Responses of Medicinal and Aromatic Plants to Engineered Nanoparticles †

: Medicinal and aromatic plants have been used by mankind since ancient times. This is primarily due to their healing effects associated with their speciﬁc secondary metabolites (some of which are also used as drugs in modern medicine), or their structures, served as a basis for the development of new effective synthetic drugs. One way to increase the production of these secondary metabolites is to use nanoparticles that act as elicitors. However, depending on the speciﬁc particle size, composition, concentration, and route of application, nanoparticles may have several other beneﬁts on medicinal and aromatic plants (e.g., increased plant growth, improved photosynthesis, and overall performance). On the other hand, particularly at applications of high concentrations, they are able to damage plants mechanically, adversely affect morphological and biochemical characteristics of plants, and show cytotoxic and genotoxic effects. This paper provides a comprehensive overview of the beneﬁcial and adverse effects of metal-, metalloid-, and carbon-based nanoparticles on the germination, growth, and biochemical characteristics of a wide range of medicinal and aromatic plants, including the corresponding mechanisms of action. The positive impact of nanopriming and application of nanosized fertilizers on medicinal and aromatic plants is emphasized. Special attention is paid to the effects of various nanoparticles on the production of valuable secondary metabolites in these plants cultivated in hydroponic systems, soil, hairy root, or in vitro cultures. The beneﬁcial impact of nanoparticles on the alleviation of abiotic stresses in medicinal and aromatic plants is also discussed.


Introduction
The 20th century was characterized by a rapid increase in the use of synthetic (often toxic) substances in various sectors of the economy, including agriculture, food, and in the pharmaceutical industry. Moreover, huge amounts of harmful solvents were often used to prepare required drugs. Due to the growing environmental contamination, which is also reflected in adverse effects on human health, "green" solutions are indispensable today. However, unlike the "Green Revolution" of the mid-20th century, which significantly increased the amounts of synthetic pesticides and fertilizers to achieve high yields [1], increased attention is now being paid to the widest possible use of biologically/pharmacologically active compounds of natural origins [2][3][4]. Medicinal plants, and in particular their essential oils (EOs), have been used in folk medicine since prehistoric times. For example, the use of medicinal plants is mentioned in the Bible. Moreover, recipes with medicinal plants can decomposition of metastable organometallic compounds are often used to prepare metalbased NPs. In the initial state of nucleation, the metal salt is reduced to form metal atoms that collide with other ions, metal atoms, or clusters, and irreversibly form "nuclei" of a stable metal core. The diameter of the cores should be below 1 nm depending on the strength of the metal bonds, the magnitude of the redox potential of the metal salt, and on the reducing agent used. Nowadays, when green synthesis of NPs is preferred, it refers to various plant or microbial extracts containing numerous biologically active compounds for the reduction of metal ions, acting as capping and stabilizing agents (increasingly being used for the synthesis of metal-based NPs). These bioactive compounds that are bound to the surface of NPs significantly improve the interaction of NPs with living cells and improve their biological efficiency [43][44][45][46].
Controlled release of active substances, the need for lower doses to achieve the same effect, and lower toxicity to non-target organisms are the main advantages of nanomaterials compared to their bulk counterparts, which contributes to reducing environmental pollution and costs [3,37,[47][48][49][50][51].
The increasing use of NPs in industry and agriculture is associated with their gradual accumulation in environmental matrices, where they could migrate, persist, or be transformed, accumulate in algae and plants, and enter the food chain. As a result, some toxic NPs can adversely affect non-target organisms and pose a serious threat to human health. To eliminate these risks, environmental monitoring is required for NPs, providing research on their fate in environmental matrices and contents in edible parts of plants. This is especially important in terms of medicinal plants that are consumed for their medicinal properties [36,49,52,53,[72][73][74][75].
This review article summarizes the current findings on the effects of metal-, carbon-, and silicon-based NPs as well as NPs derived from organic materials on medicinal and aromatic plants. Increased attention is paid to the production of important secondary metabolites exhibiting medicinal properties under in vitro conditions suitable for use in the mass production of these biologically active compounds, to be an alternative to synthetically produced ones. The beneficial effects of NP on medicinal plants exposed to some abiotic stresses are also discussed.

Effects of Metal-, Metalloid-, and Carbon-Based NPs on Vascular Plants
Metal-, carbon-, silicon-, and selenium-based compounds, applied in bulk or in nanoform, affect growth and development of vascular plants. In general, metal ions and metal NPs, including some essential metals (e.g., Cu, Zn, Fe. Ni, etc.) applied at higher concentrations, are phytotoxic, generate oxidative stress, adversely affect germination, photosynthetic processes, inhibit activities of enzymes and biosynthesis of photosynthetic pigments and non-enzymatic antioxidants, and accumulate high metal amounts in their plant organs, which is reflected in impaired plant growth and development [55][56][57][76][77][78][79][80][81][82][83]. On the other hand, some medicinal plants (e.g., Hypericum perforatum L. or Matricaria recutita L.) were found to be tolerant metal excess and accumulate in their shoots/leaves high concentrations of toxic metal, such as cadmium [83,84]. Therefore, to ensure food safety, cultivation of medicinal plants for commercial purposes under natural conditions must be performed on soils that are not contaminated with toxic metals [85].
Moreover, bulk and nanoscale metals and metal oxides can also be cytotoxic and genotoxic, cause cell-cycle arrest and apoptotic induction, as well as serious damage to DNA [57,[86][87][88][89][90]. On the other hand, their low doses may stimulate plant growth and can be used as effective fertilizers for achieving higher yields [55,56,[91][92][93][94][95][96]. Therefore, for individual plant species, it is necessary to known which concentrations have plant growth simulating effects and those with plant growth inhibiting impact.
Carbon-based nanomaterials, such as multi-walled carbon nanotubes (MWCNTs), can penetrate seed coats and, when used in low concentrations, improve water absorption, water transport, seed germination, nitrogenase, photosystem and antioxidant activities, activate water channel proteins, and stimulate nutritional absorption [97,98]. The entry of carbon-based nanomaterials into roots in an apoplastic way is thought to cause elongation of cell membrane pores [99] and the increased ability of plants to absorb water and essential nutrients when exposed to carbon nanotubes leads to stimulation of plant growth [98,100,101]. They can also affect the plant phenotype and composition of soil microbiota [102]. On the other hand, at higher concentrations, CNTs may have an adverse effect on plants due to the reactive oxygen species (ROS) generation, leading to cell death [103].
Silicon is a metalloid, which is beneficial for plant growth and development, particularly under abiotic stress conditions (e.g., high salinity, water deficit, high temperature, presence of toxic metals) because it can alleviate damages in stressed plants; it can activate some defense mechanisms and regulate some physiological processes related to defense mechanisms in plants [50,54,104,105]. Savvas and Ntatsi [106] stated that mitigation of abiotic stresses in vascular plants by Si is associated with SiO 2 deposition inside the plant tissues. Se acts as antioxidant and protects plants from oxidative damages and, depending on applied concentration; it can have both beneficial and adverse impact on plants, and modulate negative effects of abiotic stresses on plants, e.g., [107,108].
NPs affecting medicinal and aromatic plants discussed in this review are presented in

Improved Production of Secondary Metabolites in Presence of NPs
As mentioned above, NPs exhibit similar effects to their bulk counterparts; however due to their specific properties connected with their nanoscale sizes, their ultimate effects on plants can be amplified or attenuated [80,[109][110][111][112][113][114]. By green production of valuable secondary metabolites of medicinal plants elicited by various NPs, using in vitro cultures (Table 1) or HRs (Table 2), higher amounts of these pharmacologically important compounds can be achieved exceeding those observed with corresponding bulk elicitors. These secondary metabolites can be subsequently utilized by the pharmaceutical industry [115][116][117][118][119][120].  [126] [166] Elicitors are compounds that are capable of causing stress to the plant, leading to increased synthesis and accumulation of secondary metabolites, or to the induction of new secondary metabolites [116,167]. In general, secondary metabolites of plants play a role in their communication and adaptation. Plant secondary metabolism utilizes building blocks and biosynthetic enzymes derived from primary metabolic processes, and it produces large number of specialized compounds that are essential for plant survival in its environment [168][169][170]. Many of the secondary metabolites are also biologically active compounds, which are therefore desirable to produce in large quantities directly from medicinal plants [115,117,119,171]. It has been found that many NPs can be used as effective elicitors, the use of which makes it possible to avoid various other, mostly harmful/ecotoxic chemicals [115,117,118,172]. Stress often induces the production of secondary metabolites in the plant tissue culture system [116]. The accumulation of valuable secondary metabolites in micropropagated medicinal plants induced by abiotic/biotic stress using NPs is studied intensively [113,115,117,172,173]. Hairy roots are also important for the large-scale production of industrially important secondary metabolites. These are differentiated cultures of the roots of higher plants that have been injured and subsequently infected with Agrobacterium rhizogenes. Such transformed roots are capable to grow rapidly on the basal medium, show genetic and biosynthetic stability, and can produce increased amounts of secondary metabolites [119,120,171,[173][174][175][176][177].

Silver-Based NPs
A comprehensive overview focused on phytotoxicity assessment of AgNPs and their interaction with plants, including whole plant and organs, as well as interactions on the cellular and molecular levels, was presented by Tkalec et al. [178]. Coating of AgNPs can affect their toxicity.

Impact on Plant Growth
Beneficial effect of AgNP seed germination characteristics of Thymus kotschyanus was reported by Khalaki et al. [179]. Moreover, treatment with 40 mg/kg AgNPs during the germination process effectively improved germination and growth of Ocimum basilicum L. plants and their resistance to salinity stress [180]. By application of 0.2 mg AgNPs (8-21 nm) per fenugreek seedling, the root and shoot length of seedlings was approximately doubled and wet weight of treated plants was even 3-fold higher compared to the control [115]. Increasing concentrations of AgNPs (27.5 ± 4.8 nm) up to 10 mg/L showed beneficial impact on growth and development of Lavandula angustifolia propagated in vitro, which was reflected in improved formation of shoots, increased plant weight, and longer roots compared to control. Increasing AgNPs concentrations reduced the number of secretory trichomes; the greatest diameter of trichomes on the adaxial surface of the leaf blade was observed at exposure to 2 mg/L AgNPs and the smallest one at 5 mg/L AgNPs. The largest trichome diameter on the abaxial surface was formed at treatment with 1 mg/L and the lowest one using 5 mg/L AgNPs [181].
Spraying of borago (Borago officinalis L.) plants at the seed growth stage with AgNPs using doses 20, 40, and 60 ppm resulted in improved seed yield compared to the control; this beneficial impact increased with increasing AgNPs concentration, although content of polyphenols in treated plants showed a decrease [182]. Spraying of shoots of borago plants at the onset of the flowering stage (65 days after cultivation) and maintained until flowering (98 days after cultivation) with solutions containing 0.2, 0.4, and 0.6 mM AgNPs resulted in considerable increase of leaf numbers, plant, and inflorescence dry weights, and petal abscission [183].
For vanilla (Vanilla planifolia) micropropagated in a semi-solid Murashige and Skoog (MS) medium treatment, 25 mg/L and 50 mg/L AgNPs with a mean diameter of 35 ± 15 nm was found to be the most favorable for shoot multiplication. The greatest lengths, number of shoots, and number of leaves were obtained with 25 mg/L and 50 mg/L AgNPs, and such treatment effectively reduced the contamination during micropropagation of V. planifolia [184].
Ag-SiO 2 core-shell NPs (101.8 ± 8.9 nm) applied at 900 mg/L for 3 d to the HR cultures of Artemisia annua promoted their dry weight, which was 71% higher than that of the control [164]. AgNPs and AgNO 3 did not affect the in vitro multiplication of Campomanesia rufa at doses 0.385 and 0.77 mg/L, respectively, while treatments with 15.4 mg/L showed toxic effects reflected in the reduced number of shoots [185].

