Introduction

The genus Achillea is a member of the Asteraceae family and has around 130 perennial species that survive in arid and semi-arid ecosystems in temperate areas (Patocka and Navratilova 2019). Achillea fragrantissima (Forssk.) Sch. Bip (also known as Qaysoom in Arabic and lavender cotton in English) is one of these species and is considered a major element in Middle Eastern folk medicine (Abd EL-Fattah et al. 2018). It is an aromatic perennial wild herb that has been used traditionally by Bedouins in the Arabian Peninsula to relieve a range of diseases including fever, rheumatism, and diabetes. It has also been indicated to have anti-inflammatory, antioxidant, and antiproliferative characteristics (Miklasińska-Majdanik et al. 2018; Elsharkawy et al. 2021). According to recent studies, A. fragrantissima can be used to treat a range of disorders, including menstruation pains, headaches, exhaustion, smallpox, ophthalmic disorders, and pulmonary implications (Farouk et al. 2019; Patocka and Navratilova 2019). It has been demonstrated that A. fragrantissima essential oil exhibits antibacterial efficacy against many multidrug-resistant bacteria (Zeedan et al. 2014; Alsohaili 2018).

Tissue culture and somatic embryogenesis are among plant biotechnological approaches that have been successfully used to improve plant characteristics (Saadat et al. 2023). The genetic composition, physiological status, and cellular condition of the explant have a direct effect on somatic embryogenesis (Bahmankar et al. 2017). Although the physiological effect of nanoparticles on developmental processes is widely identified, less is known regarding how these nanoparticles might affect callus formation and somatic embryogenesis (Godel-Jędrychowska et al. 2023). As part of the somatic embryogenesis, non-zygotic cells undergo dedifferentiation and proliferation and the fate of the cell is reprogrammed from the molecular to the histological levels (Yang and Zhang 2010). Understanding how nanoparticles alter the process of cell differentiation and shift in the direction of differentiation is extremely important (Godel-Jędrychowska et al. 2023). Utilizing biotechnological approaches that are consistently applied to many plant species, somatic embryogenesis contributes significantly in genetic conservation and micropropagation (de Almeida et al. 2022).

Due to their characteristic physical, chemical, and biological characteristics, including their biocompatibility, environmental safety, easy fabrication, and non-toxic nature, zinc oxide nanoparticles (ZnONPs) represent one of the sustainable nanoparticles that have been widely employed in scientific studies (Ruszkiewicz et al. 2017; Alhujaily et al. 2022). In comparison to other metal oxides, ZnONPs exhibit distinctive thermochemical stability, sturdiness, and durability (Li et al. 2019). ZnONPs have been employed as a technologically superior material in catalysis for wastewater treatment, as well as in cosmetics and antimicrobial applications (Ruszkiewicz et al. 2017; Dahiya et al. 2023). Additionally, ZnONPs have been used as antimicrobial, antitumor, antioxidant, and hypoglycemic agents, as well as in the biomedical fields of tissue engineering and drug delivery (Mishra et al. 2017; Rajeshkumar et al. 2019; Greeshma and Thamizselvi 2023). Furthermore, because they have high reactivity, ZnONPs provide the plants with a more soluble and accessible form of zinc thereby addressing the issue of zinc salts not being available for plant uptake attributable to their poor solubility in the soil (Seleiman et al. 2023). However, depending on the studied species, age, and nanoparticle characteristics, ZnONPs can have different effects on plants. Many investigations have documented the beneficial impacts of ZnONPs on the growth and productivity of various crops (de la Rosa et al. 2013; Rai-Kalal and Jajoo 2021; Seleiman et al. 2023). These benefits included an increase in enzymatic activity, photosynthetic performance, seed yield, antioxidant properties, and the accumulation of many osmoregulators (Raliya and Tarafdar 2013; Faizan et al. 2018, 2021; Gauba et al. 2023).

In plants, there are predominantly three different types of markers: (a) morphological markers, which are phenotypic attributes; (b) biochemical markers; and (c) DNA-based markers, typically referred to as molecular markers. The responsiveness and genetic stability of plants generated through micropropagation have been identified utilizing many molecular markers, including RAPD (randomly amplified polymorphic DNA), AFLPs (amplified fragment length polymorphisms), ISSRs (inter simple sequence repeats), RFLP (restriction fragment length polymorphisms), and SSRs (simple sequence repeats) (Debnath et al. 2012; Rawls et al. 2015). Additionally, molecular markers can reveal information about the phylogenetic and taxonomic relationships among species. They can be employed to determine and isolate genes for genetic manipulation, as well as to recognize relevant traits in marker-assisted classification schemes (Nadeem et al. 2018). The ISSR analysis operates through amplifying DNA fragments that are positioned between two identical microsatellite repeats facing opposing directions. Because ISSR primers are longer than RAPD primers (15 to 35 bp), they have increased consistency because of a higher annealing temperature (Amom et al. 2018). This study sought to explore the response of antioxidant enzymes, secondary active metabolites, and redox activity, as well as the molecular (RAPD and ISSR) changes in A. fragrantissima explants brought on by adding different concentrations of ZnONPs to the growth medium.

