM-Induced Genotoxicity**

In vitro and in vivo characterization of the response to ENM exposure in both growth media and biological matrices have been extensively discussed in recent years [11]: uptake, pathways, biotransformation, and the mechanisms of ENM genotoxicity. In vitro and in vivo characterization of the response to ENM exposure in both growth media and biological matrices have been extensively discussed in recent years [11]: uptake, pathways, biotransformation, and the mechanisms of ENM genotoxicity. Different mechanisms can be exploited depending on the different ENM physico-chemical properties: (i) ENMs simply able to pass through the cellular membrane lipid bilayer, depending on several factors such as size, charge, hydrophobicity, composition and shape; (ii) endocytosis processes by which ENMs are taken up and accumulated in plant tissues, as well as Trojan horse mechanism and possible biotransformation processes (including corona protein interactions), lead to ENMs accumulation in plant cells; (iii) the utilization of membrane transporters which can mediate the translocation of ENMs into the plant cell, due to their affinity to the transporter itself [12,13]. As a result, ENMs response can be explicated by two different mechanisms: effects directly ascribed to the ENMs interaction with the cellular components, or its biotransformed physico-chemical forms (including ions released, depending on the ENM stability) [14] and indirectly, due to ROS production, increase mediated by mitochondrion and chloroplast functionality alteration, leading to a general cellular oxidative stress increase by triggering ENM-induced cytotoxicity and genotoxicity mechanisms [12]. The response observed is an effect of the activation defense mechanisms, including antioxidant defense mechanisms, apoptosis and secondary metabolite (e.g., phytohormone) production and antioxidant enzymes [11].

As a key metabolite, ROS are necessary in plants for many important signaling reactions, however they also constitute by-products in aerobic metabolism that can induce oxidative damage in plants [15]. It has been demonstrated that nanoparticles and ROS can directly enter the nucleus of the plant cell and, by binding chromatin and/or interacting with DNA, induce damages [16], showing potential mutagenic effect.

For nanoparticles (NPs) such as Ag NPs (coated and uncoated), carbon nanotubes, ZnO NPs, Al2O3 NPs, Fe2O3 NPs, Co3O4 NPs, and NiO NPs, the main features that determine genotoxicity have been found to be ions release, dimension, and zeta potentials [7]. As a fact, these features contribute to the penetration of the nanoparticle into the cell nucleus and the consequent damage to DNA [17]. Several assays have been developed that use higher plants to measure the mutagenic effects of chemicals in general as indicators of carcinogenicity. These assays using plants require less complex equipment and materials than many other genotoxicity tests, which is a potential advantage, particularly when research resources are limited [18]. Standard genotoxicity tests have been reviewed by the Gene-Tox program of the U.S. Environmental Protection Agency (EPA) concerning gene mutation, chromosomal effects and DNA damage repair on the following plants: *A. thaliana*, *G. max*, *H. vulgare*, *Tradescantia*, *Z. mays* [18,19]. Early studies on plants progressed to more sophisticated and complex assays on many other plants, and to many more materials including ENMs [7,17].

In this minireview, the most important genotoxicity assays applied on plants are explained, with a focus on how they can be utilized to determine the genotoxic effects for nanoparticles, which include standard techniques available and new tools and instruments. DNA damage may cause epigenetic changes, through covalent DNA modification, histones modification, and regulation of non-coding RNAs (miRNAs, lncRNAs, piRNAs). Modifications at the level of DNA methylation (global or gene-specific) may have a profound impact on chromatin remodeling and on locus-specific gene expression, respectively [20].
