NPs hold immense potential for revolutionizing various aspects of plant biotechnology, with the potential to influence various aspects of plant growth and development. The effects of NPs on plant tissue cultures can be diverse and complex, depending on various factors. The results of the study indicated that optimal concentrations of ZnONPs and iron enhanced shoot proliferation, height, and fresh biomass. In addition, synergistic effects were observed with specific concentration combinations, while higher concentrations had adverse effects. Our findings on the use of ZnONPs in vitro cultures align with previous research, demonstrating their positive effects on shoot growth and proliferation in plant tissue culture in a variety of plant species. Awad et al. (2020) investigated the percentage of shoot formation and the number of proliferated shoots in Phoenix dactylifera under the effect of ZnONPs. They observed a twofold increase in the multiplication rate of the proliferated shoot at 150 mg/L compared to the control treatment. ZnONPs have been used in tomato tissue cultures on an MS medium to induce callus production and plant regeneration (Alharby et al. 2016). Moreover, ZnONPs (1–20 mg/L) are also employed in MS medium to induce root formation of Brassica nigra plants (Zafar et al. 2016). A significant enhancement of shoot regeneration was observed when the concentration of ZnO NPs was increased to 10 mg/L. This trend aligns with the findings of Helaly et al. (2014), who demonstrated enhanced shoot regeneration in banana tissues treated with ZnONPs. Some research suggests that ZnONPs can act as catalytic cofactors for enzymes involved in key metabolic processes, such as nitrate reductase. By enhancing its activity, ZnO NPs can potentially increase plant growth (Alharby et al. 2016).
However, ZnONP also caused various detrimental effects in plants at high doses and durations that vary with different plants as well as with the size and shape of ZnONPs. For example, Wang et al. (2018) found that ZnONPs at concentrations of 400 and 800 mg/L significantly decreased the growth of the shoots and roots of tomato (Lycopersicon esculentum Mill.) plants. In addition, the ZnONPs adversely affect the growth of rape (Mousavi Kouhi et al. 2014), soybean (Yoon et al. 2014), and alfalfa (Bandyopadhyay et al. 2015) in a dose-dependent manner. Extensive research has been done to overcome the antagonist effect of ZnONPs, where low dose and duration of exposure are found to be beneficial in plants (Thounaojam et al. 2021).
It was revealed that the ZnONPs, iron, and their combination had a positive effect on pigment content, but only up to a certain point. Exceeding this threshold results in decreased pigment content. However, the positive interaction became negative at higher ZnONP levels. NPs can interact with plant photosystems and affect their photosynthesis and pigment production, either positively or negatively, depending on the type, concentration, duration, and mode of application of NPs (Ghorbanpour et al. 2021). Some NPs, such as mesoporous silica, titanium dioxide, and carbon nanotubes, can enhance photosynthesis by increasing the chlorophyll content, the activity of the key enzyme Rubisco, the efficiency of photosystem II (PSII), and the CO2 harvesting, as well as broadening the chloroplast photoabsorption spectrum (Mony et al. 2022). However, other NPs, such as iron oxide, silver, and ZnO, can inhibit photosynthesis by decreasing the chlorophyll content, the electron transport rate, the photosynthetic efficiency, and some other chlorophyll fluorescence parameters, as well as damaging the chloroplast components. For example, ZnONPs can reduce the photosynthesis regulating genes and cause oxidative stress in plants. Iron oxide NPs can impair the photosynthetic machinery and induce chlorosis (Ghorbanpour et al. 2021). The findings at lower ZnONP concentrations suggest a synergistic effect between ZnONPs and iron on pigments content. However, at higher ZnONP concentrations, this effect diminished or became antagonistic, highlighting the importance of considering the concentration-dependent nature of these interactions. Therefore, the effects of NPs on plant photosynthesis and pigments are complex and variable, and more research is needed to understand the underlying mechanisms and the optimal doses of NPs for plant cultivation.
