Introduction

Legume crops, encompassing approximately 15% of the world's cultivated land, constitute vital sources of protein for both human and animal consumption (Vance et al. 2000). Among these crops, alfalfa (Medicago sativa L.) stands out as a cornerstone of sustainable agriculture, owing to its symbiotic relationship with the soil bacterium Rhizobium and its remarkable nitrogen fixation capacity (Graham and Vance 2003). Despite its pivotal role, legume crops, including alfalfa, face a decline in productivity and quality when subjected to drought stress. In the context of evolving climate change scenarios and the pressing need to meet escalating global food and feed demands, understanding plant responses to drought stress becomes imperative. Exploring how plants adapt their metabolism and deploy defense mechanisms against adverse climatic conditions is crucial. One such defense mechanism involves the intricate reprogramming of gene expression orchestrated by microRNAs (miRNAs). MicroRNAs, small non-coding RNAs approximately 22 nucleotides in length, have emerged as pivotal regulators of genes at post-transcriptional levels across diverse organisms. Drought stress triggers the modulation of several miRNAs, demonstrating functional conservation throughout plant species. miRNAs participate in cell binding and metabolic processes, thereby enhancing resistance to various abiotic and biotic stresses and sustaining crop productivity in altered environments. miRNAs play decisive roles in regulation in biological metabolism and defense activities, including gene expression programs during callus development, somatic embryo induction, biosynthesis in vitro secondary metabolites. Therefore, miRNAs may be a crucial tool for the transcriptional reprogramming of callus cells (Xu et al. 2020). The overexpression of csi-miR156a significantly improved callus formation and the early stages of somatic embryo induction in conserved citrus callus (Zhang et al. 2017; Siddiqui et al. 2019).

These characteristics underscore the potential of miRNA-mediated genetic alterations in enhancing the drought resistance of cereal crops. The primary objective of this study is to elucidate the responses of miR159 and miR393 in alfalfa plants under drought stress. By exploring the adaptation mechanisms of miRNAs to drought stress conditions, this research contributes to the broader understanding of how molecular processes govern a plant's ability to cope with water scarcity. Insights gained from this investigation hold promise for informing innovative strategies aimed at improving the resilience of crucial crop species to environmental stressors, thereby promoting sustainable agriculture in the era of climate change.

Nanotechnology, an advancing frontier in scientific innovation, has demonstrated transformative applications across diverse domains such as packaging, biomedicine, tissue engineering, healthcare, the food sector, and space exploration (Mahanty et al. 2013). The integration of nanodevices and nanomaterials has opened new avenues in plant biotechnology and agriculture, offering promising solutions (Nair et al. 2010). This has ignited considerable interest, particularly with the successful deployment of various nanoplatforms under in vitro conditions, sparking enthusiasm for their potential contributions to agricultural practices. Callus culture is an extremely efficient vehicles for profound plant research and it has considerable advantage compared to conventional whole plant growing, which includes overcoming seed dormancy, and seed viability independent from environmental conditions (Yazıcılar and Bezirganoglu 2023).

In recent years, the focus on transition metal oxide nanoparticles as heterogeneous catalysts has gained momentum due to their exceptional structures and catalytic activities. Graphene, a notable heterogeneous catalyst, has garnered attention for its unique spherical carbon nanomaterial structure, coupled with remarkable physical and chemical properties. Recent studies have highlighted the complexity of nanomaterials, which is influenced by various factors including class, concentration, characteristics, plant species, and seed size (Siddiqui et al. 2015). Reduced graphene oxide structures, characterized by a large surface area, adjustable porosity, and impressive durability under various conditions, present promising attributes for catalytic applications (Piccinno et al. 2012; Khan et al. 2019). Supported transition metal oxide in reduced graphene oxide catalyzed reactions offers advantages such as high atomic efficiency, simplified product isolation, easy recovery, and catalyst recyclability (Kaur et al. 2016).