Elicitor Effect
AgNPs also acted as elicitor in HR cultures of Brassica rapa, causing a pronounced increase in the contents of glucosinolates and their transcripts, as well as in total phenolic and flavonoid concentrations and transcripts, compared to non-elicited HRs [186]. After treatment of in vitro grown shoots of Isatis constricta with 2 mg/L AgNPs, lasting 5 days, the indigo and tryptanthrin content were higher by 15 and 71% than in the control; however, at prolonged treatment (10 or 15 days), their content showed a decrease. On the other hand, treatments with AgNPs were found to decrease indirubin production [123].
Foliar treatment of 40 mg/L AgNPs, besides stimulating growth of fenugreek (Trigonella foenum-graecum), plants increased the total content of photosynthetic pigments by 25%, while content of indole acetic acid was even doubled compared to the control. Moreover, seeds yielded from treated plants showed pronouncedly higher content of phenolics, flavonoids, and tannins, as well as a higher percentage of proteins and carbohydrates than those of control plants [187]. Diosgenin content in fenugreek seedlings treated with 0.2 mg AgNPs (8-21 nm) per seedling considerably increased compared to the control (214.06 ± 17.07 vs. 164.49 ± 7.67 µg/mL) [115]. Pronounced enhancement of secondary metabolites, including phenols, tannins, and alkaloids, was found in borago plants sprayed with solutions containing 0.2, 0.4, and 0.6 mM AgNPs [183].
Salvia officinalis plants foliarly treated with AgNPs contained enhanced levels of phenolic compounds; the content of rosmarinic acid (RA) and salvianolic acid A and B was positively correlated with the activity of phenylalanine ammonia-lyase (PAL) and RA synthase, but not with tyrosine aminotransferase. After treatment of plants with 100 mg/L AgNPs, even 8-fold higher RA content was observed than in control plants [128]. Rosmarinus officinalis L. plants sprayed with solution containing 25, 50, 100 ppm AgNPs showed beneficial impact on the contents of secondary metabolites. Application of 200 ppm AgNPs for 12 days resulted in enhanced carnosic acid (CA) content by <11% compared to the control. In AgNP-treated plants, positive correlation was observed between TFC and CA content. Translocation of AgNPs to rosemary roots was confirmed as well [188]. The composition of EO extracted from Lavandula angustifolia propagated on MS medium containing AgNPs (27.5 ± 4.8 nm; 10 and 50 mg/L) was considerably altered, showing a decrease in compounds of lower molecular weight, which were replaced by those with higher molecular weight [130].
AgNPs more effectively elicited production of cichoric acid in cell suspension cultures of Echinacea purpurea than AgNO 3 , which reached even 9.54 mg/g dry weight (d.w.) in cell suspension culture after 48 h of treatment with 2 mg/L AgNPs and 8 mg/g d.w. in leaf suspension culture after 72 h of treatment [122]. Moreover, enhanced production of secondary metabolites, TPC, TFC, and protein content, and improved antioxidant and superoxide dismutase (SOD) activities were observed in Artemisia absinthium grown in vitro on MS medium treated with AgNPs [131]. Due to oxidative stress generated by AgNPs, artemisinin content in HRs increased and its production in 20-day-old HR cultures achieved up to 13.3 mg/L, being 3.9-fold higher than that of the control [164]. By application of 45 mg/L AgNPs to callus culture of Stevia rebaudiana, the amount of stevioside was enhanced, up to 32.34 mg/g d.w. of callus, suggesting that by using appropriate concentrations of AgNPs as an elicitor enhanced production of this secondary metabolite can be obtained [126]. AgNPs effectively elicited production of both lignans and neolignans in cell suspension culture of Linum usitatissimum [125].
Cell suspension cultures (CSC) of Momordica charantia elicited with AgNPs showed pronouncedly higher levels of Ag, malondialdehyde (MDA), and H 2 O 2 compared to nonelicited ones, and in their extracts, considerably enhanced total phenolic content (TPC) and total flavonoid content (TFC) compared to the control were observed. In CSC elicited by 5 mg/L AgNPs. the amounts of flavonols, hydroxybenzoic, and hydroxycinnamic acids greatly exceeded the amounts observed in the control [121].

Effect on Antioxidant Enzymes
Significantly enhanced callus induction of Citrus reticulata L., as well as high TPC, TFC, and antioxidant activity were observed using co-treatment with 20 ppm AgNPs and 1 mg/L thidiazuron; considerable increase of SOD, peroxidase (POD), and catalase (CAT) levels were recorded on MS medium supplemented with 30 ppm AgNPs and thidiazuron [189]. Hormetic effects of Argovit on in vitro regeneration of vanilla using a temporary immersion bioreactor system were reported by Spinoso-Castillo et al. [127]. Whereas doses 25 and 50 mg/L of Argovit increased Vanilla planifolia growth, application of 100 and 200 mg/L showed strong inhibitory effect; a dose-dependent effect on generation of ROS formation, and increasing TPC, antioxidant capacity, and lipid peroxidation was observed. AgNPs in combination with plant growth regulators added to MS medium pronouncedly enhanced callus biomass of Caralluma tuberculata. Stimulated production of phenolics, flavonoids, and increased PAL, SOD, POD, CAT, ascorbate peroxidase (APX) activities, as well as antioxidant activity were observed in the callus cultures treated with 90 µg/L AgNPs [124]. AgNPs green synthesized from leaf extracts of Swertia chirata applied on regenerating shoot cultures of S. chirata were found to generate ROS. However, due to induced antioxidant defense by antioxidant enzymes, ROS content was balanced and shoot regeneration increased, suggesting that AgNPs applied to plant tissue cultures can act as growth stimulant for improved shoot regeneration [190].
Calendula officinalis L. plants exposed to 50 ppm AgNPs and magnetic fields (3 mT), either alone or in combination, pronouncedly increased phenolic content, activities of several enzymes, such as PAL, polyphenol oxidase (PPO), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and radical scavenging in plants, suggesting positive effect of AgNPs on the defense mechanism of C. officinalis plants, combined treatment being the most effective [191]. Treatment of Calendula officinalis with AgNPs also reduced its DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity and increased formation of ROS and membrane peroxidation [192]. In the extracts of Calendula officinalis plants cultivated in hydroponic solution containing 0.4 mM AgNPs (30-50 nm), the extracts contained lower contents of chlorophyll (Chl), carotenoids, and anthocyanins than the control. Only content of flavonoids showed an increase by 14.6%; however, at higher AgNP concentrations, their content decreased as well, and saponin content increased by ca. 77% [192].
It was reported that AgNPs pronouncedly suppressed oxidative stress triggered by naphthalene toxicity in Moringa oleifera called the "Miracle Tree" [193]. AgNPs applied foliarly to the Salvia officinalis plants, penetrated via the leaf epidermis into the parenchyma cells, and after application of 50 and 1000 mg/L AgNPs, reduced levels of assimilation pigments and increased activities of antioxidants (enzymatic and non-enzymatic) were able to cope with the oxidation stress, compared to the control plants [128]. The cetyltrimethylammonium bromide (CTAB) coated AgNPs were found to be toxic to A. cepa roots, causing robust inhibition of roots and pronounced oxidative damage, whereas much bigger negatively charged citrate coated AgNPs aggregating to larger particles were least toxic. However, it could be mentioned that the toxicity of Ag + ions was higher than that of both coated AgNPs [194].

Attenuation of Abiotic Stress
AgNPs enhanced the tolerance to drought stress in germination and early growth stages of Thymus daenensis Celak and Thymus vulgaris L.; they improved germination characteristics and root length up to 200 mM salinity, although they exhibited positive impact on vigor and shoot length only up to 100 mM salinity as compared to the control [195].

Cytotoxic and Genotoxic Effects of AgNPs
AgNPs with particle sizes >100 nm were found to penetrate the root tip cells of Allium cepa and adversely affected stages of cell division resulting in formation chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks, and cell disintegration, whereby at application of 100 ppm AgNPs mitotic index (MI) decreased from 60.30% (control) to 27.62% [196]. Mitigation of genotoxic and cytotoxic effects of AgNPs in onion root tips reflected by a decrease of MI, elevated chromosome aberrations, and tail DNA caused by oxidative stress can be attenuated by using antioxidants (e.g., vitamin E), pretreatment being more effective than combined treatment with AgNPs [197]. Cytotoxic and genotoxic impact of Ag nanorods on A. cepa roots depended on their concentration, a dose of 15 µM being more toxic than doses of 5 and 10 µM, respectively [198]. Although polyvinylpyrrolidone (PVP)-coated as well as uncoated AgNPs were found to be genotoxic, cytotoxic, and induce oxidative stress in A. cepa roots, even at low concentrations; under exposure to visible-light, their aggregation state will be altered, resulting in reduced toxicity [199]. Due to application of 25 and 50 mg/L AgNPs to V. planifolia, only a small MI decrease was observed and genotoxic effects were minimal, with < 5% of the total chromatic aberrations and 3 micronuclei in 3000 cells, despite the long-time exposure to AgNPs [200].
In general, considering the beneficial impact of AgNPs on enhanced production of secondary metabolites, they could be recommended to be used, particularly for production of stevioside in callus culture of Stevia rebaudiana L. [126] and for enhanced production of rosmarinic acid by foliar spraying of Salvia officinalis L. plants [128] (Table 1).