Materials and Methods

Preparation of ZnO Nanoparticle Suspension

All the chemicals used were of scientific grade and purchased from Sigma-Aldrich, Gillingham, UK, unless otherwise stated. Zinc oxide nanoparticles (ZnONPs) were chemically synthesized and characterized according to the procedures outlined by Gaafar et al. (2020) using a co-mixture of 250 mM Zn(NO3)2, 6H2O, and 2.13 M KOH as precursors. After 4 hr of incubation at 80°C in an oil bath, the precipitated ZnONPs were repeatedly washed with deionized water and then with 95% ethanol (El-Nasr Co., Cairo, Egypt) before being dried at 105°C. The physicochemical properties of the obtained ZnONPs revealed whitish nanorods with a hexagonal quartzite crystal structure. Various concentrations of ZnONPs (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L−1) were prepared just before their administration to the growth medium by suspending in deionized water and subsequently sonication (ATS-10, Athena Tech., Mumbai, India) for 30 min.

Plant Material and Callus Induction

After attaining the maturity phase (August 2021), the seeds of Achillea fragrantissima (Forssk.) were collected from Wadi El-Shogyrate (28°56ʹ18ʺN and 34°07ʹ37ʺE) in South Sinai, Egypt (35 km east of Saint Katherine Protectorate). The seeds were surface-sterilized by submerging them in ethanol (70%) for 1 min, 50% Clorox with 5.4% NaOCl for 20 min, and then followed by five rounds of washing with sterile distilled water. Seeds were then seeded aseptically on half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962) at 25 ± 2°C to promote germination. The resulting explants were subsequently divided into sections measuring 1 cm and grown back on solid MS medium loaded with varying concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) either singly or paired with BA (6-benzyladenine) to stimulate callus development. The properly induced callus cultures were removed from the plantlets after 7 wk and grown individually until enough callus cultures were obtained.

Determination of the Produced Callus Growth Dynamics

The growth curve of the callus on the optimized growth medium (1.0 mg.L−1 2,4-D and 1.0 mg.L−1 BA) was measured. The measurement was achieved by harvesting samples of the cultures at 5 d intervals until the age of 50 d and measuring their fresh weight.

Characteristics of the Culture Medium and Treatments

Based on the results of growing medium optimization, the acquired callus cultures were grown on MS medium supplemented with 1.0 mg.L−1 2,4-D and 1.0 mg.L−1 BA coupled with ZnONPs at varying concentrations (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L−1) then incubated at 25 ± 2°C in the darkness. The callus cultures were taken out of the medium for the biochemical and molecular investigations after 7 wk.

Measurements of Antioxidant Enzyme Activities

To prepare the enzymatic extract, a mortar and pestle were used to homogenize 0.5 g of the callus samples in 4.0 mL of cold K-phosphate buffer (pH 7). The extract was centrifuged at 8000 g for 30 min at 4°C, and the resulting supernatant was employed as an enzyme source to determine the activity of POD (peroxidase), APX (ascorbate peroxidase), SOD (superoxide dismutase), and CAT (catalase) enzymes.

The activity of POD was assayed in the different callus culture extracts using the procedures established by Kato and Shimizu (1987). This assay was based on measuring the difference in the solution color at 470 nm following the increased color intensity of the measuring solution due to the oxidation of guaiacol by POD in the presence of H2O2 and the subsequent formation of tetraguaiacol.

Different callus culture extracts were assessed for their APX activity using the technique of Nakano and Asada (1981). This technique typically monitors H2O2 absorbance decline at 290 nm triggered by ascorbate oxidation via APX.

The protocol suggested by Beyer and Fridovich (1987) was applied to measure SOD activity in A. fragrantissima callus culture extracts (1987). SOD promotes formazan production photochemically through the reduction of nitroblue tetrazolium. Following dark incubation (15 min) in the presence of methionine and riboflavin, the increment in the solution optical density was monitored at 560 nm and the SOD activity was evaluated. CAT activity was assayed by the protocol adopted by Kato and Shimizu (1987) by monitoring the decrease in the absorbance of the solution resulting from H2O2 decomposition by the action of CAT at 240 nm. The activity of the investigated enzymes was represented as µmol.g−1 FM min−1 by applying the specific molar extinction coefficients of 26.6 mM−1 cm−1 for POD, 2.8 mM−1 cm−1 for APX, 21.1 26.6 mM−1 cm−1 for SOD, and 40.0 mM−1 cm−1 for CAT.

