Multi-walled carbon nanotubes interact with light intensity to affect morpho-biochemical, nutrient uptake, DNA damage, and secondary metabolism of Stevia rebaudiana

In this study, the interaction between nanoparticles (0, 50, 100, and 150 mg L−1) and light intensity (100, 200, and 400 μmol·m−2·s−1) was evaluated for effectiveness in improving stevia shoot induction by measuring morphological traits, nutrient absorption, total carbohydrates, steviol glycosides (SVglys), and DNA damage in two DNA sequence regions (promoter and sequence of the UGT76G1 gene). MWCNTs at a concentration of 50 mg L−1 in interaction with the light intensity of 200 μmol·m−2·s−1 improved the morphological traits and absorption of nutrients such as nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), iron (Fe), and Manganese (Mn), compared to other treatments. Also, under this interaction, the accumulation of total carbohydrates and SVglys was elevated. Moreover, DNA damage in both regions of the DNA sequence under light intensity at low concentrations of MWCNTs (0 and 50 mg L−1) did not show a significant change but increased with increasing MWCNT concentration at high light intensities (200 and 400 μmol·m−2·s−1). These results demonstrate that the advantages and phytotoxicity of MWCNTs in the in vitro culture of stevia are dose-dependent and are affected by light intensity. Based on this, the interaction of 50 mg L−1 of MWCNTs with the light intensity of 200 μmol·m−2·s−1 is recommended to improve stevia micropropagation and subsequent growth and metabolism.