Impact on Plant Growth and Photosynthesis
Capsicum annuum plants grown in soil amended with 500 mg/kg CuNPs, which were evaluated at the vegetative stage, i.e., 45 days post transplantation, showed pronouncedly increased length and dry weight of roots, while plant tissues accumulated lower Cu amounts compared to treatment with bulk Cu particles. On the other hand, when treated plants were evaluated in the reproductive stage, i.e., 90 days post transplantation corresponding to a full life cycle, treatment with 500 mg/kg CuNPs resulted in increased photosynthesis, stomatal conductance, and Cu leaf content compared to that observed with application of bulk Cu particles. Treatment with 62.5 mg/kg CuNPs was found to increase transpiration, but total sugar, carotenoid, Chl, and vitamin C contents in the fruit, and the root and leaf enzyme activity, were not affected 90 days post transplantation in plants exposed to 62.5, 125, or 500 mg/kg CuNPs. The results of this experiment suggested that a dose of 500 mg /kg CuNPs, ensuring higher Cu bioaccumulation in the root, and leaf tissue over long-term exposure, is suitable to be used as a nanofertilizer [201]. CuO NPs phytosynthesized using Punica granatum peel extract foliarly sprayed on Capsicum annuum plants increased the height of plants, as well as growth and the Chl content of leaves; the fruit practically did not contain residues of sprayed NPs [202]. Application of 125 mg/kg CuO NPs to Capsicum annuum plants significantly reduced leaf P content by 41%, while exposure to 500 mg/kg CuO NPs decreased Zn content in leaves and fruits by 55% and 47%, respectively, compared to the control, thereby affecting the nutritional quality of bell pepper [203].
Foliar spraying of Mentha piperita L plants performed three times of the interval of 15 days up to flowering stages with 1.0 g/L CuNPs was found to enhance Chl content and EO percentage of 35% and 20%, respectively, as compared to control [133]. Investigation of the clonal microreproduction of Mentha longifolia plant showed that addition of colloidal solutions of CuNPs (0.5 mg/L) and CoNPs (0.8 mg/L) to MS nutrient medium resulted in an increase of microplant height and growth index on 45-48.4%, the quantity of internodes on 29.4-33.9%, quantity of shoots on 55.6-66.2%, and reproduction coefficient on 30-40% [204]. Application of 5 µM CuNPs (20-40 nm) for plant regeneration of Ocimum basilicum via somatic embryogenesis resulted in 5.6-fold higher percentage of explants produced somatic embryos (84 vs. 15%) and 4.35-fold higher average number of regenerated plantlets/explant (18.7 vs. 4.3) as compared to treatment with 0.1 µM CuSO 4 5 H 2 O [205]. Exposure of Origanum vulgare plants for 60 days to CuNPs at doses 0-200 mg Cu/kg increased root and shoot growth, and water content, but reduced shoot biomass, as well as starch, total sugar, and reducing sugar in leaves; whereas shoot length, MDA, and Chl levels were not affected. However, CuNPs affected root and shoot contents of Ca, Fe, Mg, and Mn, suggesting that nutrition value of oregano could be modified [206]. Phytotoxic effect of 200, 400, and 800 mg/L CuNPs on hydroponically cultivated coriander (Coriandrum sativum) plants was reflected in reduced biomass and root length, lower levels of Chls (Chla and Chlb) compared to control plants, and increased levels of H 2 O 2 compared to untreated plants [207].

Elicitor Effect
Foliar application of 0.5 g/L CuNPs effectively enhanced the levels of camphene, α-phellandrene and limonene in Mentha piperita L. plants, while a dose of 1.0 g/L increased the concentrations of menthol, menthone, and menthofuran [133]. Allium fistulosum plants grown 80 days at greenhouse conditions in soil spiked with 150 mg/kg CuO NPs showed higher contents of Cu, Ca, and Fe in roots, and higher Ca and Mg content in bulb than plants exposed to bulk CuO or CuSO 4 . Moreover, plants of Chinese scallion plants cultivated in soil supplemented with 75-600 mg/kg CuO NPs showed higher levels of allicin in leaves (56-187%) than the control, suggesting that CuO NPs have potential to be used as nanofertilizer for onion production [208].
Crystalline monoclinic cubic CuO NPs with mean size 47 nm applied to MS media for direct organogenesis of S. rebaudiana from nodal segments at a dose of 10 mg/L pronouncedly increased shoot organogenesis (88.5%), shoot length, mean number of shoots per explant, and fresh weight, as well as the content of glycosides, rebaudioside A, and stevioside [209]. The NPs of bimetallic alloys of Cu and Au (Cu:Au = 3:1) applied at a dose of 30 µg/L together with 0.5 mg/L naphthalene acetic acid were found to stimulate biomass accumulation on the 27th day of log phases, increased the radical scavenging activity, and enhanced TPC and TFC in submerge adventitious root cultures of S. rebaudiana (Bert.) [210]. Uncapped and polyethylene glycol (PEG) or PVP capped CuO NPs applied to the growth medium of in vitro cultured S. rebaudiana plants were able to increase the production of commercially important secondary metabolites. Regarding shoot organogenesis, increased growth parameters and higher content of steviol glycosides and non-enzymatic antioxidants (TPC, TFC) in shoots were observed at treatment with polymer-capped CuO NPs compared to uncapped ones [211].
Using cell suspension cultures of Gymnema sylvestre (Retz.) R. Br and CuO NPs as elicitor (3 mg/L for 48 h), the yields of gymnemic acid II, TPC, and TFC were considerably enhanced, and the cultures exhibited significant antioxidant, antidiabetic, anti-inflammatory, and anticancer activities [136]. Bacopa monnieri (L.) Pennell herb treated in vivo with Cubased NPs (CuNP/CuO NPs) applied at doses 0-100 mg/L showed biphasic dependence of total contents of saponins, alkaloids, phenolics, and flavonoids on the concentration of used NPs. While at lower Cu/CuO NPs concentrations, the content of secondary metabolites increased; at higher concentrations, their strong reduction was observed. Besides enhancement of secondary metabolism, the CuNPs applied at sub-toxic doses also improved the antioxidant capacity in Bacopa monnieri herb via ROS-mediated defense response [212].

Genotoxicity
CdS and CuO NPs induced growth inhibition and caused cytological aberrations in both mitotic and meiotic cells in Coriandrum sativum L, the effect being like that of conventional mutagen, ethyl methanesulfonate, which was used as control [213]. Damaged root plasma membrane and altered genome of hydroponically cultivated coriander plants exposed to higher CuNP concentrations suggested that genotoxic impact of CuNPs was observed as well [207].
Considering the beneficial impact CuO NPs on the production of gymnemic acid II in Gymnema sylvestre (Retz.) R. Br cell suspension, which is 9.0-fold higher than that of the control [136] (Table 1), this method can be recommended for recovery of this antisweet compound.

Impact on Plant Growth and Assimilation Pigments
Chitosan-coated Fe 3 O 4 NPs with sizes 3-22 nm applied at doses 200 and 400 mg/kg showed beneficial impacts on seed germination and growth of Capsicum annuum L. seedlings, whereby their efficiency was better than that of bare Fe 3 O 4 NPs [214]. Priming of roots of 28-day-old chilli and marigold plants for 3 h in the suspension of FeS 2 NPs (100 µg/mL) prior to transplantation had beneficial impact on chilli yields and mean flower number of marigold, which was approximately doubled as compared to control (27 vs. 13) [215]. Introduction of 0.3 mg/L FeNPs (27.0 ± 0.51 nm) instead of FeSO 4 into a MS nutrient medium increased root length, root activity (evaluated as the reduction of triphenyltetrazolium), and leaf Chl in Capsicum annuum plants by 118,58 and 5% related to control. A 10-fold higher FeNPs concentration increased Chl content even by 27% and beneficial impact on the above-mentioned characteristics were observed also at a combination of FeNPs with CuNPs or ZnNPs. It could be mentioned that the applied FeNPs concentration of 0.3 mg/L was 18.7-fold lower than that contained in standard MS medium [216].  [91]. Zingiber officinale Rosc. plants cultivated hydroponically in Hoagland solution containing 100 ppm Fe 2 O 3 NPs exhibited higher protein levels (1.699 µg/mL) compared to plants treated with Fe-EDTA (1.108 µg/mL) and plants cultivated without Fe 3+ addition to nutrient solution (0.208 µg/mL). Similarly, in plants exposed to Fe 2 O 3 NPs, the total Chl and carotenoid content was 1.18-and 1.28-fold higher than in plants treated with Fe-EDTA, and 2.23-and 2.25-fold higher than in control plants. Fe content in rhizome of Fe 2 O 3 NPs-treated plants increased as well, suggesting that these NPs can be used to mitigate chlorosis [217].
Comparison of yield, growth, and Fe uptake of Cuminum cyminum L. plants grown on the calcareous soils and sprayed twice during the season, before and after flowering (with a 10 day interval), using three various Fe-containing fertilizers, showed that the highest weight of 1000 grains and plant growth was observed at application of Fe-nano-chelate using a dose 1 g/L, while treatment with Fe-siderophore was found to be the most suitable for seed fortification with Fe [218].

Elicitor Effect
Treatment of cell suspension cultures of Dracocephalum polychaetum Bornm with 100 ppm Fe 2 O 3 NPs, static magnetic field (SMF) of 30 mT, as well as combined treatment with Fe 2 O 3 NPs and SMF pronouncedly increased intracellular content of secondary metabolites, such as RA, naringin, apigenin, thymol, carvacrol, quercetin, and rutin; secretion of these secondary metabolites from cells exposed to the double stress (SMF and NPs) to the culture media was observed as well. Moreover, the applied treatments resulted in increased TPC, TFC, anthocyanins, lignin, and MDA contents and increased activity of PPO and PAL [143]. Exposures of HRs derived from 4-week-old leaves of Dracocephalum kotschyi Boiss and inoculated with Agrobacterium rhizogenes strain ATCC15834 to 75 mg/L Fe 2 O 3 NPs for 24 h resulted in improved biomass accumulation and activities of antioxidant enzymes, enhanced TPC and TFC, and contents of some valuable secondary metabolites, such as RA and xanthomicrol, which reached 9.7-and 11.87-fold higher values than in the control [162].
Moreover, inoculation with Agrobacterium rhizogenes of HRs derived from cotyledon explants of Hyoscyamus reticulatus L, which were elicited with 900 and 450 mg/L FeNPs using exposure time of 24 and 48 h, respectively, achieved approximately fivefold higher content of tropane alkaloids, hyoscyamine, and scopolamine, and considerably higher activities of antioxidant enzymes compared to control [64]. Fe 2 O 3 NPs acted as highly effective elicitor, increasing the growth and production of secondary metabolites of Cichorium intybus L. in HR culture induced by Agrobacterium rhizogenes strain 15834 [219]. Treatment of S. rebaudiana cultivated in vitro with 45 µg/L FeNPs resulted in beneficial impact on plant growth characteristics and production of stevioside, and rebaudioside A reached 4.2 ± 0.058 and 4.9 ± 0.068 mg/g (d.w.). At exposure to 135 µg/L FeNPs, the levels of TPC and TFC reached 3.2 ± 0.042 and 1.6 ± 0.022 mg/g (d.w.), respectively, although adverse effect on growth characteristics was observed. Exposure to 90 µg/L FeNPs resulted in increased activities of SOD, POD, CAT, and APX [220].
In cell suspension culture of Hypericum perforatum L. elicited with 100 ppb Fe 2 O 3 NPs for 72 h, enhanced concentrations of hypericin (195.62 ± 10.00 vs. 16.272 ± 4.62 µg/g (d.w.)) and hyperforin (11.181 ± 0.17 vs. 2.072 ± 0.77 µg/g (d.w.)) compared to the control were observed, whereas the dry weight was comparable with control. It could be assumed that the Fe 2 O 3 NPs may induce jasmonate production, playing a role in hypericin and hyperforin production, or can be involved in signal transduction process regulating jasmonate production genes in cells [141]. Simultaneous foliar application of 0.01% MgO NPs and 0.03% Fe 2 O 3 NPs on Hibiscus sabdariffa plants exhibited synergistic and beneficial impact on the activity of antioxidant enzymes and anthocyanin and flavonoids levels, and reduced H 2 O 2 concentration in plants. On the other hand, at combined application of 0.03% MgO NPs and 0.03% Fe 2 O 3 , ROS production increased, and the antioxidant defense system was not able to attenuate toxic effects of produced H 2 O 2 and, therefore, the observed characteristics were comparable to those of control plants [221].
Foliar treatment of Ocimum basilicum L plants grown in soil with Fe nanocomplexes Fe(His) 3 and Fe(Arg) 3 resulted in an increase of sesquiterpenes and a decrease of the content of oxygenated monoterpenes in the composition of secondary metabolites of EO, and enhanced antioxidant and antimicrobial activities of plants [222]. FeNPs applied foliarly to Mentha piperita L. at a dose of 1.5 g/L, three times of the interval of 15 days up to flowering stages, considerably enhanced contents of Chl and EO. Application of 0.5 g/L NPs increased the EO content and oil production by 60 and 50%, and menthone and menthol contents by 65% and 30%, respectively, compared to the control. On the other hand, at a threefold higher dose of FeNPs, menthofuran concentration was 2.5-fold higher than in the control [133].