Estimation of Bioactive Secondary Metabolites

Fresh callus cultures from various ZnONP treatments were extracted in 85% aqueous ethanol (1:10 w/v), centrifuged at 6000 g, and the secondary metabolites were quantified in the produced supernatant. The determination of polyphenols was carried out by the approach prescribed by Jindal and Singh (1975) using Folin-Ciocalteu’s reagent in the presence of Na2CO3 (20%). For determining the phenolic content of the extracts, gallic acid was employed as a reference phenol.

Adopting Chang et al.’s (2002) protocol, the total flavonoids in A. fragrantissima extracts were measured. The extracts were mixed with AlCl3 (10%), potassium acetate (1.0 M), and ethanol (95%); subsequently, the optical density was reported at 415 nm. The flavonoid fraction of the extracts was quantified using the reference flavonoid quercetin.

The total amount of alkaloids in the A. fragrantissima extracts was calculated utilizing the method of Shamsa et al. (2010). The alkaloids in the ethanolic extracts were allowed to interact with bromocresol green (BCG) to develop a yellowish complex, which was subsequently extracted in chloroform, and the color intensity was monitored at 470 nm. Atropine was employed as a reference for quantifying alkaloid content.

Saponins were determined through the reaction of the extracts with vanillin (8%) and sulfuric acid (72%) in a water bath at 60°C for 10 min. The developed color was measured at 544 nm, and the concentration of saponins was calculated based on a standard curve plotted by using aescin as a standard saponin (Hiai et al. 1975).

The total amount of terpenoids was estimated by reacting the extracts with chloroform, vortexing thoroughly, adding concentrated sulfuric acid, and then incubating in the dark for 2 h. After decanting the supernatant, the pellets were dissolved by adding methanol and vortexing. Terpenoid content was quantified based on measuring the absorbance (at 532 nm) and using linalool as a standard (Ghorai et al. 2012).

Antioxidant Potential Assessment

Because of their capacity to bleach the violet color of the synthetic radical DPPH (2,2-diphenyl-1-picrylhydrazyl), the free radical scavenging activity of A. fragrantissima callus extracts was investigated. The extracts and the DPPH reagent in methanol were combined, vortexed well, and incubated in the dark for 30 min; then, the decline in the absorbance was monitored at 517 nm. The level of radical scavenging activity was calculated as a percentage in relation to the blank (Brand-Williams et al. 1995).

Based on the phosphomolybdate assay (PMA) developed by Ahmed et al. (2012), the total antioxidant capacity (TAC) of the ethanolic extracts was assessed. The phosphomolybdate reagent was combined with the extracts of A. fragrantissima, and the mixture was then maintained at 100°C for 90 min. After cooling, the absorbance at 765 nm was measured, and the TAC was represented as μg.g−1 FM ascorbic acid equivalent.

Genomic DNA Extraction

The method developed by Clarke (2009) with minor modifications, such as the addition of mercaptoethanol and polyvinylpyrrolidone, was utilized to extract the genomic DNA from the callus cultures of A. fragrantissima using the CTAB (cetyltrimethylammonium bromide) DNA extraction buffer. A nanodrop spectrophotometer (ND-1000, V3.8, Thermo Fisher Scientific, Wilmington, DE) was used to calculate the DNA content and purity. The 260/280 absorption ratio was between 1.7 and 1.8, which denoted the purity of the DNA. Twelve RAPD and seven ISSR primers were employed in the genetic characterization.

RAPD-PCR Analysis

Using a 20.0 μL reaction mixture that contained genomic DNA (100 ng), 7.0 μL sterile distilled water, and 10.0 μL Dream Taq Green PCR Master Mix #k 1081 (Thermo Fisher Scientific, Glasgow, UK), RAPD fingerprinting was carried out utilizing 10.0 μL of the designated primers (Table 1). The PCR protocol was as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 1 min and 35°C for 2 min, followed by extension at 72°C for 5 min.

Table 1 The sequence of primer set used in RAPD-PCR analysis (5ʹ 3ʹ)
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ISSR-PCR Analysis

For the amplification of the ISSR regions using the PCR technique, a set of seven ISSR general primers were used (Table 2). The amplification mixture (25.0 μL) comprised the genomic DNA (15 to 25 ng), the designated primer (2.0 μM), 2 × GoTaq Hot Start Green Master Mix (Promega, Madison, WI) (6.0 μL), MgCl2 (3.0 mM), and nuclease-free water. The thermocycling conditions were as follows: an initial denaturation at 95°C for 3 min, 35 cycles at 94°C for 30 s, 48 to 52°C for 45 s (based on the annealing temperature of the primer), 72°C for 1 min, then accompanied by one cycle at 72°C for 7 min.