Phenolic compound biosynthesis in stevia tissues was affected by treatments in the culture medium, varying with treatment type and concentrations. The biosynthesis of phenolic compounds in plant tissues can be influenced by various factors, such as environmental stress, genetic modification, and elicitation (Humbal and Pathak 2023). NPs as novel elicitors that can enhance the production of phenolic compounds in plant tissues by inducing stress responses, activating signaling pathways, and modulating gene expression (Selvakesavan et al. 2023). Different types of NPs have been reported to affect the biosynthesis of phenolic compounds in plant tissues, such as metallic, bimetallic, non-metallic, carbon-based, and composite NPs (Selvakesavan et al. 2023). For example, silver NPs increased the content of phenolic acids, carotenoids, and anthocyanins in basil leaves (Shahraki et al. 2024). Copper oxide NPs stimulated the production of gymnemic acid and phenolic compounds in cell suspension cultures of Gymnema sylvestre (Chung et al. 2019). Titanium dioxide NPs led to a massive increment in the production of valuable anticancer flavonoids such as xanthomicrol, cirsimaritin, and rosmarinic acid as polyphenols in hairy root cultures of Dracocephalum kotschyi (Nourozi et al. 2021). In addition, carbon nanotubes improved the biosynthesis of phenolic compounds and flavonoids in callus cultures of Fagonia indica (Begum et al. 2023). Furthermore, some studies also showed that the composition of phenolics was affected by NPs. In lettuce seedlings, five phenolic compounds were decreased (3,4-diOH-benzaldehyde, ferulic acid, p-coumaric acid, salicylic acid, and vanillin) and two compounds (gallic acid and vanillic acid) were increased under the effect of NPs in comparison to control plants, while for sweet pepper an increase was observed for four compounds (chlorogenic acid, neochlorogenic acid, ferulic acid, and protocatechuic acid) (Kalisz et al. 2021).
However, the effects of NPs on the biosynthesis of phenolic compounds in plant tissues are not always positive. Some NPs may also have negative or toxic effects on plant growth, development, and metabolism, depending on the concentration, duration, plant species, and mode of application of NPs (Hu et al. 2022). Therefore, the effects of NPs on the biosynthesis of phenolic compounds in plant tissues are complex and variable, and more research is needed to understand the underlying mechanisms and the optimal doses of NPs for plant cultivation and improvement.
This research demonstrated a significant influence of ZnONPs and their combined treatment on PAL enzyme activity within stevia tissues. One of the ways that NPs affect plant growth and development is by influencing the activity of key enzymes. PAL is a key enzyme in the phenylpropanoid pathway, which is responsible for the biosynthesis of a variety of secondary metabolites, including flavonoids, phenolics, and lignans. PAL activity is often upregulated in response to stress, such as pathogen infection or wounding. NPs have been shown to affect PAL activity in plant tissue culture. In this respect, Ghalamboran et al. (2023), showed that the total protein and phenylalanine level in rice kernel decreased under all concentrations of chitosan NPs compared to the control, while the activity of phenylalanine ammonia-lyase was higher than that of the control. Phytochemical analysis of the callus cultures showed higher production of phenolics, flavonoids phenylalanine ammonia-lyase activity, and antioxidant activity, respectively, in the callus cultures of Caralluma tuberculate in the presence of AgNPs (Ali et al. 2019). The application of SeNPs increased the synthesis of secondary metabolites through increases in the expression of biosynthesis pathway-related genes: PAL and 4-coumaroyl CoA ligase, in bitter melon (Rajaee Behbahani et al. 2020), and pepper plant (Li et al. 2020). In addition, using SeNPs increased the activities of the PAL enzyme, which is involved in the synthesis of secondary metabolites in plants through the phenylpropanoid biosynthetic pathway (Abedi et al. 2021). Another study by Karimzadeh et al. (2019) found that the highest activity of PAL was observed in nano-ZnO treatment, whereas the effect of nano-TiO2 on PAL enzyme activity was not statistically significant. The exact mechanisms by which NPs affect PAL activity are not fully understood. However, it is thought that NPs may interact with plant cell membranes and signaling pathways to trigger the upregulation of PAL gene expression.