Attenuation of Abiotic Stress
Spraying of salt-stressed (100 mM of NaC1) Dracocephalum moldavica L. plants with Fe 2 O 3 NPs considerably increased the leaf area, content of secondary metabolites (TPC, TFC, anthocyanins), as well as activities of antioxidant enzymes (GPX, APX, CAT, GR), suggesting improved antioxidant defense. Moreover, under salinity stress, the Fe 2 O 3 NPs can serve as a source of Fe for this medicinal plant [224]. Application of Fe 3 O 4 NPs was also able to greatly attenuate Cd and Pb induced toxicity in coriander plants grown in soil artificially contaminated with these toxic metals [225]. Treatment of Mentha piperita plants under salt stress with 10-30 µM Fe 2 O 3 NPs improved the fresh and dry biomass of leaves, contents of essential elements such as P, K, Fe, Zn, and Ca, although did not affect Na content. Moreover, Fe 2 O 3 NPs pronouncedly reduced lipid peroxidation and proline contents and decreased activities of CAT, SOD and GPX in salt-stressed plants [226]. In Melissa officinalis L. plants exposed to drought stress (80%, 60%, and 40% field capacity (FC)) considerable increase in MDA, H 2 O 2 , as well as proline content and electrolyte leakage was observed with decreasing FC, while TPC, activities of antioxidant enzymes, Chl content and relative water content were reduced. Although increasing of water deficit from 80% to 60% FC resulted in improved yield of EO, further reduction to 40% already showed an opposite effect. However, application of FeNPs (5-20 µM) had beneficial impact on drought-stressed plants, which was reflected in further increase of EO yield, increased proline levels and mitigation of oxidative stress by activation of antioxidant defense system of plants and, thus, FeNPs protected the plants against oxidative stress damages [142].

Genotoxicity
Ghosh et al. [227] investigated the impact of FeNPs with different surface chemistries on the uptake, root morphology, DNA damage, oxidative stress, and cell death in Allium cepa roots after 24 h exposure. It was shown that FeNPs with low zeta potential, conductivity, and high polydispersity index, which were adsorbed on root surfaces, damaged the root tip, epidermal, and root hairs due to their colloidal destabilization and smaller size. High uptake of NPs was responsible for considerable DNA damage, chromosome/nuclear aberrations, and micronuclei formation. On the other hand, FeNPs, which were characterized with higher dissolution and substantial uptake, induced genotoxicity. Both types of FeNPs generated strong oxidation stress and in treated A. cepa roots ROS-mediated apoptotic and necrotic cell death was observed.
Elicitation of the production of hyoscyamine and scopolamine in HR of Hyoscyamus reticulatus L. by Fe 2 O 3 NPs allows achieving ca. 5-fold higher amounts than in the con-trol. This is a promising method that can be considered to produce tropane alkaloids [65] ( Table 2). Moreover, 5.4-and 12-fold higher amounts of hypericin and hyperforin, compared to the control observed in cell suspension culture of Hypericum perforatum L., using 100 mg/L Fe 2 O 3 NPs as an elicitor, suggest this method is very encouraging [141] (Table 1).

Impact on Germination and Plant Growth
ZnO nanorods with a mean aspect ratio of 7, coated in gel and mixed with the seeds of Solanum melongena L. and Capsicum annum L., applied at doses 50, 100, and 150 mg/L, accelerated germination, which was reflected in reduced germination time and improved mean germination rate compared to control plants; application of 100 mg/L ZnO NPs ensured maximal transplant length as well as fresh and dry weight [228]. Amelioration of the seed germination rate and seedling vigor of Capsicum annuum L. was observed at treatment with ZnO NPs suspensions at 100, 200, and 500 ppm, respectively. On the other hand, application of these ZnO NPs concentrations resulted in the inhibition of radical growth and stimulated accumulation of phenolic compounds [229]. Spraying of Capsicum chinense Jacq. plants during the main stages of phenological development with 1000 ZnO NPs stimulated plant height, stem diameter, and Chl content, and increased fruit yield and biomass accumulation in treated plants compared to control and treatment with ZnSO 4 , whereas the double dose of ZnO NPs adversely affected plant growth [230].
Germination of seeds of Abelmoschus esculentus (L.) Moench was positively affected by treatment with ZnNPs (50-500 ppm), and in 30-day-old treated plants, an increase in Chl, protein, proline, and carbohydrate contents were observed, along with pronounced reduction of the carbohydrate content of leaves [231]. Spraying of leaves of 10-day old chickpea (Cicer arietinum L. var. HC-1) seedlings, with solution containing 1.5 ppm ZnO NPs, stimulated shoot dry weight, but when a dose of 10 ppm was applied, root inhibition was observed. Positive impact of ZnO NPs on plant biomass related to reduced ROS generation caused less lipid peroxidation, which also resulted in lower activity of antioxidant enzymes, such as SOD and POD, compared to the control [232]. Phytotoxic effects of the ZnO NPs and Zn microparticles (MPs) (10-2000 mg/L) on Fagopyrum esculentum was reflected in biomass reduction, increased Zn bioaccumulation in plants, and higher translocation factor of Zn being observed for treatment with ZnO NPs. Moreover, reduced glutathione level and CAT estimated at application of ZnO NPs indicated oxidative stress due to ROS generation [233]. In 6-week-old callus produced from leaf explants of S. rebaudiana Bertoni grown in MS medium supplemented with plant growth regulators addition of 0.1 mg/L ZnO NPs significantly enhanced fresh and dry weight of callus [117].
ZnO NPs (40 nm) positively affected growth of the Echinacea leaf callus and showed beneficial impact on the anticancer activity of plant extracts, in contrast to the control treatment with ZnO MPs. Moreover, application of ZnO NPs increased flavonoid contents of Echinacea purpurea [234]. A dose of 2000 ppm ZnO NPs increased dry weight of Tanacetum parthenium (L.) Schultz Bip plants from 28.09 g/day (control) to 32.54 g/day, while using 1000 ppm ZnO NPs, the EO content achieved 0.9% v/w compared to 0.56% v/w observed in the control plants; an increase in Zn absorption at application of ZnO NPs was observed as well, while Fe absorption was reduced [235].
ZnO NPs at doses 0.5-2.5 mg/L applied to hormone-free MS medium inhibited root growth of ginseng root culture, deceased the lateral root number, and decreased percentage of lateral root formation was observed at exposure to 2.5 mg/L ZnO NPs. In treated plants thinner vascular tissue or cortex area and more black points in root cap area were observed compared to control. On the other hand, exposure to ZnO NPs resulted in thicker endodermis [236].
Foliar application of ZnO NPs at doses 20, 60, and 100 ppm to Momordica charantia L. plants grown in pots did not considerably affect shoot weight and anthocyanin content, but pronouncedly increased Chla content, as well as contents of secondary metabolites (phenols, flavonoids, and carotenoids), along with carbohydrate and proline content in a dose-dependent manner [132]. Growth characteristics in the callus of Punica granatum cv. Hegazy were adversely affected by 200 µg/mL ZnO NPs reflected in a stronger decrease in callus dry weight than at application of bulk ZnO (57.53% vs. 37.78%) and more elevated increment in Zn concentration over the control compared with that of ZnO bulk (335.24% vs. 247%) [237].

Elicitor Effect
Improved production of tropane alkaloids was observed using HRs obtained from two-week-old cotyledon explants of Hyoscyamus reticulatus treated with 100 mg/L of ZnO NPs after 48 and 72 h, when the levels of hyoscyamine and scopolamine reached 37.63% and 37%, respectively. The highest expression of h6h gene in HRs was observed at treatment with 100 mg/L of ZnO NPs after 24 h [67]. Zn-Ag NPs (molar ratio of 19:1) exhibiting photocatalytic and elicitor activity were able to improve the light absorption and increase oxidative stress in Withania somnifera plants, resulting in increased photosynthesis and transpiration rates along with an increase in some carbohydrates and enhanced content of withanolide, which was associated with upregulation of genes involved in photosynthesis, Calvin cycle, carbohydrate metabolism, and withanolide biosynthesis. Moreover, cyclic electron flux managing the electron flow around photosystem (PS) I was observed [63].
Spraying of Capsicum chinense Jacq. plants with 1000 ZnO NPs pronouncedly increased TPC, TFC, and content of secondary metabolites, such as dihydrocapsaicin in fruits, suggesting ameliorated nutraceutical quality of fruits [230]. Hypericum perforatum cell suspension cultures elicited with 100 ppm ZnO NPs stimulated production of hypericin and hyperforin, up to 7.87 and 217.45 µg/g d.w., providing 3 and 13-fold higher amounts compared to the control [141].
ZnO NPs capped with PEG and PVP enhanced TPC, TFC, content of steviol glycosides in in shoots of S. rebaudiana Bertoni cultured in vitro more than upcapped ones [211]. By application of 2 mg/L ZnO NPs, 91% induction of in vitro roots formation in regenerants of S. rebaudiana, and increased contents of secondary metabolites of rebaudioside A (2.96% vs. 4.42%) and stevioside (1.01% vs. 1.28%) in leaves, compared to the control, were observed. With application of increasing ZnO NPs, the content of secondary metabolites gradually decreased to 1.22% and 0.21% for rebaudioside A and stevioside, respectively, corresponding to the values observed under stress induced by 2000 mg/L ZnO NPs [135]. In contrast to the beneficial impact of 1 mg/L ZnO NPs on shoot formation of micropropagated S. rebaudiana and steviol glycosides levels, which were practically doubled compared to control, further increase of ZnO NPs was accompanied with a decrease in formation of secondary metabolites and antioxidant activities, and at application of 1000 mg/L ZnO NPs, a strong phytotoxic effect was observed [137]. Toxic impact of ZnNPs (<100 nm) applied at concentrations 400-1000 mg/L accompanied with pronounced reduction of stevioside was also observed in the nodal explants of Stevia cultured in vitro on MS medium [238]. Fertilization of Stevia rebaudiana with 75 mg/L of green synthesized ZnO NPs through roots enhanced the concentrations of TPC and TFC and Zn content up to 406.8% with respect to the control, although the biosynthetic pathway of steviol glycosides was not affected; treatment did not show adverse impact on plant growth [139].
Seedlings of flax (Linum usitatissimum) cultivated on MS in the presence of 10 mg/L ZnO NPs showed improved growth compared to control, while application of 500 mg/L ZnO NPs increased the production of secoisolariciresinol diglucoside, lariciresinol diglucoside, dehydrodiconiferyl alcohol glucoside and guaiacylglycerol-β-coniferyl alcohol ether glucoside. In another experiment, in which the stem explants were cultured on MS media supplemented with plant growth regulator and 25 mg/L ZnO NPs, enhanced rooting effect and effective accumulation of metabolites was observed [138].
In Thymus kotschyanus and T. daenensis micropropagated in the presence of 150 mg/L ZnO NPs thymol and carvacrol contents of 22.8 mg/L and 0.68 mg/L, respectively, were reported by [172]. Treatment of Glycyrrhiza glabra L. seedlings with 1 and 10 µM ZnO NPs increased the contents of glycyrrhizin, TPC, TFC, anthocyanins and proline, whereas by application of bulk ZnO at a dose of 10 µM content of tannins was enhanced [239].