Table 2 The sequence of primer set used in ISSR-PCR analysis (5ʹ 3ʹ)
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Separation and Visualization of PCR Products

Following PCR cycling, 10.0 μL of the PCR-amplified yields was electrophoresed on a 1.5% agarose gel with 1 X TAE buffer at 50 V for 1 hr in a submarine gel system (CBS Scientific, Escondido, CA). As molecular size markers, 1 kb or 100 bp Thermo Scientific Gene-Ruler DNA ladders (100 ng.μL−1) (Paisley, UK) were employed. Using a gel documentation system (Bio-1D Gel, Vilber Lourmat, Eberhardzell, Germany), the gels were photographed and visualized under UV light. To confirm the stability of the products, each run was repeated at least twice, and stable bands were scored using GelAnalyzer3 software.

Data and Statistical Analysis

The amplified RAPD or ISSR-PCR fragments were considered dominant alleles for specified loci. Each amplified fragment was scored for each primer as either present or absent, giving it a score of 1 or 0, respectively. The SAHN module of the NTSYS pc 2.11X program was used to create the dendrogram based on the unweighted pair group method with arithmetic mean algorithm (UPGMA) utilizing the RAPD and ISSR combined scored data.

Three separate repetitions of the experiment were conducted under similar circumstances, and each investigation was carried out in triplicate. The results were reported as the mean of three replicates ± SD (standard deviation). To evaluate the significance level among different treatments, the results were subjected to a one-way ANOVA; and to separate means, Tukey’s HSD test was used at a 5% level. Minitab 19 was used for all analyses. Prism 9 for Windows (version 9.0.2, GraphPad Software, San Diego, CA) was used to conduct the Pearson correlation analysis between the various biochemical characteristics and the ZnONP concentrations employed.

Results and Discussion

Callus Induction

The seeds of A. fragrantissima grown on half-strength MS medium without any PGRs (plant growth regulators) demonstrated normal germination and grew into seedlings with roots and shoots but no callus formation (Fig. 1A). To promote callus formation, stem and leaf sections from 14-d-old seedlings were maintained on MS medium with various concentrations and combinations of 2,4-D and BA. The first evidence of callus development from the explants was apparent between 15 and 20 d after the subculturing in the fresh medium, and the best callus induction rate (approximately 85%) was observed at a concentration of 1.0 mg.L−1 of the two employed PGRs (2,4-D and BA) (Table 3). The callus development proceeded above the medium’s surface and showed a friable and granular morphology (Fig. 1B).

Figure 1.
figure 1

Seed germination of Achillea fragrantissima Forskal (A) and callus induction from shoot explants on MS medium supplemented with 1.0 mg L−1 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.0 mg L−1 6-benzyl adenine (BA) (B).

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Table 3 Effect of different concentrations of 2,4-D and BA PGRs on callus induction of Achillea fragrantissima Forskal shoot explants
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For callus induction, various plant species require a specific medium that is supplemented with a suitable concentration of plant growth regulators (Zang et al. 2016). The most effective combination for inducing A. fragrantissima callus in the present investigation was MS with 1.0 mg.L−1 2,4 D and 1.0 mg.L−1 BA. For A. fragrantissima callus induction, 2,4-D was necessary; however, concentrations exceeding 1.0 mg.L−1 decreased the callus’s ability to be induced. Furthermore, the callus cultures of A. fragrantissima could not be induced by BA when it was used alone. Additionally, in this investigation, the combination of 2,4-D and BA was efficient at promoting friable and granulated callus cultures. Several plant species exhibited similar responses, supporting the synergistic influence of PGRs in callus induction (Wang et al. 2007; Zang et al. 2016; Adil et al. 2018).

The Growth Dynamics of A. fragrantissima Callus on the Optimized Medium

The growth curve of A. fragrantissima callus on the optimized medium, MS medium containing a mixture of 2,4-D and BA in a concentration of 1.0 mg.L−1, as determined on a fresh weight basis at 5 d intervals until the age of 50 d, is shown in Fig. 2. The callus development progressed directly with time until it reached its maximum fresh weight (3.6 g) after 40 d, remained steady after 45 d, and then started to slowly decline after 50 d. Between the 10th and 45th day following the culture initiation, the exponential growth phase began during which the fresh weight progressively increased. It is crucial to assess the callus fresh weight in order to determine the timescale required for the development of the maximal callus growth during in vitro propagation. Cellular damage or desiccation may have contributed to the subsequent decline in callus fresh weight after 45 d (Kintzios et al. 1999). Also, the main causes for shifting the callus into the decline phase, according to Abbade et al. (2010), are medium dryness or the formation of detrimental substances in the growth medium.