The study found that ZnONP led to an increase in the antioxidant activity of tissues. Some studies evidenced that NPs can enhance antioxidant activity in plant tissues. For example, Ishtiaq et al. (2023) found that seed priming with selenium NPs (SeNPs) increased the activity of the antioxidant enzymes, the content of the antioxidant vitamins C and E as well as reduced glutathione and oxidized glutathione content in tomatoes. In addition, it was reported that applying SeNPs upregulated the antioxidant defense enzymes in plants and the scavenging capacity of free radicals in Mangifera indica, Sorghum bicolor, and citrus (Garza-García et al. 2021; Shahbaz et al. 2023; Djanaguiraman et al. 2018; Alvi et al. 2021). ZnONPs were found to play an important role in controlling reactive oxygen species (ROS) and protecting plant cells from oxidative stress (Alharby et al. 2016). However, NPs also have negative effects on plants, including affection of antioxidant enzyme activity, oxidative stress, and increased chromosomal and micronucleus abnormalities, which may affect plant root growth, and seed germination. Disruption of ROS antioxidant mechanisms in Allium cepa and Lathyrus sativus by NPs causes cell cycle arrest, DNA damage, and cell death, resulting in cytotoxicity (Panda et al. 2017; Sun et al. 2019). Lower doses of ZnONPs were expected to have beneficial effects, but higher doses may reduce plant growth and induce stress due to increased zinc accumulation (ur Rehman et al. 2023).
The treatments including ZnONPs administered in this study had a significant impact on MDA content or lipid peroxidation within the stevia shoot tissues. Studies have reported that NPs can increase or decrease MDA levels, depending on the type of NP, its size, concentration, surface properties, and plant species. One of the proposed mechanisms by which NPs affect MDA levels is by inducing oxidative stress in plant cells. This triggers the production of reactive oxygen species (ROS), which can damage lipids and other cellular components (Zia-ur-Rehman et al. 2023). It was shown that all ZnONP treatments increased antioxidant capacity and oxidative stress, along with increased MDA content in Chenopodium murale (Zoufan et al. 2020). In eggplant (Solanum melongena L.), the NPs (NiO, CuO, and ZnO) induced a high amount of ROS, which led to a higher amount of MDA as a lipid oxidation marker (Baskar et al. 2018). However, AgNPs decreased hydrogen peroxide, ROS, and lipid peroxidation levels and thus improved the growth of rice seedlings (Gupta et al. 2018). In addition, increased activities of antioxidant enzymes and reduced levels of ROS and MDA were observed in Daucus carota L. under the effect of AgNPs (Faiz et al. 2022).