Impact on Antioxidant Enzymes
Growth of fresh and dry weight of HRs treated with 100 mg/L ZnO NPs showed a decrease compared to the control, whereas activities of CAT, GPX, and APX were considerably higher than in the control [67]. Significant increase of CAT activity was observed at application 60 and 100 ppm of ZnO NPs to Momordica charantia L. plants grown in pots, but the activities of GPX and AXP were not affected [132].
In cell suspension cultures of Linum usitatissimum L. exposed to ZnO NPs, the highest PAL activity was observed at treatment with 30 mg/L for 48 h, whereas at application of 60 mg/L ZnO NPs, the highest activity of cinnamyl alcohol dehydrogenase was achieved. On the other hand, using both above-mentioned ZnO NP concentrations, the highest TPC and lignan amounts could be obtained [140]. In seedlings of flax (Linum usitatissimum) cultivated on MS medium and in stem-derived callus, the presence of ZnO NPs (1-1000 mg/L and 10-50 mg/L, respectively) caused the dose-dependent ROS generation. A differential induction of SOD (in seedlings) vs. POD (in vitro cultures) enzymes was observed. At concentrations > 500 mg/L (seedlings) and >25 mg/L ZnONPs (callus) oxidative damage was observed [138].
Spherical green synthesized ZnO NPs with mean size of 41 nm had positive impact on root and shoot length of Borago officinalis plants, but did not affect significantly soluble sugar and anthocyanin levels, and CAT activity in the plants. Due to enhanced ROS generation by ZnO NPs, the contents of proline and Chl decreased and oxidative stress in plants was accompanied with increased H 2 O 2 content, lipid peroxidation, phenol content, and APX, GPX, PAL activities [240].
ZnO NPs capped with PEG and PVP enhanced total antioxidant capacity, total reducing power and DPPH free radical scavenging activity in shoots of S. rebaudiana Bertoni cultured in vitro more than uncapped ZnO NPs, concentration 1 mg/L being highly effective [211]. Addition of 100 mg/L ZnO NPs resulted in effective increase in TPC, TFC, total antioxidant capacity and DPPH free radical scavenging activity in 6-week-old callus produced from leaf explants of S. rebaudiana Bertoni grown in MS medium; the highest total reducing power was observed at application 50 mg/L ZnO NPs [117].

Cytotoxic and Genotoxic Effects
Cytotoxic and genotoxic impact of ZnO NPs (5 and 50 µg/mL) causing oxidative stress in roots of Allium cepa was reflected in lack of membrane integrity, reduced metabolic activity, ROS generation, chromosome aberration, DNA damage, cell-cycle arrest, and cell death in onion roots; considerably higher Zn levels were estimated in the cytoplasmic and nuclear fractions of A. cepa roots. It was confirmed that, for the toxicity of ZnO NPs suspension beside Zn 2+ ions, predominantly ZnO NPs were responsible [241]. Similar effects were observed by Ghosh et al. [242] who treated A. cepa roots with ZnO NPs having a size of 85 nm, which deregulated the components of ROS antioxidant machinery causing DNA strand breaks and cell-cycle arrest at the G 2 /M checkpoint. Mitochondrial and chromosomal damage induced by oxidative stress in A. cepa roots treated with Zn 2+ ions, bulk ZnO, and ZnO NPs (25 nm) was also reported by Ahmed et al. [243]. ZnO NPs attachment and internalization, and a considerable increase in levels of thiobarbituric acid reactive substances and antioxidant enzymes in treated roots, were observed as well. Summarizing the adverse effects of ZnO NPs on onion roots, they can be considered as a clastogenic/genotoxic and cytotoxic agent [244]. Treatment of A. cepa roots with 100 mg/L ZnO NPs reduced MI compared to the control (53.2% vs. 63.6%), which further decreased to 50% under UV irradiation. At application of 0.1 mg/L with ZnO NPs lagged metaphase and anaphase with multiple chromatin bridges in the root tip cells were recorded, although at doses 10 mg/L and 100 mg/L, respectively, both stickiness and diagonal anaphase were predominant. It was observed that, following exposure to ZnO NPs, the generation of ROS and MDA was accelerated [245].
Elicitation of the production of hyoscyamine and scopolamine in HR of Hyoscyamus reticulatus L. by ZnO NPs allows achieving 4.61-and 3.20-fold higher amounts than in the control. This is a promising method that can be considered to produce tropane alkaloids [67] ( Table 2). Moreover, 3.80-and 13.36-fold higher amounts of hypericin and hyperforin compared to the control observed in cell suspension culture of Hypericum perforatum L. using 100 mg/L ZnO NPs as an elicitor suggest this method as very encouraging [141] (Table 1).

TiO 2 -Based NPs
TiO 2 NPs may have a positive effect on photosynthetic efficiency by increasing electron transport between the PSII and PSI, positively affecting Rubisco activity in the Calvin and Benson cycle [246].

Impact on Germination, Plant Growth and Assimilation Pigments
TiO 2 NPs (10-25 nm) applied at doses 20-40 mg/L were found to accelerate the germination characteristics of Alyssum homolocarpum, Carum copticum, and Nigella sativa seeds, and increase their vigor, while most effective stimulation of germination of Salvia mirzayanii seeds was obtained with 80 mg/L TiO 2 NPs [247]. TiO 2 NPs (43 nm) coated in gel, which were mixed with the seeds of pepper using doses 50, 100, and 150 mg/L, accelerated germination and enhanced lengths, fresh and dry weights of seedlings compared to control, concentration of 100 mg/L TiO 2 NPs being the most effective [228]. Nanopriming of seeds with TiO 2 NPs (anatase) (10-40 mg/mL) considerably enhanced germination as well as morphological characteristics and Chl content of Petroselinum crispum seedlings grown in vitro in MS medium, the dose of 30 mg/L being the most effective [248]. Exposure to 40 ppm TiO 2 NPs improved fennel seed germination time by 31.8% in comparison to the untreated control, while treatment with 40 ppm TiO 2 NPs improved dry weight compared to the untreated control [249]. On the other hand, Khater and Osman [250] reported that spraying of fennel plant with 6 ppm TiO 2 NPs enhanced Chl synthesis, which was reflected in improved photosynthesis.
Foliar treatment of Mentha piperita plants with TiO 2 NPs using a dose of 150 mg/L increased fresh and dry weights by 48% and 62.6%, and nitrate reductase and carbonic anhydrase activities 150 days after planting by 17.7% and 19.1%, respectively, compared to control plants; an increase of nitrogen content, stomatal conductance, and net photosynthetic rate by 12.6%, 8.5% and 23.8%, respectively, compared to the control plants, was observed as well. Diameter and density of peltate glandular trichomes, which are important from the aspect of EO production, were increase by 77.8% and 62.5%, respectively, at treatment with 100 mg/mL TiO 2 NPs [144]. Mentha piperita L. and Salvia officinalis L. plants exposed to TiO 2 NPs either via the leaves (using TiO 2 NPs suspension) or via the root system (in soil) resulted in reduced Chl contents in M. piperita and S. officinalis plants treated through root system, but not after application of NPs suspension on leaves of sage plants, likely due to the abundance of trichomes, providing a natural barrier for the NP accumulation to the symplast. In the soil-exposed plants translocation of uptaken TiO 2 NPs from roots to shoots resulted in reduced Chl levels. The reduction of Chl correlated mainly with the loss of Mg (sage) and Mn (peppermint) in treated plants [251].
Foliar application of 0.04% TiO 2 NPs improved the contents of total Chl, carotenoids, and soluble proteins, as well as grain yield of safflower plants to greater extent than application of 0.02% TiO 2 NPs and bulk TiO 2 [252].
In contrast to smaller TiO 2 NPs of 50 nm, larger TiO 2 NPs of size 68 nm had beneficial effect on germination of Abelmoschus esculentus seeds. However, the small-sized TiO 2 NPs applied at a dose of 200 mg/kg pronouncedly increased Chla and total Chl content compared to the control; a dose of 800 mg/kg considerably increased fresh weight of plants compared to the control. On the other hand, both types of NPs reduced APX in roots and leaves, whereas GR activity in roots decreased, but increased in the leaves. A considerable raise of SOD activity in roots and leaves of plants exposed to TiO 2 NPs signalized their phytotoxic impact. Moreover, treatment with TiO 2 NPs caused a size-dependent and dose-dependent decrease in essential minerals Ca, Mg, and Fe contents in fruits, as well as adversely altered the proximate compositions (except carbohydrate) of fruits [253].
Treatment with TiO 2 NPs at 50−200 mg/L (83.7 nm) moderately increased the root and shoot fresh biomass of Coriandrum sativum L. plants cultivated in hydroponic solution. At application of 100-400 mg/L TiO 2 NPs, enhanced activities of SOD, CAT, and APX mitigated the oxidative stress; pronouncedly reduced root fresh biomass and water content was observed using a dose of 400 mg/L. At exposure to 50 mg/L TiO 2 NPs stimulated accumulation of Ca, Mg, Fe, Mn, Zn, and B in shoots, resulting in improved nutrient quality of edible plan parts was observed; internalization or translocation of TiO 2 NPs to shoots was not detected [254]. At studying of the transgenerational effects TiO 2 NPs in Ocimum basilicum plants, it was found that hydrophobic or hydrophilic TiO 2 NPs exhibited beneficial impact on plants growth, but more negative effects on photosynthesis compared to pristine TiO 2 NPs. Treatments with hydrophobic and hydrophilic TiO 2 NPs resulted in the reduction of Chlb and total Chl by 52% and 30%, respectively, while total sugar and reducing sugar contents achieved 186% and 145% of contents observed in unexposed plants in both cycles [255].
Spraying of Vetiveria zizanioides plants with 90 mg/L TiO 2 NPs, which was performed 300 d after transplantation, increased the Chl content and maximum photochemical efficiency of PS II and yield of EO by 27.2%, 23.5%, and 55.1%, respectively, compared to the control [145].