Calcium (Ca), a crucial macronutrient in plants, plays a multifaceted role as both a structural component and an intracellular second messenger. In its structural role, Ca ensures the integrity of cell walls and membranes. Insufficient Ca availability results in symptoms such as tip blight in lettuce (Lactuca sativa L.) and flower tip rot in tomato (Lycopersicon lycopersicum L.) due to limited remobilization from old to new tissue via the phloem (Hirschi 2004). Beyond its structural function, Ca serves as a second messenger in various cellular processes, responding to abiotic and biotic stressors (Michard et al. 2011; Monshausen et al. 2011; Blume et al. 2000). Calcium oxide (CaO) NPs, among metal-based nanoparticles, have emerged as a focus of attention due to their excellent properties and versatile applications (Roy et al. 2013). Notably, CaO NPs exhibit antifungal and antibacterial activities while maintaining environmental compatibility and cost-effectiveness, positioning them as valuable components in nanotechnological approaches.

While extensive research has explored the impacts of various nanomaterials on diverse crops in agricultural contexts, there remains a noticeable gap in understanding the physiological, biochemical, and molecular effects specifically associated with Calcium Oxide Nanoparticles (CaO NPs) and Graphene Oxide (GO) on drought-stressed alfalfa plants. This study addresses this critical knowledge gap by focusing on the nuanced responses of alfalfa (Medicago sativa L.) cultivars, namely 'Erzurum' and 'Konya,' to the application of CaO NPs and GO. The synthesis of CaO nanoparticles through hydrothermal green synthesis techniques represents a methodological cornerstone in this research endeavor. This eco-friendly synthesis approach aligns with the contemporary emphasis on sustainable and environmentally conscious nanomaterial production methods. The subsequent investigation aims to discern the distinctive physiological, biochemical, and molecular features elicited in alfalfa calluses subjected to different dosage modifications of CaO NPs and GO, in comparison to a control group. As drought stress poses a significant challenge to agricultural productivity, understanding the specific impacts of CaO NPs and GO on alfalfa plants under such conditions is of paramount importance. By unraveling the intricate responses at the physiological, biochemical, and molecular levels, this study contributes to advancing our comprehension of nanomaterial interactions with crops, particularly in the context of environmental stressors. The dual cultivar approach ('Erzurum' and 'Konya') further enriches the investigation, acknowledging the potential cultivar-specific responses that may emerge.

Materials and methods

Hydrothermal synthesis of CaO NPs

CaO NPs were synthesized through a hydrothermal process. In a Teflon-lined stainless steel autoclave, a reaction mixture consisting of 80 mL, 0.02 g urea, 0.3 g citric acid (CA), and a 1 mM CaCl2 solution was prepared. Following the complete dissolution of the chemicals, the mixture was thoroughly stirred and subjected to a reaction at 210 °C and 1 atm pressure for 6 h. The resultant precipitate was isolated through centrifugation at 15,000 xg for 30 min and subsequently dried in an oven at 50 °C overnight. The synthesized CaO NPs were stored at + 4 °C for utilization in other segments of the study.

GO hummers synthesis method

In the initial phase of the process, 5 g of powdered graphite, 2.5 g of NaNO3, and 115 mL of 96.4% sulfuric acid were combined in an ice bath and placed on a magnetic stirrer for 1 h. Subsequently, 15 g of KMnO4 was cautiously introduced into the mixture. After removing the ice bath, the mixture was stirred on a magnetic stirrer for an additional 2 h. Following this, 500 mL of deionized water was added, and the mixture continued to be stirred on the magnetic stirrer for another 1 h. To this, 8.4 mL of H2O2 was incorporated into the mixture and stirred for an additional 2 h. The resulting mixture underwent filtration, with deionized water being added until reaching a pH of 7. The filtrate was then incubated in an oven at 50 °C overnight, yielding powdered Graphene Oxide (GO) (Singh et al. 2015; Shoeb et al. 2015).

Characterization of CaO NPs and GO

The characterization of the acquired CaO NPs and GO was conducted at the Eastern Anatolia High Technology Application and Research Center (DAYTAM) of Atatürk University. Scanning Electron Microscopy (SEM)-Energy Dispersive X-ray analysis, XRD analysis, and FTIR analysis were employed for the characterization of CaO NPs. This facilitated the acquisition of information about the size and morphological properties of the synthesized nanoparticles.

