Silver Nanoparticles Reduce the Toxic Effects of Cadmium on Datura stramonium Callus Culture

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Introduction
Cadmium (Cd) is a nonessential metal, and its excess amount hinders normal growth and development, even causing death at high concentrations [1,2].Its danger stems from its difculty to biodegrade and long-term persistence, creating a chronic environmental threat that negatively impacts human and animal health [3][4][5].
Its negative efect on plants is represented by a decrease in photosynthesis [6], a weak ability to absorb the necessary nutrients, and an increase in the production of reactive oxygen species (ROS), which leads to damage to cellular components as a result of increased damage due to oxidative stress [7,8].
Adapting plants to resist heavy toxins is one of the most sustainable methods for removing these pollutants while preserving environmental diversity.Te integrated plant system provides a unique opportunity to preserve the environment from heavy metal contamination through its ability to withstand this toxicity and transform it into a safe biological state, which cleans the environment of toxins.Te enzymatic and nonenzymatic antioxidant systems meet this challenge [9].
Nanoparticles (NPs) are nanomaterials with a diameter ranging from 1 to 100 nm [10].Tey have unique physical and chemical properties that make them highly efective in various felds, such as agriculture, food, and industry [11,12].In addition, nano-elements contribute to the mitigation or detoxifcation of chemical pollutants produced by the action of trace metals [13].Terefore, they are one of the most promising methods to reduce the risk caused by toxic metals, including Cd [14].
Silver nanoparticles (AgNPs) have signifcant physiological potential.Tey have been widely used in resisting various abiotic stresses because they can control a plant's cell enzymatic and nonenzymatic oxidative systems.AgNPs have been reported to reduce ROS and increase the stability of cellular membranes, contributing to the increase in growth and the accumulation of biomass [15].
Te genus Datura is one of the most famous plants of the Solanaceae family and includes several important species, including stramonium.It is a rich source of biologically active chemical compounds, including various chemical components, such as alkaloids, steroids, phenols, amides, and acyl sugars [16].Tese compounds have therapeutic properties, including antioxidant, anti-infammatory, antiulcer, and analgesic [17], as well as antibacterial, antifungal, anticancer, and antiviral properties [18,19].
When searching the literature, we found no study addressing the role of AgNPs in reducing the efect of Cd metal on the tissue culture of plants.Te current study aimed to explore the contribution of laboratorymanufactured AgNPs to enhance metal tolerance and accumulation based on physiological and biochemical parameters and the bioaccumulation capacity of the D. stramonium cultures grown under cadmium stress conditions.

Plant Material and Callus Induction. D. stramonium
seeds were obtained from the Center of Desert Studies, University of Anbar.Tey were washed with water and surface sterilized with 70% ethanol for one minute.Te sterilization process was then completed with a 2.0% NaOCl solution for 15 min in a laminar fow cabinet.Ten, it was rinsed with sterile distilled water three times.Tey were cultured on MS medium-containing sucrose 30 g and agar 7.0 g free of plant growth regulators.Te seeds were incubated to obtain sterilized seedlings at 25 °C and photoperiod 16/8 h dark.For callus induction, the cotyledon of the obtained D. stramonium seedlings was grown in the MS medium with the same components and conditions indicated for seed germination with the addition of growth regulators 1.0 mg•L −1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg•L −1 α-naphthalene acetic acid (NAA), and 1.0 mg•L −1 6-benzylaminopurine (BA).

Establishment of Callus Culture.
A homogeneous callus culture was obtained from several subcultures on the MS media supplemented with the same components of the medium indicated for seed germination, taking into account the addition of growth regulators, and incubated at 25 °C in the dark.Experimental treatments were prepared by dividing fresh three-month-old calli into two groups.

Preparation of AgNPs.
AgNPs colloidal solution was prepared using the laser ablation in liquid phase (LAL) technique in DDDW.A rotating (2 cycles/min) Ag plate (1.5 × 1.5 cm) was shot by a 1064 nm laser wavelength with 6 Hz, 100 pulses, and 100 mJ in 15 mL of double deionized distilled water (DDDW).Te height of water above the Ag billet surface was 8.0 mm.However, the color appearance of the colloidal material changed from transparent to a yellowish liquid.Te fask was covered by foil to protect it from light.Te estimated concentration of Ag in the colloidal was about 20 ppm.