Elicitor Effect
Spraying of S. rebaudiana Bertoni plants with 400 ppm TiO 2 NPs showed pronounced beneficial effect on fresh and dry weights of shoots, while application of 200 ppm mostly enhanced stevioside glycoside and reduced MDA levels [147]. In addition, application of 100 mg/L TiO 2 NPs also increased EO content and contents of important secondary metabolites. Moreover, TiO 2 NPs ameliorated agronomic traits and increased antioxidant enzyme activities in D. moldavica L. plants grown under salt stress, and considerably reduced H 2 O 2 levels [149].
The beneficial impact of 150 mg/L TiO 2 NPs on foliarly treated M. piperita plants was reflected in an increase of EO yield up to 105.1% and application of 150 mg/L TiO 2 NPs increased the yields of menthol, menthone, and menthyl acetate by 124.1%, 169.4%, and 130.5%, respectively, over the control, whereby contents of these secondary metabolites were enhanced as well [144]. Concentration of 0.06% TiO 2 NPs was reported as most favorable for foliar spraying of Cuminum cyminum L. to achieve the highest EO yield of cumin [256]. Treatment of black cumin plants in the early flowering stage with 100 mg/L TiO 2 NPs promoted the geranyl diphosphate synthase (GPPS) expression 24 h after treatment more than application of a half dose, which resulted in improved production of thymoquinone [257]. TiO 2 NPs applied foliarly at a dose 100 mg/L acted as an elicitor by improving secondary metabolism in S. officinalis plants resulting in enhanced biosynthesis of natural antioxidants and at application of 100 mg/L TiO 2 NPs highest dry weights of roots and shoots were observed as well [146]. Spraying of rosemary leaves with TiO 2 NPs (7-times, using doses 20-400 ppm) pronouncedly affected quantity of EO, mostly increasing the content of secondary metabolites, although at concentrations >200 ppm some of these compounds (e.g., β-pinene, 3-pinanol, myrcene, 1,8-cineol) showed a decrease [258].
Ebadollahi et al. [154] compared the effect of TiO 2 /perlite nanocomposites (15.50-24.61 nm) applied at doses 25-200 mg/L on production of secondary metabolites in the calli obtained from in vitro, as well as from field grown plants of Hypericum perforatum. They found that, at application of 25 mg/L TiO 2 /perlite NCs, the enhancement of alkaloids production in the calli obtained from in vitro grown plants related to control was almost by one order higher than in calli obtained from field grown plants. Exposure of Tanacetum parthenium L. grown in greenhouse to TiO 2 NPs affected the expression of genes involved in biosynthesis pathway of parthenolide and β-caryophyllene; TiO 2 NPs upregulated the costunolide synthase (COST) and feverfew (T. parthenium) germacrene A synthase (TpGAS) but downregulated the feverfew (T. parthenium) (E)-β-caryophyllene synthase (TpCarS) with increasing time from 6 to 24 h. Exposure to salinity increased the expression of these three genes with increasing time, suggesting that TiO 2 NPs and salinity can be used as elicitors for achieving more effective production of secondary metabolites [259].
Khusimol content and yield of Vetiveria zizanioides plants sprayed with 90 mg/L TiO 2 NPs 300 d after transplantation increased by 24.5% and 93.2%, respectively, compared to control [145]. In hydroponically cultivated Nigella arvensis plants treated for 21 days with TiO 2 NPs increased amounts of secondary metabolites, such as glaucine and quercetin, as well as higher TPC levels were observed compared to the control, although the concentrations of the elicitor providing the highest levels of individual bioactive components differed each from other [148].

Attenuation of Abiotic Stress
Treatment of Verbascum sinuatum plants exposed to artificial drought conditions (−0.6 MPa) with 20 ppm TiO 2 was found to mitigate negative impact of water deficit on growth characteristics and contents of assimilation pigments by stimulation of antioxidant defense systems, resulting in improved drought tolerance [260]. Foliar application of 10 ppm TiO 2 NPs increased shoot dry mass and EO content of Dracocephalum moldavica L. plants and in plants exposed to drought stress. Plants grown under water deficit, which were treated with 10 ppm TiO 2 NPs, had more proline and considerably lower content of H 2 O 2 and MDA compared to untreated plants, suggesting that TiO 2 NPs can mitigate water deficit-induced oxidative damages. On the other hand, assimilation pigments in leaves were pronouncedly reduced in Dracocephalum moldavica plants treated with 10 ppm TiO 2 NPs [261]. Whereas spraying with TiO 2 NPs positively affected growth of Thymus vulgaris cultivated under drought stress, it did not affect the components of EO [262].
TiO 2 NPs (2 and 5 ppm) increased tolerance of Glycyrrhiza glabra L. plants to cold stress, reduced MDA and H 2 O 2 levels in cold-stressed (4 • C) plants, resulting in lesser oxidative damage, and increased the contents of phenolics, total protein, and osmolytes [263]. Treatments of sensitive and tolerant chickpea genotypes with TiO 2 NPs did not induce oxidative damage in plans, which were able to mitigate membrane damage and electrolyte leaching, and pronouncedly reduce MDA content also under cold stress conditions via improved redox status of the genotypes, with a concentration of 5 ppm TiO 2 NPs being the most effective [264].
Everyday spraying with TiO 2 NPs at doses 25 and 50 mg/L was found to prevent Crocus sativus L. plants cultivated hydroponically against harmful effects on morphological characteristics of plants caused by UV irradiation (30 and 45 min daily for one month). Combined treatment reduction of dissolved sugars, increased levels of total anthocyanins and MDA in leaves, as well as higher radical scavenging activity were observed; a raise in TPC and TFC in saffron stigmas was estimated as well, which could contribute to enhanced nutritive value of saffron [265].
TiO 2 NPs (anatase; 10-25 nm) were able to increase concentrations of assimilation pigment in leaves of flax under normal and water deficit conditions, reduce the levels of H 2 O 2 and MDA, resulting in lower degree of lipid peroxidation, particularly at a dose of 10 mg/L, and enhance seed oil and protein contents in treated plants [266]. In cell suspension cultures of flax exposed to 50, 100, and 150 mg/L TiO 2 NPs, increased activity of cinnamyl alcohol dehydrogenase and maximum TPC content was observed at application of 150 mg/L TiO 2 NPs; TiO 2 NPs at tested concentrations increased the total lignan content as well [140].

Genotoxicity
Using A. cepa root tip test, it was shown that TiO 2 NPs (>50 nm) applied at 0.1 and 100 g/L reduced the MI from 63.6% (control) to 59.5% and 53.5%, respectively, and at additional UV stress further decrease to 58.0% and 51.4%, respectively, was observed. Whereas at treatment with 0.1 mg/L TiO 2 NPs (anaphase) multiple chromatin bridges, at exposure to 10 mg/L and 100 mg/L TiO 2 NPs distributed and fragmented chromosomes can be observed. The chromosomal aberration of A. cepa root cells can be associated with accelerated generation of ROS and higher MDA levels at the presence of TiO 2 NPs [245]. Similar results were observed also by Pakrashi et al. [267] or Ahmed et al. [268] who reported that due to oxidative stress induced by of TiO 2 NPs the ROS levels as well as SOD and CAT activities of A. cepa roots increased by 10%, 20.8%, and 12.4%, respectively, and the levels of O 2 − in roots showed a dose-dependent increase. TiO 2 applied at a dose 1000 mg/L caused DNA damage in A. cepa root meristem cells, which, expressed as %DNA tail, achieved 56.54 ± 3.82% and 37.57 ± 3.60% for TiO 2 NPs of 50 and 21 nm, respectively [269].
Considering 2.4-fold enhancement of glaucine in Nigella arvensis L. plants cultivated in hydroponic system in the presence of 2500 mg/L TiO 2 NPs, this method seems to be suitable to achieve enhanced levels of this aporphine alkaloid in N. arvensis plants [147] ( Table 1).

Attenuation of Abiotic Stress
Mn compounds are able to stimulate crop growth and mitigate abiotic stress, whereby at application of nanosized Mn particles, higher efficiency in attenuation of abiotic stresses and less toxic effects can be achieved compared to bulk Mn particles. In addition, plant Mn deficiency can be overcome by additional supply of Mn, resulting in induction of manganese superoxide dismutase at the transcriptional level, to suppress ROS production and promote Mn-dependent proteins for preservation of cell integrity [271].

Genotoxicity
Chromosomal aberrations and a decrease in MI compared to the control, with a pronounced raise in H 2 O 2 and O 2 − production and lipid peroxidation, was observed in Allium cepa roots treated with 12.5-100 µg/mL MgO MPs and MgO NPs, the toxicity of NPs being higher [272].

Elicitor Effect
Artemisia annua cell suspension culture exposed to 5 mg/L CoNPs for 24 h achieved artemisinin content of 113.35 mg/g (d.w.), being 2.25-fold higher than that of the control. Increased artemisinin production was assumed to be associated with decreased expression levels of SQS and DBR2 genes playing essential roles in the regulation of artemisinin pathway [134].

Impact on Plant Growth
Exposure of Coriandrum sativum L. plants to NiNPs with mean particle size 20 nm, applied at doses 20, 40, and 80 ppm for 22 days, resulted in reduced water content, root, and shoot growth, decreased levels of photosynthetic pigments, lower antioxidant activity and increased NiNPs content in plants, suggesting toxic effect of NiNPs on this medicinal plant [273]. In vitro grown seedlings of Abelmoschus esculentus treated with NiO NPs (100-1000 mg/L) showed impaired plant growth, lower Chl content, pronouncedly altered levels of anthocyanin contents, TPC, and TFC and increased ROS and MDA levels, suggesting phytotoxicity of NiO NPs [274].

Elicitor Effect
Nigella arvensis plants treated with 50 mg/L NiO NPs resulted in 2.2-and 1.8-fold higher quercetin content in the shoots and roots compared to the control plants. On the other hand, following exposure of black cumin plants to 1000 mg/L NiO NPs up to 3.2-fold higher glaucine content in shoots, 2.9-fold higher kaempferol content in roots, and considerably higher levels of TPC and TFC were observed compared to the control plants, suggesting that NiO NPs can be used as an elicitor for effective in vitro production of secondary metabolites [148].

Genotoxicity
Treatment of Allium sativum L., Allium schoenoprasum L., Allium porrum L., and Allium fistulosum L. growing root tips with NiO NPs, even at a dose of 10 mg/L induced genotoxicity in plants. NiO NPs caused perturbation of biochemical homeostasis and disrupted normal physiology of the cells, which was reflected in enhanced lipid peroxidation, MDA levels, and activities of antioxidant enzymes (CAT, SOD, and GPX) [275].

Cr 2 O 3 NPs
Genotoxicity Chromosomal aberrations and cytogenetic effects in root tip cells of A. cepa due to exposure to Cr 2 O 3 NPs (0.01-100 µg/mL) were reported by Kumar et al. [276] and in A. cepa roots treated with 25-100 ppm Bi 2 O 3 stickiness chromosome laggards, disturbed anaphasetelophase, and anaphase bridges were observed in anaphase-telophase cells, pro-metaphase, and c-metaphase in other cells, as well as a pronounced rise in DNA damage [277].

CeO 2 NPs
Coriandrum sativum L. plants germinated and cultivated for 30 days in soil amended with 125 mg/kg CeO 2 NPs were characterized with longer roots, pronouncedly increased CAT activity in shoots, and APX activity in roots, and changed the chemical environment of carbohydrates in shoots, suggesting impact of CeO 2 NPs on the nutritional properties of cilantro [278]. Green synthesized organometallic CeO 2 nanostructures with mean size 38 ± 5 nm applied at a dose of 4 mg/L increased callus induction of Berberis lycium Royle to 90%, whereas using a dose of 20 mg/L direct shoot regeneration was improved to 79% [279]. On the other hand, CeO 2 NPs (≈20.28 nm) and bulk CeO 2 (≈4.24 µm) particles applied at doses 12.5-100 ppm for 4 h exhibited cytotoxic and genotoxic effects on the root meristem cells of A. cepa reflected in decreased MI values, considerably increased chromosomal aberrations including chromosome laggards, disturbed anaphase-telophase, stickiness, bridges, and caused DNA damage [280].

Al 2 O 3 NPs
Due to oxidative stress induced in roots of A. cepa, which were exposed to Al 2 O 3 NPs-ROS, SOD, and CAT activities were enhanced by 30%, 17.1%, and 13.2%, respectively, as compared to the control and the levels of O 2 − , which showed a dose dependent increase [268].