Adding ZnONPs to the culture medium enhanced the uptake of both iron and zinc by the shoots. However, the increase in uptake wasn't proportional to the increase in ZnONP concentration. It was revealed that NPs can affect mineral accumulation in plants by influencing their uptake, translocation, and distribution within plant tissues. Some NPs can enhance mineral absorption and transport by plants, while others can interfere with or inhibit these processes (Yang et al. 2020). The effect of NPs on mineral accumulation in plants depends on several factors, such as the type, size, shape, concentration, and surface coating of the NPs, as well as the plant species, growth stage, and environmental conditions (Mishra et al. 2014). According to some studies, NPs can have positive effects on mineral accumulation in plants. For example, the ZnONPs addition was found to increase the zinc accumulation in tomato callus tissue. Meanwhile, no significant differences were found between the ZnONPs and control treatment for K, N, and P content (Alharby et al. 2016). Soybean (Glycine max L.) exposed to amendments of Fe2O3 NPs during an eight-week growing period enhanced potassium, zinc, iron, and nitrogen content in the plant (Yang et al. 2020). The higher N, P, Zn, and Cu concentrations were recorded under TiO2NPs treatment in the wheat (Triticum vulgare L.) plant (Dağhan et al. 2020). Treatment with Fe3O4 NPs led to noticeable increases in the leaf Fe, P, and K content in wheat (Triticum aestivum) plants (Feng et al. 2022). The work of Sundaria et al. (2019) demonstrated that seed priming by iron oxide Fe2O3 NPs in two contrasting wheat genotypes induced germination, improved growth parameters, and enhanced accumulation of Fe in the grain. Different concentrations of Fe3O4NPs increased significantly some nutrient contents of moringa (Moringa oleifera) leaves (N, P, K, and K/Na) compared with untreated control plants, meanwhile decreasing Na contents (Tawfik et al. 2021). The Zn content in rice leaf and seed was higher in ZnO nano-treated plant samples compared to ZnSO4 treatment (Rameshraddy et al. 2017). The exposure to ZnONPs increased leaf fresh and dry weight and leaf Zn content in red perilla as compared with untreated control (Salachna et al. 2020). Similar outcomes were achieved in earlier studies in beans and tomatoes grown in the soil enriched with 3, 20, or 225 mg ZnONPs kg− 1 (García-Gómez et al. 2020).
Meanwhile, nano-Si application significantly increased concentrations of K, Mg, and Fe in rice grains and rachises, but had no significant effect on concentrations of Ca, Zn, and Mn in them (Chen et al. 2018). Application of a foliar spray of SiO2 NPs to rice seedlings in hydroponics decreased Ca and enhanced Mg, Fe, and Zn in shoots and roots. However, this decrease or increase in uptake of Fe, Cu, and Mn by plants depended on plant organ and nutrient type (Wang et al. 2015a; Wang et al. 2015b). Therefore, the effect of NPs on mineral accumulation in plants is complex and variable, and it requires further research and evaluation to understand the underlying mechanisms and potential applications or risks of nanotechnology in agriculture.
The results showed that ZnONPs and iron individually and in combination influenced the production of glycosides in stevia-cultured shoots. Several studies have shown that NPs can increase the production of secondary metabolites including glycosides in tissue cultures of stevia and other plants. Javed et al. (2017) found that under 1 mg L− 1 of ZnONPs increased the glycoside content in Stevia rebaudiana shoot cultures by up to 100%. However, the formation of other secondary metabolites and the physiological parameters showed a sudden decline after crossing a threshold of 1 mg L− 1 concentration of ZnONPs and falling to a minimum at 1000 mg L− 1, elucidating the maximum phytotoxic effect of ZnONPs at this concentration. The selenium and titanium dioxide NPs increased the concentration of stevioside and rebaudioside A in stevia plants (Sheikhalipour et al. 2021). Although zinc oxide and copper oxide NPs maximized levels of total phenolic content, total flavonoid content, and total antioxidant capacity in stevia callus cultures, surprisingly, none of the cultures produced steviol glycosides (Javed et al. 2018).
Golinejad et al. (2023) found that cells treated with gold NPs had the highest levels of phenols after 8 hours. The highest amount of taxanes, both inside and outside the cells, was found in cells treated with a lower dose of NPs after 24 hours. Hyoscyamus reticulatus transformed roots treated with SiO2 NPs offer a promising approach to significantly enhance the production of hyoscyamine and scopolamine, while the same method showed less potential in Hyoscyamus pusillus (Hedayati et al. 2020). Shahhoseini et al. (2020) showed that ZnONP treatment significantly increased essential oil content and zinc absorption in the Feverfew (Tanacetum parthenium (L.) Schultz Bip.) plant while decreasing parthenolide levels. This suggests a potential trade-off between maximizing essential oil production and maintaining the plant's natural chemical profile.