Figure 2.
figure 2

Growth dynamics curve of the callus of Achillea fragrantissima Forskal explants over 50 d of culture.

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Influence of ZnONP Supplementation on Antioxidant Enzymes

The addition of different ZnONPs to the growing medium had a substantial impact on the activity of the four antioxidant enzymes that were being examined (Fig. 3). The results showed that, in comparison to the ZnONP-free growth medium (control), ZnONP supplementation considerably boosted POD, APX, and SOD activities. The dosage of 10.0 mg.L−1 of ZnONPs was the most significant in boosting POD and APX activity as it induced 13.5- and 12.2-fold increases in the activity of both enzymes, respectively. Regarding SOD activity, its activity was increased 3.0 folds by ZnONPs at the concentration of 15.0 mg.L−1. However, as the concentration of ZnONPs increased, the activity of CAT gradually decreased. The highest ZnONP dosage prompted a 4.2-fold reduction in CAT activity.

Figure 3.
figure 3

The impact of different ZnONP concentrations on the peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities in the callus cultures of Achillea fragrantissima Forskal. Significant differences at 5% level are represented by different superscripted letters.

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The increase in POD, APX, and SOD as a result of ZnONP treatment in this study was consistent with the results of Ahmad et al. (2022) in wheat, Khan et al. (2022) in maize, and Ramzan et al. (2022) in flax. It was believed that plants would be able to neutralize the extra ROS generated in response to stressful stimuli attributable to the enhanced antioxidant enzyme activity in response to ZnONPs (Ramzan et al. 2022). Furthermore, Singh et al. (2018) demonstrated that at lower concentrations of ZnONPs the improvement in the antioxidant enzymes activities contributed to enhancing plant development and productivity, whereas at higher concentrations, an additional increment in these enzymes would be employed to combat the detrimental effects brought on by increased production of oxidative stress.

The concentration-dependent decline in CAT activity with the increased ZnONP concentration observed in this study was also reported in cotton (Venkatachalam et al. 2017b) and in cucumber (Kim et al. 2012). The authors ascribed this outcome to the fact that ZnONP-treated plants displayed decreased ROS formation, suggesting less phytotoxicity to the grown plants. However, the findings of Mukherjee et al. (2014) showed that the main factor behind the decline in CAT activity was the increased release of Zn2+ at high ZnONP concentrations.

Influence of ZnONPs on Secondary Metabolite Accumulation

Global climate change results in a reduction in the availability of pharmaceuticals and bioactive substances that are utilized in many medical aspects to treat disorders and address malnutrition issues. The most reliable method to address such an issue is by employing nanoparticles to promote the natural phytochemicals’ metabolic pathways, especially when applied to the callus cultures, which grow quickly and are independent of environmental factors, such as growth time. The results of the current study revealed that different ZnONP concentrations had significant consequences on the production of secondary active metabolites in the callus culture of A. fragrantissima (Fig. 4). The amount of phenolics, flavonoids, alkaloids, saponins, and terpenoids present in the callus cultures of A. fragrantissima was considerably influenced by the applied concentrations of ZnONPs. In comparison to the control treatment, all concentrations of ZnONPs achieved a significant enhancement in phenolics, flavonoids, alkaloids, and saponins, with 20.0 mg.L−1 yielding the highest phenolics content (157.0%), 15.0 mg.L−1 yielding the highest flavonoids (98.7%), 10.0 mg.L−1 yielding the highest alkaloids (120.0%), and 10.0 mg.L−1 yielding the highest saponins (309.4%). In terms of terpenoids, the low ZnONP (5.0 and 10.0 mg.L−1) concentrations promoted their accumulation with 10.0 mg.L−1 dosage being the most significant (134.0% increase) in comparison to the control level. Nevertheless, higher doses, particularly 20.0 mg.L−1, had lowered terpenoid concentration in A. fragrantissima callus culture.

Figure 4.
figure 4

The impact of different ZnONP concentrations on the accumulation of phenolics, flavonoids, alkaloids, saponins, and terpenoids in the callus cultures of Achillea fragrantissima Forskal. Significant differences at 5% level are represented by different superscripted letters.