Genotoxicity
Exposure of A. cepa roots to Al 2 O 3 NPs showed chromosomal aberration, whereby application of low Al 2 O 3 NPs concentration (0.1 mg/L) disturbed metaphase. At treatment with 10 and 100 mg/L Al 2 O 3 NPs-abnormal anaphase and sticky metaphase were observed; reduction of MI and generation of ROS was observed as well [245]. Genotoxic effects and oxidative damage on A. cepa caused by Al 2 O 3 NPs were pronouncedly higher than those of bulk Al 2 O 3 , and a considerable raise of GPX accompanied with the depletion in CAT activity was observed in plants treated with both Al 2 O 3 NPs and bulk Al 2 O 3 , respectively [281].
Considering 3.2-fold enhancement of glaucine in Nigella arvensis L. plants cultivated in hydroponics in the presence of 1000 mg/L NiO NPs, this method seems to be suitable to achieve enhanced levels of this aporphine alkaloid in N. arvensis plants [136] (Table 1).

Silicon and Silica NPs
Silicon has beneficial impact on plants, particularly under stress conditions, and is considered a non-essential beneficial plant nutrient. It ameliorates not only the vigor of plants, but also their resistance to exogenous stresses [50,283,284].

Impact on Germination and Plant Growth
SiO 2 NPs applied at a dose of 400 mg/L pronouncedly improved seed germination, enhanced the length and fresh/dry weight of plant organs of Nigella sativa L., and increased levels of photosynthetic pigments, total protein, total amino acid, and proline [285]. Irrigation of hawthorn (Crataegus aronia L.) seedlings with SiO 2 NPs at doses 10, 50, and 100 mg/L for 45 days before exposure to drought stress resulted in improved plant biomass, xylem water potential, and MDA content, especially under drought conditions, and had positive influence on photosynthetic pigments; relative water content and membrane electrolyte leakage were not affected and carbohydrate and proline content showed a decrease under all water regimes, especially under water stress [286]. Plants grown on MS medium from Matricaria recutita seeds treated for 1 h with SiO 2 NPs (4 or 6 g/L) were characterized with improved growth characteristics, higher relative water content, TPC, and TFC, but reduced levels of H 2 O 2 and MDA [153].

Elicitor Effect
Pretreatment of Calendula officinalis L. seeds with 200 mg/L SiNPs resulted in maximal increase of quercetin content in plants grown under 50% FC [287]. Treatment of Tanacetum parthenium L. plants with 25 mM SiO 2 NPs increased the expression of TpGAS, COST, and TpCarS genes, which are involved in the biosynthesis pathway of the secondary metabolites, parthenolide, and β-caryophyllene, when the time increased from 6 to 24 h, and a similar effect on the expression of genes exhibited salinity stress [259]. Mentha piperita L. plants treated with 50 and 100 mg/L SiNPs considerably enhanced the density and diameter of the peltate glandular trichomes, Chl content, net photosynthetic rate, TPC, and EO content, as well as content of secondary metabolites at 150 days after plantation [152].
Dracocephalum kotschyi HRs elicited by 100 mg/L SiO 2 NPs after 48 h exposure time showed considerable increase in biomass compared to the control and pronounced enhancement of the expression of pal and ras genes, as well as a strong increase in several secondary metabolites was observed as well [163]. SiO 2 NPs and TiO 2 promoted the expression of GPPS, a key gene involved in thymoquinone biosynthesis pathway in Nigella sativa L. plants, TiO 2 NPs being more effective, and a dose of 100 mg/L raised the gene expression more than that of 50 mg/L. The tested NPs acting as elicitors increased the biosynthesis of thymoquinone in plants via upregulation of related metabolic pathway genes [257]. Continuous production of metabolites from plant cultures using nanoharvesting, in which NPs are designed to bind and carry biomolecules out of living cells, was described by Khan et al. [288] who treated Solidago nemoralis HR cultures in MS medium with mesoporous SiO 2 NPs (165 nm diameter) functionalized with both TiO 2 (425 mg/g particles) for coordination binding sites, and NH 2 (145 mg/g particles) to stimulate cellular internalization in order to obtain increased production of polyphenolic flavonoids. Moreover, observed post-nanoharvesting growth indicating the viability of the roots after nanoharvesting, and thus their ability to synthesize henceforward the flavonoids, suggested that application of NPs can facilitate continuous isolation of many biologically active compounds from living and functioning plant cultures.

Attenuation of Abiotic Stress
SiNPs effectively reduced adverse effect of saline stress on leaf dry and fresh weight and Chl content in Ocimum basilicum plants and increased proline levels at exposure to SiNPs suggested induction of the tolerance in basil plants [289]. Exposure of Allium cepa roots to non-irradiated suspensions of SiO 2 NPs and those irradiated with UV-A light did not lead to the adsorption of SiO 2 NPs on the root surface and no considerable adverse impact was observed even at high exposure concentrations. Consequently, it can be supposed that cell wall shielded the cell membrane from direct contact with NPs. It could be mentioned that similar results were obtained also with application of TiO 2 NPs showing photocatalytic potential [290]. Priming of Calendula officinalis L. seeds with 200 mg/L SiNPs greatly enhanced antioxidant activity and TFC of plants exposed to drought stress of 25% FC [287].
Production of rosmarinic acid elicited by SiO 2 NPs in HR of Dracocephalum kotschyi achieving up to 8.26-fold higher yield compared to control can be considered as very promising [163]. However, also beneficial impact of foliar treatment of Mentha piperita plants with 100 mg/L SiNPs resulting in nearly double enhancement of menthol, menthone and menthyl-acetate in plants seems to be convenient for application also in the field [152] ( Table 1).

Selenium NPs
Selenium is a member of the chalcogen family. It is classified by some researchers as metalloid showing intermediate properties between metals and nonmetals [291,292]. It resembles chemically to sulfur and shares a similar pathway for uptake and translocation in plants.

Impact on Plant Growth and Elicitor Effect
Impact of SeNPs and nitric oxide on growth, metabolism, antioxidant machinery, gene expression, and flowering Cichorium intybus L. plants was discussed by Abedi et al. [293]. Stimulating effect on Capsicum annuum grown in vitro in MS medium was observed after addition of 0.5 and 1.0 mg/L SeNPs; considerable induction in nitrate reductase activity was observed as well [294]. The low doses of 0.5 and 1 mg/L of SeNPs showed growthstimulating effects of growth of Capsicum annuum grown in vitro in MS medium, while higher SeNPs concentrations (10 and 30 mg/L) were found to be phytotoxic due to DNA hypermethylation, upregulation of the bZIP1 transcription factor as well as upregulation of the expression of the WRKY1 transcription factor and inhibition in the differentiation of xylem tissues [294]. Exposure of Ocimum basilicum plants to CdSSe quantum dots (QDs) and Cd 2+ ions resulted in an initial adverse impact on plant growth, but after 3 and 6 weeks, the roots of treated plants were comparable with those of the control group. Although the Ocimum basilicum plants exposed to CdSSe QDs accumulated Cd predominantly in roots, when they were applied at doses 25 and 50 mg/kg of plant material, higher translocation of Cd to shoots was observed compared with application of Cd 2+ ions during 3 and 6 weeks. This can be related to the functional groups on the surface of QDs, which make easier their movement by the cationic transporters or permit free movement of QDs through the cells, whereas due to interaction of Cd 2+ ions with CO 3 2− and PO 4 3− species inside the plant, insoluble salts are formed, which can be immobilized in the apoplastic or symplastic compartments [295].

Elicitor Effect
Treatment of Capsicum annuum grown in vitro in MS medium with SeNPs enhanced the activity of phenylalanine ammonia-lyase and concentrations of soluble phenols as well [294]. Similar results were obtained at in vitro treatment of Momordica charantia with SeNPs [296].

Effects of Carbon-Based NPs on Medicinal and Aromatic Plants
In addition to biochar, which is widely used to improve soil quality, nanoscale carbonaceous materials, such as carbon dots (CDs), graphene (GR), graphene quantum dots (GR QDs), graphene oxide (GO), reduced GO (rGO), fullerenes, single-walled (SWCNTs), and multi-walled carbon nanotubes (MWCNTs) on plants are also increasingly used in agriculture [40,[297][298][299]. CDs are quasi-spherical carbon-based NPs (>10 nm) with a core that can be either amorphous or nanocrystalline. GR is a two-dimensional allotropic form of carbon, formed by individual layers of sp 2 -bonded carbon atoms arranged in a hexagonal lattice with a distance of 0.142 nm between adjacent carbon hexagon atoms. GO is achieved by introducing carbonyl, hydroxy, and epoxide groups on planar surfaces and edges of graphene sheets, while rGO is obtained by oxidation of GR, followed by reduction and exfoliation. Carbon nanotubes (CNTs) are cylindrical structures with open or closed ends and, depending on the number of concentric layers of rolled graphene sheets, are classified as SWCNTs (outer diameter 0.8-2 nm) and MWCNTs (outer diameter 5-20 nm) with a length ranging from 100 nm to several centimeters. Fullerenes are composed entirely of carbon and exist in the form of hollow balls (buckyballs), ellipsoids, or tubes (buckytubes or CNTs), e.g., [40,300,301].

Impact on Photosynthesis
Li et al. [81] comprehensively overviewed the interactions of CDs on plant growth, internal physiological processes, and further external factors affecting plant growth. Amine functionalized CDs, effectively conjugated over the surface of the chloroplast after absorbing light or photons can transfer electrons towards chloroplasts, thereby positively affecting photosynthetic electron transport resulting in faster conversion of light energy to the electrical energy and finally to the chemical energy [302]. Li et al. [303] prepared far-red (FR) CDs that are able to efficiently convert UV-A light to 625-800 nm FR emission, i.e., wave lengths suitable to be directly absorbed by photosynthesizing organisms. In an in vitro experiment, coating of chloroplasts of Lactuca sativa L. var. longifolia with FR CDs resulted in a hybrid photosynthetic system, ensuring improved efficiency of electron transport between photosystem PSII and PSI, leading to increased ATP production. Moreover, in an in vivo experiment, the FR CDs-treated lettuce achieved higher electron transfer rate by 28.00% compared to the control, resulting in increased fresh and dry weights by 51.14% and 24.60%, respectively, suggesting that FR CDs could be used for improved conversion efficiency from solar energy to chemical energy.

Graphene Quantum Dots and Graphene Nanosheets
Treatment of coriander and garlic seeds with 0.2 mg/mL of GR QDs for 3 h before planting resulted in improved growth rate of plant organs of both plants, including fruits, suggesting that GR QDs could be used as plant growth regulators [304]. Spraying of graphene nanosheets (GNS) on Capsicum annuum L. and Solanum melongena L. plants during two seasons at doses 0.1, 0.2 and 0.3 GNS/g induced metabolic regulation of the leaves physiological status and resulted in an increased number of branches and number of fruits per plant, as well as fruit yield. GNS were localized on plastids, cell walls, and intercellular spaces of both plants, and GNS situated inside chloroplasts were found to activate assimilation pigments resulting in promotion of fructose, sucrose, and starch. Application of GNS triggered activity of antioxidant enzymes CAT, APX, GPX, and glutathione-S-transferase, and induced SOD and antioxidant molecules, reducing the levels of OH and O 2 − radicals and, thus, prevented the lipid peroxidation and electrolyte leaching. Moreover, increased PAL activity due to GNS treatment stimulated the formation of valuable secondary metabolites [305].

