Introduction

Water deficit, as one of the most significant environmental stresses, has been negatively affecting the productivity of plants universally, and it hinders plants to reach their full yield potential (Mozafari et al. 2019). This type of stress is normally more serious in arid and semi-arid locations, but it might also be the case in other parts where periodic lack of rain is observed (Pirasteh-Anosheh et al. 2016). Polyethylene glycol (PEG) used to simulate the water stress in laboratory conditions has been used in numerous studies to assess the effect of drought stress on plants. PEG-induced water stress involves inadequacy of the moisture required for plant growth and development (Riasat et al. 2019). Water shortage can cause a range of chain reactions from transcriptome changes to different phenotypic responses. Therefore, drought stress is a complex issue involving molecular, biochemical, and physiological responses, and alters signaling cascades and pathways in plants (Ghaderi et al. 2015; Basu et al. 2016; Mozafari et al. 2018a, b; Zandalinas et al. 2018). Different plant hormones such as jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA) and particular metabolites including reactive oxygen species (ROS) are key components on plant defense signal transduction pathways that react synergistically and/or antagonistically (signaling crosstalk) to modulated stress-induced adverse effects (Verma et al. 2016). A significant horticultural plant, strawberry (Fragaria × ananassa Duch.) is a small fruit susceptible to short intervals of water shortage mainly due to its shallow root system (50 to 90% of the root is located within 0 to 15 cm from the soil surface) and large leaf area (Klamkowski and Treder 2008).

Nutritional balance is perturbed under water stress (Servani et al. 2014), causing problems in photosynthesis, chlorophyll formation, cell wall development, respiration, vascular permeability, and activity of enzymes involved in plant synthesis (Hsiao et al. 1976). Different studies have confirmed the suitability of exogenous application of nutrients as a strategy adopted to alleviate the negative effects of drought (Sharma et al. 2019). Iron is categorized as a micro-nutrient in plants, but it is a key element in cell metabolism, and is involved in photosynthesis (playing a role in biosynthesis and chlorophyll activation), respiration, and enzyme activity (Rout and Sahoo 2015). Although not readily absorbable in its ordinary form due to low solubility, iron is used in nano-fertilizer forms, where it can be considered as a resolution. A nano-fertilizer is capable of making nutrients available to plants in a gradual, controlled manner. Furthermore, nanotechnology enhances fertilizer efficiency, and reduces soil contamination and environmental hazards caused by conventional fertilizers (Manjunatha et al. 2016). Therefore, iron nano-particles, which are smaller than iron oxide, can make iron particles more readily available to plants (Liu and Lal 2015).

Plant tissue culture has been used widely as a great technique for producing healthy and vigorous plants under laboratory conditions, and is now a vital tool in modern agriculture (Manjunatha et al. 2016). In vitro cultivation of explants is considered as a success in strawberry micro-propagation and production of healthy, virus-free plants (Soundararajan et al. 2013). In the current study, therefore, the response of strawberry explants to PEG-induced water stress under the application of iron nano-particles was assessed using different morpho-physiological and biochemical characteristics.

Materials and Methods

Plant Materials

Strawberry (Queen Elisa cultivar) shoot explants were cultured vertically in sterile jars containing Murashige and Skoog (MS, Murashige and Skoog 1962) medium, as in vitro-raised plants, in a laboratory. All the media were supplemented with 3% sucrose, 2 mg L−1 benzyladenine (BA), and 0.01 mg L−1 indoleacetic acid (IAA) that were provided from Merck Co. (Darmstadt, Germany). For the treatments, the corresponding compounds in specific concentrations of polyethylene glycol (PEG 6000, Merck) were added to basal MS media. Before the autoclaving process (for 20 min at 121.5°C and 1.3 bar), the medium pH was adjusted to 5.8 using HCl or NaOH (Bichem and Biotech Co, Tehran, Iran). Due to the presence of PEG 6000, an agar-containing medium could not be solidified, so synthetic cotton (Pristive Co, Tehran, Iran) (0.5 g per jar) was used instead of agar. For the establishment of in vitro cultures, 45 mL of each prepared medium was decanted into 250-mL glass jars (experimental units). The temperature of the growth chamber (MTR30, Conviron, Toronto, Canada), where the jars were kept, was maintained at 25 ± 1°C under 16-h/8-h (day/night) photoperiod conditions (42 ΜE m−2 s−1), along with a relative humidity of 55 ± 3%. Polyethylene glycol was used at concentrations of 0, 5%, and 7% w v−1 for PEG-induced water stress treatments. The experimental treatment factor included iron sulfate (FeEDTA (Na2EDTA = 37.3 mg L−1 and FeSO4·7H2O = 27.8 mg L−1) in a concentration of 27 mg L−1, used as control, and iron nano-particles (35 to 48 nm) in the form of Fe3O4 (Nanozino, Tehran, Iran), coated with l-cysteine (Sigma-Aldrich, St. Louis, MO) in concentrations of 1 and 1.2 mg L−1). Each treatment was repeated 9 times (9 jars per treatment and 3 explants per jar). Fully expanded leaves were used for trait measurement. The experiment was continued for 59 d.