Introduction
Engineered nanomaterials have been widely used in various fields of plant sciences, such as improving quality, increasing growth and nutritional value, gene preservation, and so on. Recent reports indicate properties such as growth enhancement, growth inhibition, as well as specific toxic effects on plants. However, the translocation, growth responses, and stress modulation mechanisms of engineered nanomaterials in plant systems need to be better and more deeply understood . Nanoparticles (NPs) are generally demarcated by having at least one dimension between 1 and 100 nm in diameter (Auffan et al. 2009), and recent advances in this field have affected various industries, including agriculture (Ma et al. 2015). Carbon nanotubes (CNTs) are cylindrical nanoparticles of carbon atoms that are organized in a hexagonal framework and are classified into single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) based on the number of concentric layers of rolled graphene sheets in the structure (De Volder et al. 2013). MWCNTs have an outer diameter of 2-100 nm and an inner diameter of 1-3 nm and a length of 100 nm to a few centimeters (He et al. 2013). MWCNTs have higher density, tensile strength, and electrical conductivity compared to SWCNTs (Shoukat and Khan 2022).
Because of their unique mechanical and physicochemical nature, CNTs have been tested as a potential solution Responsible Editor: Gangrong Shi to various biologically important problems. In this regard, Samadi et al. (2020) reported increasing in biomass, seedling height, and antioxidant activity of Thymus daenensis Celak under MWCNTs. Sorcia-Morales et al. (2021) reported that the use of low concentrations of MWCNTs (50 and 100 mg L −1 ) had positive effects on growth, total chlorophyll, carbon, and macronutrient and micronutrient contents during sugarcane micropropagation. Ghasempour et al. (2019) showed that MWCNTs improve the secondary metabolism and yield of Catharanthus roseus callus cultures in a good way.
However, various toxic effects associated with plants and nanoparticles have been reported. MWCNTs have been stated to have lower toxicity effects than SWCNTs for plant cells (Samadi et al. 2020). The molecular mechanisms underlying the toxic effects of CNTs on plants are still unclear. Yang et al. (2021) reported that leaf growth nanoparticles and root elongation were greatly inhibited by these nanoparticles in Arabidopsis and that overproduction of oxidants (H 2 O 2 and O 2 − ) and malondialdehyde was recorded as a result of the application of these nanoparticles. Combinatorial transcriptome and m6A methylome studies by these researchers also showed that photosynthesis and auxin signaling are inhibited by MWCNTs Luo et al. 2020). It has been shown that the expression of stress-related enzyme genes is increased by the application of nanoparticles (Frazier et al. 2014). Because of this, it is important to know how nanoparticles and CNTs hurt plants in different ways.
Nowadays, because of the flexibility of light-emitting diodes (LEDs), the ability to tailor the spectral composition and intensity of light is provided, as is the opportunity to select the most desirable light spectra for photosynthetic and photomorphogenic evaluations for agricultural purposes (Brazaitytė et al. 2015). In a recent study, Farrokhzad et al. (2022) showed the interaction between light intensity and silver nitrate nanoparticles in the Phalaenopsis in vitro culture on traits such as fresh weight, shoot induction, protein content, and total phenol. Accordingly, the interaction of different nanoparticles, especially CNTs, with different light parameters can be an interesting field of study in plant sciences.
Stevia (Stevia rebaudiana Bertoni) has been used commercially as a natural sweetener in a wide range of products in the food and beverage industry for decades because its leaves contain stevioside and rebaudioside (Prakash and Chaturovedula 2016), and its cultivation has expanded in recent years because of its increasing use in the food industry (Kovačević et al. 2018). Stevia can be propagated by seeds or cuttings. However, tissue culture is one of the most effective methods for producing disease-free and uniform plants for large-scale cultivation (Yucesan et al. 2016). About 25% of the dry mass of stevia leaves containing diterpene is sweet glycosides or steviol glycosides (SVglys) (Geuns 2003). Glycosyltransferase enzymes are responsible for glycosylation of the steviol molecule by transferring the glucose moiety to form various SVglys, with different units of D-glucose attached to the steviol ring (Ceunen and Geuns 2013). Accordingly, a significant amount of carbon produced by photosynthesis is used for the biosynthesis of SVglys (Hajihashemi et al. 2018;Yadav 2010). In this way, feeding with CNTs and a possible interaction with light intensity may help to improve the amount of SVglys in Stevia leaves and other parts of growth and metabolism.
In view of the above, the aim of this study was to test the interaction between MWCNTs and light intensity to improve shoot induction in in vitro cultures of stevia. Given the role of light in photosynthesis and carbon stabilization, we hypothesized that light intensity could interact with the carbon nanotubes in nutrient uptake and subsequent growth of stevia. No study has reported the interaction of light intensity with CNTs in plant growth and development, and there is little information concerning their effects on plant tissue culture. Therefore, this study aimed to assess the possible capabilities of MWCNTs in interaction with light intensity during shoot induction in stevia plants. Also, this study may provide useful information about the effects of light intensity on the phytotoxicity of MWCNTs. Accordingly, the most important features that were evaluated under these conditions are: (i) the content of stevioside, rebaudioside A and total carbohydrates, (ii) uptake of various nutrients by plants, (iii) the amount of DNA damage in two gene regions by PCR-based method, and (IV) morphological characteristics of stevia in the shoot-induction stage.

Materials and methods
This study was done in the plant tissue culture lab at the Faculty of Agriculture, University of Zabol, Zabol, Iran.

MWCNT characterization
The study used MWCNTs formulated by US Research Nanomaterials, Inc. (Houston, USA). These nanoparticles had 20-to 30-nm optical density and a 95% purity rate. The physicochemical properties of MWCNTs (the fine powder) were verified through a double beam UV-Vis spectrophotometer (Beijing Rayleigh Analytical Instrument Corporation; UV-2100; China) ( Fig. 2), scanning electron microscope (SEM) (Cambridge Stereoscan 360; England) ( Fig. 1A, B) and transmission electron microscopy (TEM) (Hitachi HF3300; Japan) ( Fig. 1C). To complete the culture medium, MWCNTs were dispersed at the desired concentration by sonication in a water bath for a total duration of 2 h by exposing them to 20 kHz (100 W) ultrasound waves based on the Ghorbanpour and Hadian (2015) procedure. After dispersion, the concentration of the samples was determined spectrophotometrically (Fig. 2).