Application experiments of CaO NPs, GO, and drought to alfalfa callus

In this investigation, alfalfa (Medicago sativa L., cvs. 'Erzurum' and 'Konya') seeds sourced from the Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University were employed as the plant material. The seeds underwent surface sterilization in 22% sodium hypochlorite for 15 min, followed by three washes in distilled water. Leaves from three-week-old plants were removed and placed onto a Murashige & Skoog (1962) MS medium supplemented with 1 mg L−1 of 2,4-D and 1 mg L−1 of kinetin, along with 2 mg L−1 of glycine, 100 mg L−1 of myo-inositol, 0.5 mg L−1 of nicotinic acid, 0.5 mg L−1 of pyridoxine HCl, 0.1 mg L−1 of thiamine HCl vitamins, 1.95 g of MES, 50 mg L−1 of ascorbic acid, and 20 g of sucrose. The medium was solidified using 7 g of agar, and the pH was adjusted to 5.8 prior to autoclaving. These leaf explants were incubated in complete darkness at 25 ± 1 °C for one month, and callus formation was assessed.

For subsequent studies, drought acclimation (0.0, 50, and 100 mM mannitol), CaO NPs (1 and 2 ppm), and GO (0.5 and 1.5 ppm) were employed with 1 mg L−1 2,4-D and 1 mg L−1 kinetin MS medium. The samples were maintained in an acclimatization chamber for a total duration of 1 month, under conditions of 16 h of light and 8 h of darkness, exposed to a luminous intensity of 1500 lx, at a temperature of 25 °C, and with a humidity level of 50%.

Analysis of physiological and biochemical parameters

Proline content

To quantify proline content, a 100 mg callus sample was taken and homogenized using liquid nitrogen. Subsequently, 10 mL of 3% sulphosalicylic acid was introduced to the homogenized samples. The resulting homogenate underwent centrifugation at 15,000 rpm for 15 min, with the pellet discarded. Next, 2 mL of the supernatant was transferred to a separate tube, and 2 mL of acid ninhydrin was added. The mixture was incubated in a water bath at 90 °C for 1 h, followed by cooling in an ice bath. Subsequently, 4 mL of toluene was added, and vortexing was performed. The absorbance of the samples was measured at a wavelength of 520 nm in triplicate. The proline content in the samples was determined by referencing a standard chart prepared using pure proline (Ahmad et al. 2012).

Determination of soluble sugar content

The determination of water-soluble sugars was conducted using the 3,5-dinitrosalicylic acid (DNSA) method. In this approach, the protocol for reducing sugar determination proposed by Krivorotova and Sereikaite in 2014 was adapted.

Determination of lipid peroxidation content

The level of lipid peroxidation (LPO) serves as a common indicator for assessing oxidative damage in cells or tissues, with Malondialdehyde (MDA) frequently employed as a marker for lipid peroxidation levels. In our study, a 0.2 g callus sample from M. sativa, treated at each dosage, was weighed in triplicate. The callus samples were homogenized with 2 mL of 0.1% Trichloroacetic acid (TCA) and centrifuged in 2 mL Eppendorf tubes at 15,000 rpm for 15 min. Subsequently, 2 mL of the resulting supernatant was transferred to tubes, and 1 mL of 20% TCA and 1 mL of 0.5% Thiobarbituric acid (TBA) were added. The samples underwent incubation in a 90 °C water bath for 40 min. Following this, 100 µL of each sample was extracted in triplicate, and added to a 96-well plate, and absorbance measurements were recorded at 450, 532, and 600 nm (Heath and Packer 1968; Erdal 2012). The MDA content was calculated using the formula provided below.

$$MDA i{\text{c}}eri{\text{g}}i\left({~}^{nmol}\!\left/ \!{~}_{mL}\right.\right)= \left[\left[\left({A}_{532}- {A}_{600}\right)- \left({A}_{532-}{A}_{600}\right)x\mathrm{ 0,9571}/157000\right]x{10}^{6}\right.$$