Morphological Traits

After the plants were separated from the media, root fresh weight, shoot fresh weight, and the dry weights were measured using plant samples held in an oven (Noor Sanat Ferdows, Yazd, Iran) at 70°C for 48 h.

Physiological Traits

Relative water content (RWC) was calculated for the leaves based on the procedure proposed by Galmés et al. (2007), in which fresh weight (FW), turgid weight (TW), and dry weight (DW) were specified. RWC was finally reported as a ratio (in percent) of the above measured weights based on the following formula.

$$\mathrm{RWC}\ \left(\%\right)=\frac{\mathrm{FW}-\mathrm{DW}}{\mathrm{TW}-\mathrm{DW}}\times 100$$

The membrane stability index (MSI) was assessed based on Sairam, Rao, and Srivastava’s (2002) method. In this method, the electrical conductivity (EC) of the water in bath tubs containing leaf samples was measured after 30 min at 40°C (C1) and after 10 min at 100°C (C2) using the EC meter (RS232 conductivity meter 8302). The final value of MSI was reported after the application of the following formula.

$$\mathrm{MSI}\ \left(\%\right)=\left[1-\left(\mathrm{C}1/\mathrm{C}2\right)\right]\times 100$$

The pigment contents, consisting of total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid, were evaluated based on the method published by Lichtenthaler and Buschmann (2001). Accordingly, acetone and magnesium oxide were used for extraction of pigments, and their contents were then measured spectrophotometrically (UV-2100 model SUV, Paterson, NJ) at wavelengths of 470 nm (chlorophyll a), 646 nm (chlorophyll b), and 663 nm (carotenoid).

$$\begin{array}{l}\begin{array}{c}{\mathrm{Chl}}_{\mathrm a}\;\left(\mathrm{mg}.\mathrm g^{-1}\right)=\left(12.25\times{\mathrm A}_{663}\right)-\left(2.79\times{\mathrm A}_{646}\right)\\{\mathrm{Chl}}_{\mathrm b}\;\left(\mathrm{mg}.\mathrm g^{-1}\right)=\left(21.5\times{\mathrm A}_{646}\right)-\left(5.1\times{\mathrm A}_{663}\right)\end{array}\\{\mathrm{Ch}}_{\mathrm{total}}\;\left(\mathrm{mg}.\mathrm g^{-1}\right)={\mathrm{Chl}}_{\mathrm a}+{\mathrm{Chl}}_{\mathrm b}\\\mathrm{Carotenoid}\;\left(\mathrm{mg}.\mathrm g^{-1}\right)=\left(\left(1000\times{\mathrm A}_{470}\right)-\left(1.8\times{\mathrm{Chl}}_{\mathrm a}\right)-\left(85.02\times{\mathrm{Chl}}_{\mathrm b}\right)\right)/198\end{array}$$

Biochemical Traits

The method described by Irigoyen, Einerich, and Sánchez-Díaz (1992) was used for assessment of total soluble sugar (TSS). The method proposed by Bates et al. (1973) was applied for the measurement of free proline contents in the leaf samples. The malondialdehyde (MDA) content was measured as a marker of lipid peroxidation, based on Liu and Huang (2000), using trichloroacetic acid (TCA) and thiobarbituric acid (TBA) as extractors (Nanozino), and light absorbance was then obtained after extraction at 532, 450, and 600 nm. Along the same lines, trichloroacetic acid (TCA) was used along with a potassium phosphate buffer (pH = 7) and potassium iodide (KI), based on Alexieva et al. (2001), for spectrophotometric measurement of the hydrogen peroxide (H2O2) content (390 nm). Total soluble protein (TSP) was measured at a wavelength of 595 nm according to Bradford’s (1976) method. The activity of the superoxide dismutase (SOD) and peroxidase (POD) enzymes was specified using the methods described by Dhindsa et al. (1981) and Hemeda and Klein (1990) at 560 and 470 nm, respectively (change in absorbance after 120 s).