Culture condition
Stevia rebaudiana plants were prepared from a commercial greenhouse (Karaj, Iran) as pot plants and kept in a growth chamber for explant preparation. Shoot tip explants of stevia with a size comprised between 1 and 1.5 cm were collected, followed by 20 min of washing with a slow flow of running water. After washing, the samples were shaken in 2% (w/v) carbendazim fungicide solution for 30 min and then rinsed with distilled water. Then they were shaken with a 0.5% solution (v/v) of sodium hypochlorite for 10 min. The explants were then washed in a laminar hood three times with autoclaved distilled water and immediately treated with 70% ethanol for 30 s, followed by three rinses with distilled water. The last two steps of washing were performed with autoclaved distilled water. MS (Murashige and Skoog 1962) medium at half strength supplemented with 2 mg L −1 6-benzylaminopurine (BAP), 0.8% agar (w/v), 3% sucrose (w/v) was used to establish the shoot tips of stevia. After 4 monthly subcultures of explants and obtaining sufficient and free-pathogenic plant material, different concentrations of MWCNTs (0, 50, 100, and 150 mg L −1 ) were added to the same medium, and then culture vessels were kept for 35 days under three different light intensities of LED fixture (100, 200, and 400 μmol·m −2 ·s −1 ) with a photoperiod of 16 h. After 35 days, as the shoot induction was accomplished and the plants had a good development, size, and number of leaves (Fig. 3), the  Optical density

Wavelength (λ nm)
regenerants were taken out of the culture containers and put in a freezer at − 20 °C until they could be studied further. Figure 3 is a representative figure of plants under different treatments in the stage of shoot induction.

Growth analysis
Growth analysis was performed 35 days after shoot culture. The number of new regenerated shoots and leaves per explant was recorded as the shoot number and leaf number, respectively. After removing the residues of culture medium, the weight of regenerated plantlets was considered as fresh weight. The regenerants were then dried at 60 °C for 48 h to obtain a constant weight and a dry weight. The length of shoots longer than 0.5 cm was also determined with a ruler.

Diterpene glycoside determination and total carbohydrates
Contents of stevioside and rebaudioside A were determined using an HPLC system equipped with a Waters Alliance 2695 separation module (Waters, Milford, MA, USA) and a Waters 2996 diode array detector and controlled by Empower Pro software. According to the JECFA (2010) method, the separations were conducted at 40 °C on the Luna C-18 reverse phase column (250 mm × 4 mm) provided by Phenomenex (Alcobendas, Spain). A combination of acetonitrile: phosphate buffer 10 mM, pH 2.6 with a flow rate of 1 mL min −1 was used as the mobile phase. Detection of SVglys in plant extracts was conducted by comparing UV spectra and retention times with the spectra of a standard rebaudioside A. Chromatograms were achieved at 210 nm and quantification of SVglys in plant extracts was done using rebaudioside A as a standard (Sigma-Aldrich) (Fig. 4A, B). The amount of diterpene glycoside was stated as rebaudioside A μmol equivalent per gram (dry matter). Total soluble carbohydrates were quantified based on the phenol-sulfuric-acid procedure (Dubois et al. 1956).

Analysis of mineral elements
In this study, the concentration of nine nutrients, including nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) was analyzed. After treatments, plant samples (new well-expanded leaves) were dried at 60 °C and ground in a blender to measure the concentration of macro-and micro-nutrients. According to the Alcántar and Sandoval (1999) method, the samples were wet-digested in a mixture of HNO 3 -H 2 O 2 at a 2:1 (v/v) ratio followed by the next steps in microwave digestion: (i) 145 °C, 75% radio-frequency power (RF), 5 min; (ii) 180 °C, 90% RF, 10 min; and (iii); 100 °C, 40% RF, 10 min. Then, the extracts were analyzed by an inductively coupled plasma-optical emission spectrometer (ICP-OES, Optima 8300, USA) to measure the concentrations of macro-(P, K, Ca, and Mg) and micro-nutrients (Fe, Zn, Mn, and Cu). The procedure described by Bremner (1965), which is the semi-micro-kjeldahl method, was used to determine the N content of the samples.