Characterization of AgNPs.
Te primary test of AgNPs to confrm the formation of nanoparticles was the measurement of the UV-Vis absorbance to determine the surface plasmon resonance peak using a PG80 UV-Vis spectrophotometer with wavelengths from 300 to 800 nm.Te concentration of Ag was estimated using atomic absorption spectroscopy (Perkin-Elmer, 300), and the topographical properties were determined by transmission electron microscopy (TEM) analysis.Te surface plasmon resonance peak was located at a wavelength of 410 nm (Figure 1), which is in the reported range [20,21].
Te formed AgNPs appeared as spherical particles, as shown in the TEM image in Figure 2 (2.5 to 28 nm of the size range with an average of 9 nm).

AgNPs Treatment.
A group was grown on the media with diferent levels of Cd, including 0, 150, 300, 450, and 600 μM, which were performed by adding CdCl 2 to the media solution.Te second group was treated with the same Cd concentrations, and 100 μg•L −1 of AgNPs was added to the MS medium.Te callus was harvested after 28 days and analyzed.

Measurements of Parameters. Te studied parameters
were calculated for all treatments after 28 days of cultures in the previously mentioned conditions; these traits included the following: 2.6.1.Fresh and Dry Weight.Te fresh weight (FW) of callus samples was determined after separating them from the culture medium and removing the remnants of the media attached to the callus.Te dry weight (DW) was calculated after drying the samples in an oven at 50 °C for 72 h.

Relative Water Content.
Te relative water content (RWC) of the callus was calculated for all treatments based on the following formula: where (TW) is the weight of the fresh callus immersed in distilled water at room temperature for 4 h in the growth chamber under constant light.

Total Chlorophyll Content.
Total chlorophyll (Chl.T ) was determined using 250 mg callus samples according to the method proven by Lichtenthaler [22].Briefy, the samples were centrifuged to obtain the extract using an 80% (v/v) acetone solution at 2500 rpm for 10 min.Ten, the 2 International Journal of Agronomy (2) Te report of Sergiev et al. [23] was followed to estimate the hydrogen peroxide (H 2 O 2 ) content of the callus samples.Briefy, it was centrifuged at 12,000 rpm for 15 min to produce 500 mg of homogeneous callus tissue supplemented with 0.1% TCA.Ten, 500 μl of the supernatant was added to 500 μl of potassium iodide (1 M) and 500 μl of phosphate bufer (10 mM).Te absorbance of the solution was recorded at 390 nm.

Determination of Enzyme Activities.
For the purpose of extracting antioxidant enzymes, a report was based on Gapinska et al. [24].In short, 500 mg of fresh callus were homogenized in 50 mM K-phosphate bufer (pH 7) containing 1 mM EDTA and 1% PVP.After centrifugation (10,000 × g, 15 min, 4 °C), the supernatant fraction was used to determine enzyme activities.

Catalase (CAT).
Te CAT activity was determined based on the Aebi [25].In short, 15 mM of H 2 O 2 was mixed with 50 mM K-phosphate bufer at pH 7.0 and 25 °C with 50 μl of enzyme extract.Te absorption at 240 nm for 30 s was achieved using 39.4 mM −1 •cm −1 as an extinction coefcient.

Guaiacol Peroxidase (GPX).
Te GPX activity was quantifed by adding 10 mM of guaiacol, 50 mM of phosphate bufer, and 40 mM of H 2 O 2 .Te assay, using 2.5 ml of the reaction mixture and 75 μl of the enzyme extract at 470 nm for 3 min, was determined, and the extinction coefcient (25.5 mM −1 •cm −1 ) was used to calculate enzyme activity [26].
2.6.8.Accumulation of Cd.Te Cd concentration (Cd con.) in 100 mg dried callus samples was estimated for acid digestion according to the method modifed by Hernández Arteaga et al. [27].Briefy, 10 ml of a mixture of hydrochloric acid (HCl) and nitric acid (HNO 3 ) in a ratio of 1 : 3 was added to the homogenized sample in a hot plate stirrer at 100 °C.Te product was diluted with volumetric 10 ml deionized water.Te sample was analyzed by atomic absorption spectrometry.
2.6.9.Tolerance Index.Te tolerance index (TI) of biomass after exposure to Cd metal was calculated as follows [28]:

Results
Te growth of the D. stramonium calli culture was negatively afected by increasing Cd treatment, which caused all growth characteristics to decrease signifcantly.Te higher concentration of Cd treatment of 600 μmol reduced these characteristics compared to the control treatment by about 29.64%, 24.32%, 22.48%, and 54.06% for FW, DW, RWC, and Chl.T , respectively.However, a signifcant improvement of these parameters was observed once treated with a concentration of 100 μg•l −1 of AgNPs, to reduce that efect at the same concentration to 7.88%, 11.53%, 9.72%, and 16.63%, respectively (Figures 3 and 4).
To verify the efect of Cd concentrations on membrane damage, H 2 O 2 was monitored.Te Cd-treated cultures showed a continuous increase in the mean values with the increase in the treatment concentration.Te increase reached 1.7-fold for the 600 μM concentration compared to the control treatment.Relative stabilization occurred in the level of H 2 O 2 with treatment with AgNPs (Figure 5(a)).
Tis stability may be due to the increase in the activity of CAT and GPX enzymes, as treatment with AgNPs contributed signifcantly to this increase.Treatment with a high level of Cd with AgNPs achieved the highest enzyme activity with an increase of 2.4-and 1.6-fold compared to the control not treated with silver particles (Figures 5(b) and 5(c)).
Cd treatment showed an increase in the metal concentration in callus cultures with increasing treatment, reaching its highest level for the tissue cultures treated with a concentration of 600 μM.Te pretreatment of AgNPs allowed increasing Cd in callus cultures under low concentrations of 150 and 300 μM.At the same time, the nanomaterial contributed to reducing the tissue Cd content under high concentrations of 450 and 600 μM (743.3 and 846.7 mg/kg DW) compared with untreated counterparts with nanoparticles (Table 2).6 International Journal of Agronomy Despite the continuous decrease in the tolerance to the efects of Cd treatment by callus culture, the pretreatment of callus culture of D. stramonium with AgNPs showed a signifcant tolerance towards Cd.Te prominent role of pretreatment with AgNPs was increasing the tolerance of plant tissues to this stress by 2.20%, 14.65%, 13.18%, and 6.96% more than their counterparts untreated with nanoparticles, respectively.
In this study, a prominent role was observed for the treatment with AgNPs in the phytoremediation of Cd metal.At low levels of 150 and 300 μM, phytoremediation of 2.24 and 1.939 were found, respectively.However, Cd levels of 450 and 600 μM did not difer signifcantly from the lowconcentration products of the untreated Cd treatments (Table 2).