Statistical Analysis

The current study involved the application of a two-way factorial experiment (drought and iron nano-particles) arranged in a completely randomized design (CRD) with nine replications. Three levels of PEG-induced water stress (0, 5%, and 7% w v−1 of PEG 6000) and three levels of iron nano-particles (0, 1, and 1.2 mg L−1) as treatments were applied in this study. The Agricolea library, in R 3.5.2, was applied for analysis of variance (ANOVA) and comparison of means according to Duncan’s multiple-range test (0.05). Pearson’s correlation coefficient was employed for the generation of correlation plots using the ggcorrplot library, principal component analysis (PCA) was made based on the Euclidean distance, and loading plots were constructed using the factoextra library, in R.

Results

Morphological Traits

The morphological traits experienced negative trends in response to PEG-induced water stress (Table 1). Root fresh weight (RFW), root dry weight (RDW), shoot fresh weight (ShFW), and shoot dry weight (ShDW) amounted to their lowest mean values for maximum PEG-induced water stress (7% PEG), while their means were not significantly different from the application of 5% PEG. The control conditions concerning PEG-induced water stress (0 PEG) exhibited mean values in an approximate range of 25% (ShFW) to 65% (RFW), higher than their medium levels. The application of iron nano-particles had positive impacts on the morphological traits, all of which (including RFW, RDW, ShFW, and ShDW) were significantly higher in the iron nano-particle treatments than in the control treatment (with no iron nano-particles). The application of 1 mg L−1 and 1.2 mg L−1 iron nano-particles resulted in no significant difference in any of the morphological traits. The interaction effect of PEG-induced water stress and iron nano-particle application indicated that the absence of drought along with the application of 1.2 mg L−1 iron nano-particles resulted in the highest mean values of growth-related and morphological traits, while the maximum drought level (7% PEG) without application of iron nano-particles brought about the lowest mean values for these traits.

Table 1. Mean comparison for morphological traits under polyethylene glycol (PEG)–induced water stress and application of iron nano-particles in Fragaria × ananassa Duch. plants
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The application of iron nano-particles changed the response of strawberry plants to drought-induced treatments, making them relatively tolerant of the stress, given the morphological traits. Therefore, a comparison was made using correlation plots between the application and non-application of iron nano-particles in terms of the relationships between the morphological traits, independently for each set of conditions (Fig. 1). The coefficients of correlation between RFW and ShDW and those between RDW and ShDW were not significant (P = 0.05) under the control conditions (no iron nano-particle application), while they were significant under the iron nano-particle application conditions. It should be noted that no change in the signs of the correlations was observed in the comparison of the two conditions. The result was verified using principal component analysis (PCA), which was implemented independently for each set of conditions of iron nano-particle application (Fig. 2). The first components (PC1) accounted for 81% and 95% of the variance, and the second ones (PC2) explained 18% and 4% under the absence and presence of iron nano-particle application, respectively. ShDW exhibited an acuter angle with ShFW without iron nano-particle application, while acuter angles were observed with RFW and RDW under iron nano-particle application.

Figure 1.
figure 1

Comparing correlation coefficients between control (a) and iron nano-particles (b) application on Fragaria × ananassa Duch. plants according to morphological traits (cross sign indicates non-significant correlation; alpha = 0.05).

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Figure 2.
figure 2

Comparing relationship of physiological traits in control (a) and iron nano-particle (b) application on Fragaria × ananassa Duch. plants according to principal component analysis.