Data analysis
Experimental factors including MWCNTs (0, 50, 100, and 150 mg L −1 ) and white-LED light quantities (100, 200, and 400 μmol·m -2 ·s -1 ) organized into a triplicate CRD (completely randomized design). Each culture vessel contained four explants and was considered as one replicate (3 replicates in general for each treatment). A two-way factorial ANOVA using the GLM procedure and the least significant difference (LSD) test at the 0.05 probability level was carried out on the data using SAS 9.4. The PROC UNIVARIATE within SAS was used to test the assumptions of variance analysis, and residuals were normally distributed.

Growth analysis
Light intensity and MWCNTs and their interaction showed a significant difference in the number of shoots, shoot length, and fresh and dry weight of in vitro grown shoots ( Table 2). The maximum number of shoots was observed in the treatment of 50 mg L −1 MWCNTs under light intensities of 200 and 400 μmol·m −2 ·s −1 . As the concentration of MWCNTs increases, the number of shoots increases and then decreases. The main effects of light intensity and MWCNTs affected the number of leaves so that the highest number of leaves was found under the treatment of 50 mg L −1 MWCNTs and 100 μmol·m −2 ·s −1 light intensity ( Table 2). The maximum shoot length was recorded under the combination of 0 or 100 mg L −1 MWCNTs and 100 μmol·m −2 ·s −1 . The fresh and dry weight of stevia was the highest under the combination of 50 mg L −1 MWC-NTs and 200 μmol·m −2 ·s −1 (Table 2). In general, these results indicate that a combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 is the most suitable condition for in vitro growth of stevia.

Mineral uptake
The main effects of MWCNTs and light intensity and their interaction were significant on the contents of total N, P, K, Ca, Mg, and Fe in stevia (Table 3). The total nitrogen content was the highest under the combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 and 50 mg L −1 MWC-NTs and 400 μmol·m −2 ·s −1 , while under the combination of 150 mg L −1 MWCNTs and 400 μmol·m −2 ·s −1 was the lowest (Table 3). In general, Table 3 shows the positive effect of supplementation of stevia medium with a low concentration of MWCNTs (50 mg L −1 ) on the uptake of nutrients such as N, P, K, Ca, Mg, and Fe. However, with increasing the concentration of MWCNTs in the culture medium, the tendency of nutrient uptake shows a decrease. As shown in Table 3, the major absorption of the studied nutrients was greater at a light intensity of 200 μmol·m −2 ·s −1 than at the other two light intensities. Changes of Mg content at 100 μmol·m −2 ·s −1 follow a first-order linear relationship (R 2 = 0.95) (Fig. 6A). The regression plot showed that Mg content at 100 μmol·m −2 ·s −1 decreases linearly with increasing MWCNT levels so that per unit of MWCNTs, the amount of Mg decreases by 0.3085. Overall, the combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 followed by 50 mg L −1 MWCNTs × 400 μmol·m −2 ·s −1 showed a positive and statistically significant effect on the nutrients like N, P, K, Ca, Mg, and Fe. Two-way ANOVA showed that the Zn content in in vitro-grown stevia was closely related to the main effects of light intensity and MWCNTs, so that the highest content of this element was noticed at a concentration of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 (Table 3). Regression analysis for Zn at the level of 100 μmol·m −2 ·s −1 followed the second-order polynomial (R 2 = 0.99) and, based on calculations, its concentration in plant tissue was predicted by 3 mg L −1 MWCNTs (Fig. 6B). In addition, the results revealed substantial differences in the rates of Mn adsorption due to the completion of the culture medium with MWCNTs (p ˂ 0.05), light intensity (p ˂ 0.01), and their interaction (p ˂ 0.01) ( Table 3). The treatment of 0 mg L −1 MWCNTs × 400 μmol·m −2 ·s −1 resulted in maximum Mn concentration in plant tissues. Regression analysis at the level of 200 μmol·m −2 ·s −1 for Mn changes followed the second-order polynomial (R 2 = 0.99), so that the highest level of Mg was achieved at the level of 95 mg L −1 MWCNTs (Fig. 6C). On the other hand, the concentration of Cu in plant tissue affected only by light intensity (p ˂ 0.05) with a maximum value under 100 m −2 ·s −1 followed by 200 μmol·m −2 ·s −1 (Table 3). These results indicate the importance of optimal light intensity in plant nutrient uptake by plants. Based on the amount of nutrient uptake (Table 3), a combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 is recommended for the micropropagation of stevia.