Hydrogen peroxide (H2O2)

To quantify H2O2 content, 0.2 g of M. sativa callus was subjected to homogenization in liquid nitrogen, followed by the completion of homogenization with 2 mL of acetone at -18 °C. Subsequently, the resulting mixture was centrifuged at 10,000 × g for 10 min, and the pellet was discarded. A 1.5 mL aliquot of the supernatant was retrieved and combined with 150 µL of 5% Ti(SO4)2 (titanium disulphate) and 0.3 mL of 19% NH4OH (ammonium hydroxide). The resulting mixture underwent centrifugation again at 10,000 × g for 10 min. The pellet was dissolved in 3 mL of 2 M H2SO4 and thoroughly mixed. The absorbance of the final mixture was measured at 415 nm. The results are expressed as the quantity of H2O2 per gram of callus (µg/g of callus) (Hao et al. 2014).

Scanning electron microscope (SEM)

Each sample derived from M. sativa callus was immersed in a 0.05 M buffer (comprising 0.2 M Na2HPO4 and 0.2 M NaH2PO4) at 25 °C for 1 h and subsequently in a fixative buffer (0.05 M tampon, 3% glutaraldehyde) for 2 h. It was then subjected to incubation in a 0.05 M buffer on ice for an additional 1 h. Following this, the sample underwent immersion in sterile pure water for 30 min. Subsequently, a sequential series of ethyl alcohol concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) were employed at 25 °C for 15 min each (Fowke 1995). Imaging was conducted utilizing a Quante FEG 250 (ZEISS) microscope located at YUTAM (High Technology Application and Research Center).

Laser scanning confocal microscope (CLSM)

M. sativa callus samples were stained with Rhodamine B (N-[9–2-carboxyphenyl-6-(diethylamino)-3H-xanthene-3-ylidene]-N-ethylethanolaminium chloride). Microscopic images were captured using a Laser Scanning Confocal Microscope (Nikon Eclipse TE2000) situated at DAYTAM (Eastern Anatolia High Technology Application and Research Center) at Atatürk University (Tang et al. 2013).

Evaluation of changes in mtr-miR159 and mtr-miR393 gene expressions by RT-qPCR

A two-step qPCR analysis was conducted to assess changes in the gene expressions of mtr-miR159 and mtr-miR393 in response to CaO NPs and GO applications on callus samples of local alfalfa varieties subjected to drought stress.

RNA isolation

A 0.3 g callus sample was weighed from M. sativa callus treated at each dose. Subsequently, 500 µL of the callus sample for each dose was homogenized with Trizol. After allowing it to stand at room temperature for 5 min, 100 µL of chloroform was added and slowly inverted. The samples were then left at room temperature for 3 min and centrifuged at 12,000 rpm at 4 °C for 15 min. A 250 µL aliquot of the supernatant was extracted, and 250 µL of isopropanol was added and gently inverted 10 times. After keeping the samples at room temperature for 10 min, they were centrifuged at 12,000 rpm at 4 °C for 10 min. Following this, 1 mL of 70% ethanol was added to the pellet, and it was centrifuged at 7,500 rpm at 4 °C for 5 min. Subsequently, 50 µL RNase Free was added to the pellet, dissolved in pure water, and stored at -20 °C. The concentration and purity of RNA samples were determined using the Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA) (Ma et al. 2015).

cDNA synthesis and RT-qPCR

The microRNA-specific primers for mtr-miR159 and mtr-miR393 were designed using the mirBase program (Chen et al. 2005) (Table 1). The cDNA synthesis was carried out using the Hydra kit, which includes 5X buffer, dNTP, primers, cDNA RT, and water, following the manufacturer's protocols. For the expression analysis of mtr-miR159 and mtr-miR393, the 5 × HOT FIREPol EvaGreen® qPCR Supermix commercial kit was employed.