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Physiological Traits

Figure 3 shows the relationships between RWC and MSI and the applied iron nano-particles under different levels of PEG-induced water stress. The interaction effect of drought and iron nano-particles for RWC indicated rising trends in response to iron nano-particle application under all the drought levels. Upon the application of iron nano-particles, no stress treatment experienced a steeper slope than the other two PEG-induced water stress levels. Moreover, no significant difference was observed between 1 and 1.2 mg L−1 iron nano-particles for medium (5% PEG) and maximum (7% PEG) drought treatments. Under all the levels of iron nano-particle application, no PEG-induced water stress conditions exhibited a higher RWC than the other two levels. Similar responses were observed for MSI, but the slopes of the regression lines representing the MSI response to iron nano-particle application were not significantly different under different drought levels.

Figure 3.
figure 3

Response of relative water content (RWC) (a) and membrane stability index (MSI) (b) to polyethylene glycol (PEG)–induced water stress under application of iron nano-particle on Fragaria × ananassa Duch.

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The pigment contents consisting of chlorophyll a, chlorophyll b, chlorophyll a+b, and carotenoid were significantly higher in absence-of-stress conditions than in stress conditions (Table 2). The application of 1.2 mg L−1 iron nano-particles resulted in greater contents for all the measured pigments than under the other levels (0 and 1 mg L−1). For all the pigments, the differences between the medium levels of iron nano-particles (1 mg L−1) and the control levels were significant, where the lowest contents were observed in the 0 mg L−1 treatment.

Table 2. Mean comparison for plant pigments under polyethylene glycol (PEG)–induced water stress and application of iron nano-particles in Fragaria × ananassa Duch. plants
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Correlation plots are outlined in Fig. 4 for comparison between presence and absence of iron nano-particle application. Chlorophyll b content exhibited significant positive correlations with RWC, MSI, and chlorophyll a content under iron nano-particle application, but the correlations were not significant under the control conditions (p = 0.05). There were also altered correlations in response to iron nano-particle application with respect to those involved in its absence. Along the same lines, the scree and loading plots concerning PCA exhibited different percentages of variance explanation by the PCs and different angles between the physiological variables (Fig. 5). The first two PCs accounted for 93% (PC1 = 82% and PC2 = 11%) and 94% (PC1 = 88% and PC2 = 6%) of the variance in the absence and presence of iron nano-particle application, respectively. The loading plots clearly indicated varying trends among the physiological traits under iron nano-particle application and the control conditions, where the greatest changes were observed for chlorophyll b content.

Figure 4.
figure 4

Comparing correlation coefficients between control (a) and iron nano-particle (b) application on Fragaria × ananassa Duch. plants according to physiological traits (cross sign indicates non-significant correlation; alpha = 0.05). RWC, relative water content; MSI, membrane stability index, chlo a, chlorophyll a; chlo b, chlorophyll b; chlo T, chlorophyll (a+b).

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Figure 5.
figure 5

Comparing relationship of physiological traits in control (a) and iron nano-particle (b) application on Fragaria × ananassa Duch. plants according to principal component analysis.

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Biochemical Traits

PEG-induced water stress led to higher activity in the SOD and POD enzymes, where the highest and lowest activity was recorded for 7% PEG (i.e., maximum drought) and the control conditions (Fig. 6). The differences between the three levels of PEG application were significant for both SOD and POD. The use of iron nano-particles in the experiment increased the activity of SOD and POD, except for POD activity in the absence of PEG application, where no significant difference was observable between the iron nano-particle levels. Moreover, PEG-induced water stress brought about a greater production of H2O2 and free proline contents. The application of iron nano-particles caused a decline in H2O2 generation, along with an increase in proline production. The higher the iron nano-particle concentration, the lower the H2O2 and the higher the proline contents. Total protein content decreased as the PEG-induced water stress level rose (Table 3). Iron nano-particle treatment raised protein production under all the PEG-induced water stress levels. TSS and MDA generation was increased by the higher PEG-induced water stress than in the control conditions. On the contrary, TSS and MDA responded differently to iron nano-particle application. Higher concentrations of iron nano-particles motivated higher TSS but lower MDA in strawberry plants.

Figure 6.
figure 6

Response of superoxide dismutase (SOD), peroxidase (POD), hydrogen peroxide (H2O2), and proline to polyethylene glycol (PEG)–induced water stress under application of iron nano-particle on Fragaria × ananassa Duch.