Discussion
In this study, although Cd improved the parameters of FW and DW under concentrations 150 and 300 μM, the negative efect of this mineral was evident at concentrations 450 and 600 μM.At the same time, Cd metal concentrations caused a signifcant reduction in physiological parameters, including RWC and Chl.T .Te inhibition of Cd for the mentioned indicators could be due to its efectiveness in preventing the elongation and division of cells that occur due to the inhibition of the proton pump [30,31].Moreover, the concentration of Cd caused a decrease in the water absorption of calli cultures due to the decrease in the osmotic potential in the media.Also, the efect of Cd on the damage International Journal of Agronomy of pigments, including chlorophyll pigment, was negatively refected in the growth of calli cultures [32].Tis result agreed with previous reports on diferent plant species such as Brassica juncea [28], Brassica napus L. [33], and Plantago major [34].
Te current study showed the positive role of AgNPs in enhancing growth parameters, such as FW and DW.Tis may be due to the improvement of RWC, which is the main factor in its main role in the formation of polysaccharides [10].As well as improving the metabolic and physiological 8 International Journal of Agronomy state of calli cultures through the role of AgNPs in maintaining the hormonal balance negatively afected by stress factors.Previous literature indicated that AgNPs enhance the formation of essential pigments such as chlorophyll as the basis for photosynthesis and thus improve the rate of photosynthesis [35].Tis result is similar to that reported by Ali et al. [15] on Caralluma tuberculata and de Andrade et al. [36] on Helianthus annuus L. and.
In this study, treatment with Cd signifcantly increased ROS generation, leading to increased H 2 O 2 content in calli cultures.Tis contributes to the damage of various cellular components, including proteins, lipid membranes that form cell walls, and nucleic acids [37].Te activity of the antioxidant defense enzyme systems represented by CAT and GPX represents one of the defense mechanisms that can reduce the risk of oxidative stress and thus protect plant tissues.Tese results are similar to previous reports that showed the negative efect of H 2 O 2 in decreasing cell viability, such as Daud et al. [38] in Gossypium hirsutum L. Also, previous literature showed a progressive activity in H 2 O 2 degradation by enzymatic antioxidants [39][40][41][42][43].
Te results of the current study showed that AgNPs had an efective role in protecting the plant cell from the negative efect of Cd stress, as they limited the increase of H 2 O 2 by increasing the antioxidant enzymatic activities represented by CAT and GPX.Tis can be explained by the fact that AgNPs stimulate the vital pathways responsible for building antioxidant enzymes.Moreover, the role of AgNPs in the production of secondary metabolites has been studied [44].Te antioxidant activities contribute to reduce the efect of ROS by neutralizing the superoxide radicals (O 2 ) [45].Te results showed an increase in the activity of CAT treated with AgNPs with an increase in the level of stress induced by Cd.On the contrary, GPX activity was balanced with all levels of Cd, which may be explained by the convergence of mean values of GPX activity untreated with AgNPs.Tese results are similar to the results of previous studies that showed the role of the antioxidant enzyme system in counteracting various environmental infuences [15,46].
Te accumulation of Cd, Ti, and BCF was estimated to verify heavy metal uptake by plant tissues and bioconcentration behavior [47].Ti and BCF can be used to determine plant potential for phytoremediation purposes [48].From the data, the mean values of BCF in Cd+ 100 treatment levels of AgNPs ranged from 1.528 to 2.24, which is indicative of signifcant bioaccumulation of Cd in plant tissues, which indicates a strong tolerance against heavy metals despite the strong accumulation compared to the control treatment of the nanomaterial [49].In general, pretreatment with AgNPs led to a signifcant increase in the accumulation of Cd with an increase in the tolerance of Ti and BCF, thus contributing to the improvement of growth parameters for the efciency of Cd phytoremediation.

Conclusions
Tere is an urgent need to understand the mechanisms by which plant cells can tolerate the efects of oxidative stress caused by trace metals, including Cd. Terefore, this study suggests a synergistic relationship between the parameters that would qualify the plant to play a role in phytoremediation.Te current article showed that the inclusion of Cd in the cultivated media harmed most of the study parameters.However, it was found that the external application of AgNPs synthesized in vitro signifcantly improved all parameters through their stimulating efect of the antioxidant enzymatic system, which preserved the integrity of the cells.Moreover, the increase in the activity of the CAT and GPX enzymes contributed to regulating various physiological processes, which was positively refected in the growth of callus culture.Tus, this contributes to prevent the risks of oxidative stress caused by Cd.Tis is the frst study investigating the ability of AgNPs to mitigate Cd stress on the tissue cultures of plants, thus determining their role in plant bioremediation.Terefore, further studies can be suggested to understand the chemistry of compounds and food safety.International Journal of Agronomy 9

Figure 2 :Figure 1 :
Figure 2: AgNPs treatment: (a and b) TEM image of the prepared AgNPs; (c) the size distribution of particles.

Figure 5 :
Figure 5: Te efect of Cd treatment and AgNPs on: (a) H 2 O 2 content; (b) CATactivity; and (c) GPX activity of D. stramonium callus culture after 28 days.Values represent the mean ± SE; diferent letters indicate a signifcant diference between the treatments (p < 0.05, Duncanʼs test).
Experimental Design and Statistical Analysis.Te experiment was conducted in a completely randomized design (CRD).Te data were analyzed for three replicates using a two-way ANOVA to analyze variance (Table1).Te experimental error value (±SE) was calculated based on the mean values of the three replicates.Duncanʼs multiple range test (DMRT) was conducted to determine the diferences between the mean values of the statistically signifcant coefcients (p ≤ 0.05).

Table 2 :
Te efect of Cd treatment and AgNPs on Cd accumulation, Ti, and BCF of D. stramonium callus culture after 28 days.Values represent the mean ± SE; diferent letters indicate a signifcant diference between the treatments (p < 0.05, Duncanʼs test).