Impact on Germination and Plant Growth
Exposure of Salvia mirzayanii seed to GO retarded and reduced the germination as well as growth characteristics of plants due to lower water uptake and oxidative stress, the effects being greater in demucilaged compared to intact (mucilaginous) seeds. On the other hand, the dependence of growth characteristics on the applied concentration of GO/polyaniline (PANI) NC was bi-phasic, showing growth stimulation at low composite doses and growth inhibition at high composite doses. In shoots of S. mirzayanii plants exposed to GO, and high concentration of GO/PANI, elevated H 2 O 2 levels were estimated. GO/PANI NC also showed beneficial impact on germination and ensured a regular porosity pattern in roots resulting in ameliorated water uptake and, therefore, they could be utilized in drought-prone ecosystems [306].

Attenuation of Abiotic Stress
The addition of 800 µg/L nanosized GO to Plantago major L. leaf-derived calli grown on the 1/2 MS medium, in which drought stress conditions were induced by PEG, caused a 78.5% decrease in relative growth rate and 48.2% reduction of osmotic potential value, while dry matter and H 2 O 2 contents increased by 35.1% and 54.2%, respectively. Under normal water availability in the presence of 800 µg/L nanosized GO, a pronounced increase of phenolic and flavonoid contents by 40.9% and 35.3%, respectively, compared to the control, was observed, while proline content showed a considerable decrease by 26.9% [307].

Attenuation of Abiotic Stress
Exposure of Hyoscyamus niger seeds under drought stress (0.5-1.5 MPa) to low concentrations of SWCNTs for 14 days resulted in improved water uptake and upregulation of mechanisms involved in starch hydrolysis, while oxidative stress and electrolyte leakage were suppressed. In the presence of SWCNTs, plant defense system was activated, resulting in increased activities of antioxidant enzymes, such as APX, CAT, POD, and SOD as well as improved biosynthesis of phenolics and proline, which finally contributed to the mitigation of drought stress [308].
6.5. MWCNTs 6.5.1. Impact on Germination and Plant Growth Beneficial impact of MWCNTs on growth rate, germination, and morphological characteristics of Salvia sclarea and Salvia macrosiphon was also reported by Mehrjardi et al. [309].

Elicitor Effect
In vitro cultivation of Thymus daenensis seeds in MS medium containing MWCNTs, which were found to pass across the plant cell wall and enter the cellular cytoplasm, affected early germination of plants, and stimulated the production of plant biomass up to a dose of 250 µg/mL, while further increasing of MWCNTs concentration reduced the biomass. Moreover, MWCNTs upregulated the activities of enzymes and antioxidants and elicited biosynthesis of some secondary metabolites and antioxidants, the optimum dose of MWCNTs being 250 µg/mL [157]. Treatment of Satureja khuzistanica Jamzad plants grown in vitro with 250 mg/L MWCNTs resulted in RA content of 140.49 mg/g (d.w.), while at in vivo exposure of S. khuzistanica to MWCNTs for 24 h, the RA content of 7.13 mg RA/g (d.w.) was achieved [310]. Stimulation of callus induction, biosynthesis of secondary metabolites, and improved antioxidant capacity in medicinal plant S. khuzestanica grown in vitro by MWCNTs was also reported previously by Ghorbanpour and Hadian [158].
Spraying of the leaves of two-month-old Salvia verticillata L with MWCNTs (0-1000 mg/L) resulted in their absorption via the epidermal cells layer into the parenchymal cells of the exposed leaves; the treatment reduced levels of assimilation pigments and caused oxidative stress in a dose-dependent manner. However, at application of 50 and 1000 mg/L MWCNTs, an (approximate) 4-fold increase of RA compared to the control was observed, whereby the activity and gene expression patterns of RA synthase correlated with the RA accumulation. While lower ROS levels at application of lower MWCNT concentrations contributed to improved production of secondary metabolites, treatment with higher concentrations was phytotoxic due to increased oxidative stress [155]. The γ-irradiation of Salvia nemorosa callus, using a dose 70 Gy, resulted in a high yielding cell line able to produce up to 18.53, 5.21, 1.9, and 7.59 mg/g d.w. of RA, salvianolic acid B, ferulic acid, and cinnamic acid, respectively. The cell suspension culture prepared of irradiated callus and elicited with 100 mg/L MWCNT-COOH (outer diameter of 20-30 nm) achieved fresh and dry biomass of 268.47 g/L and 22.17 g/L, respectively. Both treatment with γ-irradiation and elicitation using MWCNT-COOH pronouncedly improved the antioxidant activity of cultures. These findings showed that improved production of secondary metabolites in S. nemorosa achieved by combination of cell line selection via γ-irradiation with elicitation with MWCNT-COOH could be utilized for large-scale production of phenolic compounds [159].
MWCNTs increased the production of biomass production in callus culture of Catharanthus roseus cultivated in the dark, promoted the biosynthesis of total produced alkaloids, whereby strong improvement in the production of vinblastine and vincristine alkaloids was observed, not only with callus cultivation at dark, but also in light conditions [311].

Attenuation of Abiotic Stress
The addition of 50 mg/L of MWCNTs functionalized with carboxylic acid groups (MWCNT-COOH) in hydroponic solution was able to improve the adverse impact of salt stress (50 and 100 mM NaCl) on hydroponically cultivated O. basilicum L. plants, resulting in increased contents of assimilation pigments and the levels of non-enzymatic antioxidants and enhanced activities of antioxidant enzymes, including APX, CAT, and GPX. Whereas application of 25 mg/L MWCNT-COOH was found to be beneficial for plant growth and EO content and compound profile; treatment with a 4-fold higher dose was found to be phytotoxic. Hence, MWCNT-COOH could be considered as plant growth promoting and stress protecting agents, which have potential to be used in agriculture [312].

Genotoxicity
Uptake of MWCNTs in Allium cepa root cells modified cellular morphology, adversely affected membrane integrity and mitochondrial function, and caused considerable DNA damage, micronucleus formation, and chromosome aberration, as well as formation of internucleosomal fragments, which is indicative of apoptotic cell death. Accumulation of cells in the sub-G 0 phase of the cell cycle, pronounced increases in CpG methylation, and in the levels of 5-methyl-deoxycytidine verified the cyto-genotoxic effect of MWCNT [313].
The use of MWCNT-COOH as an elicitor of rosmarinic acid and salvianolic acid B in a cell suspension culture prepared from γ-irradiated (70 Gy) Salvia nemorosa L callus, which allows to reach >13-and 14-fold higher concentrations of these secondary metabolites than in the control, can be recommended for obtaining these bioactive acids on a larger scale [159] (Table 1).

Effects of Organic Material-Derived NPs on Medicinal and Aromatic Plants
Chitosan/tripolyphosphate NPs supplemented to culture media modified the root architecture and differentiation in micropropagated Capsicum annuum, and at toxic doses 5-20 mg/L caused interruption of plant growth and development. On the other hand, at a dose of 1 mg/L, it acted as an effective elicitor to trigger organogenesis via micropropagation, causing considerable increases in contents of secondary metabolites, including soluble phenols, proline, and alkaloid [314]. Exposure of seeds of Capsicum annuum to aqueous suspensions of nanoscale chitin (0.001-0.05% w/v) or hydropriming reduced mean germination time at 25 • C on blotter paper to 4.9-5.3 days compared to 5.4-6.7 days observed with untreated seeds, seeds treated with 1% acetic acid, or 1% Captan fungicide. Application of 0.05% chitin NPs or hydropriming ameliorated seedling emergence at 19 to 30 • C and chitin NPs was able to reduce fungal growth as well [315].
Root meristems of A. cepa obtained from seeds exposed to nanoplastics (50 nm) at doses 0.01-1 g/L showed reduction of MI and caused induction and of cytogenetic anomalies and micronuclei suggesting their cytotoxicity and genotoxicity. This adverse impact of nanoplastics is caused by the mechanical surface contact of nanoplastics with root external layers and the internalization of nanoplastics in different cellular compartments. Consequently, nanoplastics internalized into crop plants could enter in different trophic levels of the food chain and, so, they represent a hazard for the health of the human population [316]. Polystyrene NPs applied at doses 25-400 mg/L considerably inhibited growth of A. cepa roots, induced the production of OH and O 2 − radicals, and increased DPPH scavenging activity and lipid peroxidation, causing a decrease in MI compared to the control. They also caused various chromosomal and nuclear aberrations and downregulated the expression of the plant cyclin-dependent kinase (CDKA) encoding gene: cdc2, an important cell cycle regulator. The above results suggest the cytotoxic and genotoxic potential of polystyrene NPs [317].

Conclusions
Aromatic and medicinal plants grown in the field require low energy input for cultivation, can contribute to increasing biodiversity in agroecosystems, and their cultivation can also be used to restore degraded areas. Secondary metabolites of aromatic and medicinal plants represent a rich source of phytochemicals that can be used not only for medicinal purposes or as nutraceuticals, but also as "lead compounds" for the design of new drugs. Medicinal plants should generally not be grown in contaminated soil, and chemicals used in the growth or protection of crops should be kept to a minimum. On the other hand, it is desirable to ensure favorable conditions for their germination and plant growth. In this context, the use of nanoparticles, which allow a controlled release of the active ingredient and the use of a smaller amount of active ingredient to achieve the same (or even better) biological effect than in the case of bulk, has a great perspective. The use of seed nanopriming can significantly improve plant germination and growth and can be considered a suitable approach for the revegetation of medicinal plants in harsh environmental conditions. In agricultural soils with low nitrogen content, mesoporous SiO 2 NPs loaded with urea can contribute to achieving the desired levels of this macronutrient. On the other hand, in calcareous soils characterized by a lack of micronutrients (especially Zn and Fe), mineral fertilization of medicinal and aromatic plants using nanoscale particles of these essential nutrients or their oxides can be favorable. In particular, foliar spraying with nanosized metal fertilizers allows maximum use of plant nutrients and contributes to the fortification of plants with essential micronutrients. In general, low-concentration nanoparticles applied in such treatment do not contribute to environmental contamination and do not pose a risk to non-target organisms. Medicinal plants, especially their essential oils containing secondary metabolites, show many biological activities, e.g., anti-inflammatory, antimicrobial, antiviral, antidiabetic, anticancer, etc. Oxidative stress induced by NPs stimulates the production of these valuable compounds, not only in medicinal and aromatic plants grown in the field, but also with higher efficiency in tissue and hair root cultures of these plants. Therefore, these "green" in vitro methods, which do not use harmful solvents, can be promising for the efficient production of these beneficial bioactive metabolites. Utilizing the ability of nanoparticles to mitigate the adverse impact of abiotic stresses on medicinal and aromatic plants can be particularly useful in changing climatic conditions, where longer periods of drought and elevated temperatures can be expected in the near future. Increased plant tolerance to salts due to the application of nanoparticles will facilitate the cultivation of medicinal and aromatic plants in soil with higher salinity without adversely affecting their performance. Medicinal and aromatic plants have been associated with the human population since ancient times; their popularity as alternatives to synthetic drugs is currently growing, and the use of appropriate nanoparticles can significantly contribute to their better yield, production of higher concentrations of valuable metabolites, and better nutritional value.