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Table 3. Mean comparison for biochemical traits under polyethylene glycol (PEG)–induced water stress and application of iron nano-particles in Fragaria × ananassa Duch. plants
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The correlation between the biochemical traits for presence and absence of iron nano-particle application was plotted for comparison (Fig. 7). TSS exhibited significant correlations with protein, SOD, POD, and H2O2 under iron nano-particle application, but the corresponding correlations were not significant under the control conditions. Moreover, the correlations of H2O2 with protein content and SOD were significant under iron nano-particle application but not under the control conditions. The other correlations were somewhat altered in the comparison between the two conditions. The influence of iron nano-particle application on the relationships between the biochemical traits was more tangible in their comparison on the loading plots of PCA, as given in Fig. 8. The first PC explained 74% and 81% of the variance for control and iron nano-particle application, respectively, while the second accounted for the same percentage of the variance (12%) for the two. Changes were clearly observed on the loading plots in the angles between the proline and H2O2 contents and almost all the other biochemical traits of strawberry plants. H2O2 and MDA were closer together in the presence of iron nano-particle application than in its absence.

Figure 7.
figure 7

Comparing correlation coefficients between control (a) and iron nano-particle (b) application on Fragaria × ananassa Duch. plants according to biochemical traits (cross sign indicates non-significant correlation; alpha = 0.05). SOD, superoxide dismutase activity; POD, peroxidase activity; H2O2, hydrogen peroxide; MDA, malondialdehyde; TSS, total soluble sugar.

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Figure 8.
figure 8

Comparing relationship of biochemical traits in control (a) and iron nano-particle (b) application on Fragaria × ananassa Duch. plants according to principal component analysis.

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Discussion

Mean comparison for the morphological traits in the current study demonstrated that PEG-induced water stress significantly decreased RFW, RDW, ShFW, and ShDW, while the application of iron nano-particles compensated for the negative effect of the stress on strawberry plants. Comparison was made between the presence and absence of application of iron nano-particles using correlation plots and PCA. The results of the advanced statistical methods indicated that the relationships between the morphological traits changed as a response to the application of iron nano-particles. For the morphological traits examined in this study, the difference between high levels (1.2 mg L−1) and medium levels (1 mg L−1) of iron nano-particle application was not significant; therefore, is seemed to be appropriate up to a point to apply medium levels of iron nano-particles, approximately at 1 mg L−1, to compensate for the negative effect of PEG-induced water stress on strawberry plants.

The most substantial observable effect of drought stress is declined growth and lower mass production (Yaghubi et al. 2016). According to Hsiao et al. (1976), delay in growth is the first step taken in response to water shortage in plants. There are also numerous studies that have indicated decrease in the growth and mass production of plants in conditions of PEG application and drought simulation under in vitro conditions (Masoabi et al. 2018, Razavizadeh et al. 2019). As a result of either unavailability or inaccessibility of water due to low water potentials, water shortage leads to osmotic stress in plant cells, contributing to higher production of deleterious compounds, such as reactive oxygen species (ROS), and suppression of both respiration and photosynthesis (Saed-Moucheshi et al. 2014a). The reduced rate of photosynthesis and increased demand for photosynthetic compounds, along with the requirement for osmo-regulatory, inhibit growth parameters (Iqbal 2018). The results of this study in regard to the impact of PEG-induced water stress on morphological parameters are consistent with those of previous research on strawberries (Klamkowski and Treder 2008) and other horticultural plants (Singh et al. 2013).

The application of amendments for increasing the tolerance of plants to stresses has long been an important research topic. Among the available amendments, micro-nutrients are very effective nutrients necessary for metabolic activities due to their low requisition in plants, contributing to different aspects of plant growth (Govindaraj et al. 2011). As a micro-nutrient, iron is involved in photosynthesis and the activity of certain enzymes such as phosphoenolpyruvate carboxylase and ribulose diphosphate carboxylase (Roosta et al. 2018). Under stressful conditions, the activity of different enzymes on the photosynthetic pathway is declined; as a result, availability of iron can be of great resolution to increase photosynthesis rate and retain plant growth. The results of the current study clearly indicated that the application of iron, in the form of a nano-oxide, increased the tolerance of strawberry plants and their growth parameters under PEG-induced water stress conditions. The importance of the compound was demonstrated through the observation of changes in the relationships among the growth parameters under iron nano-particle application as opposed to the control conditions. Similar results have been reported in regard to the application of iron for improvement of plant tolerance to drought stress (Devadasu et al. 2019).