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The use of nanoparticles in plant biotechnology has been demonstrated to have a distinctive impact on in vitro plant growth and development with positive consequences on micropropagation, callus induction, somatic embryogenesis, cell suspension culture, and secondary metabolite production (Al-Qudah et al. 2022). In various plant species and culture techniques, it has been shown that ZnONPs can efficiently trigger the biosynthesis of secondary metabolites. Consistent with the present study’s findings, Zaeem et al. (2020) showed that ZnONPs promoted phenol and flavonoid accumulation in flax callus cultures. Additionally, Stevia rebaudiana callus culture demonstrated an improved accumulation of phenols and flavonoids with ZnONP treatment, according to Javed et al. (2018). Likewise, ZnONPs were shown to generate a substantial effect on the accumulation of phenols and tropane alkaloids in Hyoscyamus reticulatus root hair culture (Asl et al. 2019). Karamian et al.’s (2020) study revealed a significant accumulation of saponins in the callus culture of Verbascum sinuatum treated with titanium oxide NPs. However, Chahardoli et al. (2020) revealed a substantial increase in saponins in Nigella arvensis callus cultures with aluminum oxide NPs. To the present authors’ knowledge, based on an extensive literature survey, this is the first report on the impact of ZnONPs on the level of saponins and terpenoids in the callus culture system.

Secondary metabolites were reported to be elicited by cells as part of their defensive mechanism when they are subjected to the oxidative stress triggered by ZnONPs (Javed et al. 2018). The advantages of tiny size, larger surface area, and the facility of apoplastic or symplastic mobility of the designated NPs within the cells may result in more electrostatic interactions with the cellular membranes, promoting and regulating certain metabolic routes to produce secondary metabolites and to promote their accumulation (Javed et al. 2017). According to Venkatachalam et al. (2017a), ZnONPs prompt the overexpression of the antioxidant metabolite pathways, which substantially affect the plant metabolic routes and the synthesis of bioactive metabolites. Therefore, the results of this work implied that low ZnONP concentrations are a feasible approach for promoting secondary metabolite accumulation in A. fragrantissima callus cultures.

Influence of ZnONPs on the Antioxidant Potential

The administered concentrations of ZnONPs significantly affected the redox status of A. fragrantissima callus cultures (Fig. 5). The radical scavenging activity (DPPH) of the resultant callus cultures was significantly boosted by each of the ZnONP concentrations that were applied. The DPPH activity grew progressively until 15.0 mg.L−1 of ZnONPs, which achieved the maximum DPPH activity (6.97%) after which it showed some decrease but remained higher than the untreated control. The total antioxidant capacity (TAC), reported as ascorbic acid equivalent, likewise gradually increased with the application of ZnONPs until the concentration of 10.0 mg.L−1, which produced the highest TAC (36.5 μg.g−1 FM), before being gradually decreased with the higher concentrations of ZnONPs; nevertheless, their level was still higher than that of the untreated control.

Figure 5.
figure 5

The impact of different ZnONP concentrations on the radical (DPPH) scavenging activity and the total antioxidant capacity (TAC) in the callus cultures of Achillea fragrantissima Forskal. Significant differences at 5% level are represented by different superscripted letters.

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Concomitant to the present study’s results, the induction of antioxidant activity in the callus culture with lower concentrations of ZnONPs and their reduction with higher concentrations were reported in Stevia rebaudiana (Javed et al. 2018), Nicotiana tabacum (Mazaheri and Soleyman 2020), Fagonia indica (Khan et al. 2021), and Silybum marianum (Shehzad et al. 2021). A recent development in the retrieval of abiotic stress involved the application of ZnONPs to influence callus metabolism and secondary metabolite synthesis within the callus culture. These nanomaterials confronted the callus tissues to oxidative stress, which activated their metabolic activity and increased their antioxidant potential (Choi and Hu 2008). However, whenever oxidative stress overwhelmed the natural antioxidant potential and capacity to detoxify free radicals, callus development became impaired and antioxidant activities substantially declined (Javed et al. 2018).

Metal accumulation in plant tissue triggered detoxification-related enzymes activity (for example, SOD, POD, APX, and CAT). Furthermore, secondary metabolites contained in callus cells, including phenols, flavonoids, alkaloids, saponins, and terpenoids, were reported to have antioxidant properties. These secondary metabolites helped plants protect themselves from oxidative damage by eliminating free radicals (Khan et al. 2021). Furthermore, the recently identified transcription factor known as zinc finger protein (ZFP) activated the Zn-assisted defense of plant cells, enabling them to endure a variety of stress-related stimuli by raising their antioxidant potential (Noman et al. 2019).