Diterpene glycoside determination and total carbohydrates
The total carbohydrate and content of SVglys (rebaudioside A and stevioside) were related to the levels of MWCNTs (p ˂ 0.01), light intensity (p ˂ 0.01), and their interaction (p ˂ 0.01) (Table 4). Similarly, the highest amount of total carbohydrates and rebaudioside A was found in the combination of 50 mg L −1 MWC-NTs and 200 μmol·m −2 ·s −1 and 50 mg L −1 MWC-NTs × 400 μmol·m −2 ·s −1 . According to Table 4, the content of total carbohydrates and rebaudioside A increased with the addition of 50 mg L −1 MWCNTs to the culture medium first and then decreased with respect to control. In addition, the maximum stevioside content was also obtained under the combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 (Table 4). In this regard, supplementing culture medium with a low concentration of MWCNTs (50 mg L −1 ) was able to increase the amount of total carbohydrates and SVglys in stevia leaves under optimal light intensity (200 μmol·m −2 ·s −1 ) ( Table 4).

DNA damage evaluation
In this study, the extent of DNA damage was also evaluated in the UGT76G1 gene region and its promoter site under MWCNTs and light intensity. The UGT76G1 gene is responsible for the conversion of stevioside to rebaudioside A, which enhances the organoleptic properties of SVglys (Yang et al. 2014). Treatment of MWCNTs significantly induced DNA damage at high concentrations, although this depended on the level of light intensity (Fig. 5A, B). The promoter region of the UGT76G1 gene under the combination of 150 mg L −1 MWCNTs and 400 μmol·m −2 ·s −1 showed the most damage following the combination of 100 mg L −1 MWCNTs and 400 μmol·m −2 ·s −1 and 150 mg L −1 MWCNTs × 200 μmol·m −2 ·s −1 (Fig. 5A). The lowest DNA damage was achieved at concentrations of 0 and 50 mg  (Fig. 5A). Damage to the UGT76G1 gene sequence was also correlated with both factors. The rate of damage to this area was highest under the combination of 150 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 and 100 mg L −1 MWC-NTs × 200 μmol·m −2 ·s −1 (Fig. 5B) The lowest damage was also recorded under the treatment of 0 and 50 mg L −1 MWC-NTs under all three light intensities (Fig. 5B). As shown in Fig. 6D, regression analysis for damage to the promoter region of the UGT76G1 gene at 200 μmol·m −2 · −1 level followed a first-order linear relationship (R 2 = 0.95). Damage to this site increases linearly at 200 μmol·m −2 ·s −1 with increasing MWCNT levels so that for each MWCNT unit, it increases by 0.013 (fold) (Fig. 6D). Regression analysis at the level of 100 μmol·m −2 ·s −1 for damage to the gene sequence followed the second-order polynomial (R 2 = 0.99) so that at the level of 100 μmol·m −2 ·s −1 the lowest level of damage was noticed at the level of 57 mg L −1 MWCNTs (Fig. 6E). Therefore, it can be concluded that concentrations of more than 50 mg L −1 MWCNTs induced DNA damage in the tissue of stevia leaves, and this damage was higher under higher light intensities (200 and 400 μmol·m −2 ·s −1 ).