As two important indices for drought tolerance in plants (Mozafari et al. 2018a, b), RWC and MSI were significantly reduced in response to PEG application (PEG-induced water stress). This is consistent with the results of Ghaderi et al. (2015), in which drought stress reduced RWC and MSI in all the three utilized cultivars of strawberry. The findings also indicated that both of the traits responded positively to application of iron nano-particles, and their contents rose as the concentration of the iron nano-particle increased. The pigment content of strawberry plants decreased through PEG-induced water stress, but the application of iron nano-particles reduced the negative impacts of PEG-induced water stress on the pigments. Application of iron in nano form, which is easy to absorb and transform in plants, can expand the content of osmo-regulators, maintaining cell turgor and increasing RWC (Mozafari et al. 2018a, b). Maintenance of the relative water content of plant cells directly improves the cell wall structure and membrane stability (Mozafari et al. 2019), which is in line with the results of the current study, in which iron application increased MSI in strawberry plants with respect to the value in the control conditions. Similar studies have also reported higher RWC and MSI in plants under treatment with iron (Metwally et al. 2018). As a cofactor on the photosynthetic apparatus and electron pathways, iron is an important element in the specification of photosynthesis rate and pigment structure. Application of iron in nano form can readily contribute to the photosynthesis mechanism and improve functionality, as it is easy to use and move (Mozafari et al. 2018a, b). The increase observed in this study in pigment contents as a result of the iron application is in line with that encountered in other research on different plants (Askary et al. 2017). Moreover, the effectiveness of iron nano-particle application on the physiological traits asserted in this study was brought about by changes in the relationships among the traits as a response to the application of iron nano-particles.

PEG-induced water stress increased SOD and POD activity, proline content, TSS, H2O2, and MDA, while decreasing total protein content. Except H2O2 and MDA, the above biochemical traits increased in response to the iron nano-particle application. A number of studies have demonstrated that iron application under stressful conditions can stimulate mechanisms by which H2O2 is relatively detoxified along with other radicals (Saed-Moucheshi et al. 2013), maintaining cell membrane integrity under drought stress. These mechanisms are involved in enzymatic and non-enzymatic antioxidant activities (Saed-Moucheshi et al. 2014a). Diaz-Mendoza et al. (2016) reported that drought stress induced the gene expression of a protease that degraded different types of protein, and reduced TSS and total protein, which is in line with the results of this study. It has also been reported in consistence with the current results that iron application increased TSS content in different plants (Nemati Lafmejani et al. 2018).

Evolutionary events cause plants to implement antioxidant defense systems to cope with different environmental stimuli (Saed-Moucheshi et al. 2014b). This category of systems consists of enzymatic antioxidants such as SOD and CAT (Caverzan et al. 2016). Enzymatic barriers opposing drought stress require microelements as cofactors to improve their functionality (Kim et al. 2013). Iron nano-particles directly improve these defense systems both by contributing in enzymatic and non-enzymatic antioxidants (carotenoids) and by increasing the plant capability of producing the required compounds through improvements made in photosynthesis rate (Mozafari and Ghaderi 2018). Iron application in nano form involves even more merit than the application of the common form of iron, including easy movement and absorption. The suitability of iron in the form of a nano-oxide was demonstrated in this study using advanced statistical methods, where changes were made through application of iron nano-particles in the relationships among the biochemical traits. These particles reduce the effects of drought stress on growth parameters. With increase in the concentration of iron nano-particles, the effects of drought stress on growth parameters were moderated efficiently.

Conclusion

In this study, the suitability of iron nano-particles in the induction of drought stress tolerance in strawberry plants was confirmed through comparison of measured traits, and these results were confirmed by the assessment of the relationships among these traits. Moreover, the results indicated that the use of iron in the form of a nano-particle provides a plausible method of coping with the negative effects of PEG-induced water stress in strawberry production. It was also demonstrated that a high concentration of iron nano-particles is not obligatory, and a medium-level application of this element (approximately 1 mg L−1) suffices to improve tolerance in strawberry culture under in vitro conditions.