DNA Fingerprinting of A. fragrantissima Callus Cultures Supplemented with ZnONPs

To evaluate the genetic variation among formed A. fragrantissima callus cultures, which may be correlated with ZnONPs, RAPD and ISSR markers were employed. In the PCR-RAPD analysis of A. fragrantissima callus cultures, 12 primers were screened for genetic variability based on the DNA fragments that were amplified, which may be produced within the callus cultures because of treatment with various ZnONP concentrations (Table 4; Fig. S1). The amplified bands generated by each primer were consistent and scoreable. Throughout the five ZnONP levels, 48 bands were produced of which 40 were polymorphic and 8 were monomorphic. The amplified bands of the RAPD profiles of all callus cultures ranged from 80 bp (primer O-17) to 1322 bp (primer L-20). OPG-01, L-13, and OPG-03 primers each produced three fragments whereas L-20, OPP-10, Z-05, Z-18, OPP-01, and X-06 each produced four fragments; and the primers OPC-17, L-12, and O-17 produced five fragments each. The primer OPP-01 yielded the least level of polymorphism (25%) while the primers OPG-01, L-13, OPP-10, Z-05, Z-18, and O-17 produced the highest level of polymorphism (100%).

Table 4 RAPD-PCR profiles amplified by 12 random primers demonstrating the effect of ZnONP supplementation on genetic stability of Achillea fragrantissima Forskal callus cultures
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Medium supplementation with different ZnONP concentrations triggered the appearance of two bands of 286 and 95 bp with the L-12 primer and a single band of 1322 bp in length using the L-20 primer. Additionally, the primer OPP-10 produced three fragments of 1100, 500, and 370 bp, which were distinct from the three fragments produced by primer O-17 (390, 282, and 162 bp). The primer OPG-03 revealed the induction of 503 and 307 bp DNA fragments whereas primer OPP-01 revealed the appearance of a 608-bp fragment. The lowest and highest dosages of ZnONPs (5.0 and 20.0 mg L−1) resulted in distinct bands of 310 bp and 163 bp using primers OPP-10 and L-20, respectively. Nonetheless, in the case of O-17 primer, the dosage of 20.0 mg L−1 caused fragments of 121 and 80 bp to appear, which disappeared at the other ZnONP concentrations.

The disappearance of normal DNA fragments in the RAPD profiles could possibly be due to DNA degradation or chromosomal changes brought on by heavy metal–induced genotoxicity while the presence of new DNA fragments could be the result of mutations (Atienzar et al. 2000). According to Ghosh et al. (2016), the bands detected at the highest concentration of ZnONPs (30.0 mg.L−1) could be attributed to the genotoxic activity of ZnONPs at these high concentrations, inducing DNA mutation at specific sites. They observed that onion root meristem cell membrane integrity was impaired and that the cells also underwent chromosomal abnormalities and experienced DNA strand breakage. Genotoxicity of ZnONPs arose from the damage that was either not fixed or improperly fixed by parental cells (Leme and Marin-Morales 2009). The ISSR analysis was also used to examine how ZnONP exposure at various doses affected the genetic composition of A. fragrantissima in callus culture. Table 5 and Fig. S2 provide a summary of the results of the ISSR fingerprinting of the investigated samples using 7 ISSR primers. Among various ZnONP treatments, 33 amplicons were consistently determined using the seven ISSR primers. In total, the primers identified 23 monomorphic and 10 bands were amplified. The primers 89A and HB-11 had the highest reproducibility, scoring 6 fragments for each primer. However, with only 3 fragments, primer 49A had the lowest reproducibility. Primers 89A, HB-9, and HB-15 all achieved 50% polymorphism, but primer HB-11 achieved the lowest polymorphism rate (16.7%). According to the provided data, 5 and 10.0 mg.L−1 ZnONPs produced two distinct fragments: one (670 bp) with 89A primer and another (350 bp) with HB-9 primer. With HB-15 primer, two fragments (820 and 420 bp) were nevertheless produced at doses of 10.0, 15.0, and 20.0 mg.L−1 ZnONPs. A 1200 bp amplified with the primer HB-11 totally disappeared because of the ZnONP concentrations applied. Three DNA segments, 365 bp with primer 49A, 265 bp with primer HB-9, and 620 bp with primer HB-13, abruptly disappeared at a concentration of 15.0 mg.L−1 ZnONPs. Generally, the rate of polymorphism obtained by the RAPD analysis was higher than that obtained by the ISSR analysis.

Table 5 ISSR profiles amplified by 7 primers demonstrating the effect of ZnONP supplementation on genetic stability of Achillea fragrantissima Forskal callus cultures
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In general, the RAPD analysis revealed a higher rate of polymorphism than the ISSR analysis. The same result was obtained in quinoa by Saad-Allah and Youssef (2018) and in rice by Alam et al. (2016). ISSR is a DNA-based marker that anticipates the recognition of polymorphisms in inter-microsatellite regions, and the produced DNA patterns might diverge due to the presence of new fragments, the disappearance of existing fragments, or alterations in fragment density (Izzatullayeva et al. 2014). According to Osman et al. (2020), treatment with nanoparticles led to a DNA modification or nucleotide exchange, which caused specific DNA fragments to either appear or disappear. Likewise, Fouda et al. (2021) indicated that the presence of new DNA bands and the absence of normal ones in the ISSR profile may be characterized as a genetic variation, which is definitely the consequence of DNA damage or conformational changes brought on by nanoparticles. In this context, Sreelekshmi et al. (2022) stated that the high level of monomorphism among the ISSR markers confirms the genomic consistency and homogeneity of cultures raised using the nanoparticles at low concentrations. Accordingly, the results of the present investigation showed that under the impact of ZnONPs, the monomorphic bands of ISSR primers displayed significant homogeneity between the A. fragrantissima callus cultures.