Discussion
In the present study, it was shown that the combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 synergistically increased shoot induction and growth of stevia (Table 2). In line with our results, Hu et al. (2021) and Joshi et al. (2018) noted the increase in biomass (fresh and dry weight) in maize (Zea mays) seedlings fed with 100 mg L −1 MWCNTs. Although the number of leaves was not significantly affected by the interaction of both factors, under the combination of 50 mg L −1 MWCNTs and 200 μmol·m −2 ·s −1 were slightly higher than other treatments (Table 2). In addition to improving photosynthesis, the increased fresh and dry weight of plantlets may be related to increased chlorophyll accumulation, stomatal conductance, and intercellular CO 2 (Hu et al. 2021). However, with increasing MWCNT levels from 50 to 400 mg L −1 , shoot proliferation and plant growth showed a decreasing trend (Table 2). Stem length under low light intensity (100 μmol·m −2 ·s −1 ) and no MWCNT application (control treatment) was longer than under other treatments. Shoot elongation, which is one of the responses to low light (Snowden et al. 2016), has been reported under low light intensity in lettuce (Johkan et al. 2012), which is in parallel with our results (Table 2). The increased uptake of nutrients such as N, Ca, S, Fe, and Zn under low levels of MWCNTs during sugarcane micropropagation has been shown by Sorcia-Morales et al. (2021). However, they have noted a decrease in N, Fe, and Zn contents under the high level of MWCNTs (200 mg L −1 ). In the present study, the application of 50 mg L −1 MWCNTs improved the adsorption of elements including total N, P, K, Ca, Mg, and Fe, compared to the control treatment (Table 3). The increase in fresh weight of the regenerants in the present study (Table 1) is an indicator of the increase in water absorption under MWCNT treatments and may be a justification for increasing the nutrient uptake. Joshi et al. (2018) studied the anatomy of wheat and reported that the priming of wheat seeds with nanotubes leads to the elongation of cells in the xylem and phloem regions, which leads to an improvement in the absorption of water and nutrients such as P and K, which taken together increases the wheat biomass and grain number. However, increasing the concentration of MWCNTs from 50 to 150 mg L −1 a decreasing trend was observed in nutrient absorption (Table 3). Decreased nutrient uptake at the high levels of MWCNTs (100 and 150 mg L −1 ) may also be due to damage to stem tissues and result in low capacity for nutrient uptake. In the present study, as we had hypothesized, the light intensity had a significant positive interaction with the MWCNTs in the absorption of the considered elements (Table 3). Nutrient uptake at higher light intensities (200 and 400 μmol·m −2 ·s −1 , respectively) was higher than at a low light intensity (100 μmol·m −2 ·s −1 ) ( Table 3). This finding may indicate the importance of optimal light intensity in nutrient uptake, especially in the greenhouse and indoor farming, to achieve optimal yield. Many studies have studied the role of light signals in the absorption and metabolism of nutrients by plants (Zhai et al. 2019;Lu et al. 2020). Despite considerable advances in nutrient uptake mechanisms and metabolism, how light interferes with nutrient uptake is not well understood (Xu et al. 2021). In the literature, the following reasons have been shown to improve nutrient uptake and increase light intensity: (i) increased sugar and carbohydrate synthesis as a result of increased photosynthesis; (ii) increased expression levels of genes encoding ion transporters and nitrate transporters (Xu et al. 2021). In our study, the highest total carbohydrate content was recorded under the treatments of 50 mg L −1 MWCNTs × 200 μmol·m −2 ·s −1 and 50 mg L −1 MWCNTs × 400 μmol·m −2 ·s −1 (Table 4). Increasing the concentrations of P and Zn, which play an important role in carbohydrate metabolism (Kumar et al. 2021), induced by MWCNTs may be a justification for increasing the content of carbohydrates in plants. Hu et al. (2021) also recommended that the improvement of carbohydrate content under MWCNT application in Zea mays seedlings is related to the increase in the activity of enzymes involved in carbon metabolism such as sucrose synthase (SS), sucrose phosphate synthase (SPS), and phosphoenolpyruvate carboxylase (PEPC). There are also results that show that excess light also has a negative effect on the element uptake. Almansa et al. (2017) reported that a light intensity of 175 W m −2 reduces the amount of N uptake by tomatoes, compared to a light intensity of 105 W m −2 . The effect of excess light on reduced plant nutrient uptake may be due to oxidative stress and damage to photosynthetic organelles (Patelou et al. 2020)  This shows that any plant species (as well as varieties and cultivars) needs to have the best lighting conditions possible.
In the present study, total carbohydrate levels and SVglys showed a positive response to the treatments of 50 mg L −1 MWCNTs × 200 μmol·m −2 ·s −1 and 50 mg L −1 MWC-NTs × 400 μmol·m −2 ·s −1 (Table 4), which is associated with the occurrence of the highest dry weight under these treatments (Tables 2 and 4). Increased carbohydrate content may be associated with increased photosynthesis (Zhou et al. 2011) induced by increased irradiance, light interception, and the role of elitist MWCNTs in plant metabolism. However, with increasing the concentration of MWCNTs from 50 to 150 mg L −1 MWCNTs, the content of carbohydrates and SVglys shows a decreasing trend (Table 4). Fig. 5A and B show an increase in damage to the promoter site and the sequence of the UGT76G1 gene, which is an important gene in the synthesis of SVglys, with increasing application of MWCNTs in the culture medium. This damage interacts with the light intensity so that under high light intensity (200 and 400 μmol·m −2 ·s −1 ) the amount of damage is greater. DNA damage has been shown in meristem cells of Allium cepa root induced by ZnO nanoparticles (Kumari et al. 2011), and in eggplant seedlings induced by cobalt oxide nanoparticles (Co 3 O 4 -NPs) (Faisal et al. 2016). As noticed by Faisal et al. (2016) and Panda et al. (2017), the degree of DNA damage is correlated with ROS induction and oxidative stress. In agreement with our findings, the results of Ghosh et al. (2011) revealed that MWCNTs could cause chromosomal aberrations, DNA fragmentation, and apoptosis in Allium cells, which were correlated with the internalization of nanotubes. On the other hand, Farrokhzad et al. (2022) during in vitro culture of Phalaenopsis showed that high light intensity alone and in interaction with silver nanoparticles at high concentrations leads to oxidative stress (increase in H 2 O 2 production and MDA content). So, the high light intensity may interact with MWCNTs by increasing the level of oxidants in DNA damage

Conclusions
The findings of this study showed that the use of MWCNTs significantly increases shoot induction, biomass yield, nutrient absorption (macro-and micronutrients), carbohydrate content, and steviol glycosides in a dose-dependent manner during in vitro cultures of stevia. However, the positive role of MWCNTs is affected by the interaction with light intensity. According to the results, the interaction of 50 mg L −1 MWCNTs × 200 μmol·m −2 ·s −1 during the micropropagation of stevia is recommended. On the other hand, increasing the concentration of MWCNTs from 50 to 150 mg L −1 increases the damage to DNA, which is intensified by increasing the light intensity from 100 to 200 μmol·m −2 ·s −1 . This study showed the role of light intensity in increasing the toxicity of MWCNTs. For more research, it is suggested to use labeled MWCNTs to monitor their translocation in the plant/tissue and if they might be used in metabolic processes to find out more about underlying mechanisms.