Cluster Analysis

Cluster analysis based on RAPD and ISSR genotyping data separately or as RAPD and ISSR combined data was performed using the DICE coefficient similarity matrix (Fig. 6; Fig. S3). The cluster analysis divided the A. fragrantissima callus cultures based on combined data from RAPD and ISSR into two main distinct groups (Fig. 6). One group included callus cultures produced without ZnONP supplementation (0.0 mg.L−1 ZnONPs), and the second group contained callus cultures produced with medium supplemented with other ZnONP concentrations (5.0, 10.0, 15.0, and 20.0 mg.L−1). This indicated that the different ZnONP concentrations induced genetic variability in the A. fragrantissima callus cultures formed on a medium containing ZnONPs.

Figure 6.
figure 6

Dendrogram of cluster analysis of Achillea fragrantissima Forskal callus cultures formed on MS medium supplemented with different ZnONPs (0, 5, 10, 15, 20 mg L−1) based on (A) RAPD markers, (B) ISSR markers, and (C) combined data of RAPD and ISSR markers.

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The present findings in this study suggested that ZnONPs can increase the genetic variability of callus cultures by nucleotide exchange, which may consequently affect the expression of growth genes and genes that are responsible for the synthesis of secondary metabolites in A. fragrantissima callus cultures. Like the present findings, Saeed et al. (2021) found that ZnONPs have been shown to have effects on the genetic variability of callus cultures of Silybum marianum. In addition, in vitro experiments with Artemisia annua cell suspension culture exposed to cobalt nanoparticles resulted in a decrease in the expression of artemisinin-related genes, which allowed an increase in artemisinin accumulation (Ghasemi et al. 2015).

Correlation Analysis Assessment of ZnONP Effects and Biochemical Traits

To explore potential relationships among the biochemical traits under investigation, a correlation analysis was conducted involving distinct concentrations of ZnONPs and various biochemical traits that were measured (Fig. 7). The analysis revealed high positive associations between different concentrations of ZnONPs, and the accumulation of phenols and flavonoids where Pearson correlation coefficients (r) were + 0.93 and + 0.86, respectively. Additionally, a positive correlation emerged between the levels of peroxidase (POD) and ascorbate peroxidase (APX) in conjunction with saponins (r values were + 0.94 and + 0.93, respectively). However, there seemed to be a possible negative correlation between catalase (CAT) and both peroxidase (POD) and ascorbate peroxidase (APX) (r values were − 0.68 and − 0.66, respectively). In addition, the level of CAT also had a negative association with the other variables suggesting that an increase in the concentration of ZnONPs might be accompanied by a reduction in the level of CAT. This finding was in accordance with Abdel-Wahab et al. (2020) who found an insignificant increase in the activity of CAT in the Solanum nigrum callus supplemented with 50.0 mg.L−1 ZnONPs. Moreover, a general rise in saponin value brought on by ZnONP treatment may be accompanied by a rise in value for POD, APX, or the other tested variables, apart from CAT.

Figure 7.
figure 7

Pearson correlation matrix showing the relationships among different measured biochemical traits and the different concentrations of ZnONPs used in the callus cultures of Achillea fragrantissima Forskal. Positive numbers represent a positive correlation, and negative numbers represent a negative correlation between the tested parameters. Color intensity determines the strength of the correlation. Blue color represents positive correlation, and red color represents negative correlation.

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As stated above, ZnONP supplementation into callus cultures showed a positive correlation with phenolic and flavonoid compounds. This observation was notable in other studies, including callus cultures of Stevia rebaudiana Bertoni (Javed et al. 2018), Verbena officinalis and Verbena tenuisecta (Afridi et al. 2018), and Silybum marianum callus cultures treated with ZnONPs, which accumulated substantial total phenolic and flavonoid compounds (Shehzad et al. 2021).

Conclusion

The findings of this study suggested that ZnONP exposure increases the genetic variability of Achillea fragrantissima callus cultures, which may consequently affect the expression of growth-related genes that enhance the defense systems and accumulation of phytochemicals in the produced callus cultures. However, further studies on the molecular and biochemical levels must be considered to validate the role and safety of ZnONPs in the production of therapeutically active molecules.