Table 1 mtr-mRi159, mtr-miR393, and 6U primer sequences
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The RT-qPCR conditions were completed over 40 cycles, comprising 15 s at 95 °C, 20 s at 58 °C, and 20 s at 72 °C, with a total duration of 92 min. RT-qPCR was performed on a RotorGene (Qiagen) real-time thermal cycler using standard parameters. Each experiment was conducted in triplicate, and differences in expression levels were assessed using the 2−ΔΔCT method (Chen et al. 2005).

Statistical analysis

The comparison of the results from the project trial plan involving 2 samples with 3 replications each was conducted through a two-way analysis of variance (ANOVA) utilizing the SPSS 22.0 software package. The determination of significant differences was achieved using Duncan's Multiple Comparison Test at a significance level of P ≤ 0.05.

Results

SEM, FT-IR, and XRD analyze results of CaO NPs

The hydrothermal synthesis method was employed for the preparation of CaO NPs. The SEM analysis, conducted using a Zeiss Sigma 300 model, reveals a particle size range of 66–145 nm. Importantly, the SEM images depict that the CaO NPs exhibit desirable characteristics, such as a porous structure, minimal clumping, and a multilayered arrangement. This information is crucial in understanding the physical properties of the synthesized nanoparticles.

The XRD spectrum (Fig. 1) provides insights into the crystalline structure of the CaO NPs. The peaks at specific angles (17.98°, 31.71°, 34.13°, and 50.75°) indicate the presence of Ca(OH)2 units, offering valuable information about the crystallographic composition of the synthesized nanoparticles. This analysis contributes to the overall understanding of the material's structure.

Fig. 1
figure 1

Scanning electron microscopy (A), Fourier-transform infrared spectrum (B) and X-ray diffraction pattern of CaO NPs (C)

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The F-TIR graph (Fig. 1) highlights important functional groups in the CaO NPs. The broad peak in the range of 3643–3450 cm-1 is associated with -O–H groups, suggesting the presence of water in the nanoparticle structure. The strong absorption band at 952 cm-1 is attributed to lattice vibrations of CaO. Additionally, the absorption bands at 1,382 cm-1 and 1,600 cm-1 are linked to the symmetric stretching vibration of non-identical carbonate. The explanation for the formation of carbonate species with –OH on the CaO surface during calcination adds context to the observed IR absorption features.

The interpretation provided in the results section is clear and connects the observed characteristics to the synthesis and exposure conditions. The formation of carbonate species with –OH on the surface due to the exposure of the highly reactive CaO surface area to air during calcination is a valuable insight that adds depth to the understanding of the material's properties.

SEM, FT-IR, and XRD analyze results of GO

Graphene oxide (GO) was synthesized using the Hummers method. The layered GO structures were visualized by zooming in on GO to 1 µm (10.42kx) using the SEM device (Fig. 2). The SEM image revealed that the layered GO structures exhibited overlapping layers and, at times, scattered configurations.

Fig. 2
figure 2

Scanning electron microscopy (A), Fourier-transform infrared spectrum (B) and X-ray diffraction pattern of GO (C)

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The XRD pattern of both GO and graphene nanosheets is presented in Fig. 2. The pattern aligns with graphite, featuring the characteristic peak (002) structure at 26.67°. The peak at approximately 11.6° is indicative of GO. The observed increase in the distance between GO layers suggests the presence of oxygen functional groups and water molecules within the carbon layer structure. Additionally, peaks at 23.9° and 26.5° suggest incomplete bonding of GO layers with oxygen atoms.

The FTIR spectrum of GO is depicted in Fig. 2. The peak around 3.000 cm-1 corresponds to the stretching peak of –OH groups in the reduced GO structure, which has undergone deoxygenation. The peak at 1.700 cm-1 reflects stresses in the C = O structure, while the peak around 1.150 cm-1 is associated with C-O stress peaks in the subsisting graphene structure. Peaks within the range of 1.200–1.060 cm-1 signify bond vibrations, indicating the presence of the unoxidized graphitic skeleton structure.

These results collectively provide a comprehensive understanding of the structural and chemical features of the synthesized GO, as elucidated through SEM, XRD, and FTIR analyses (Fig. 2).

Assessment of physiological and biochemical parameters

To investigate physiological and biochemical changes in two distinct local clover ecotypes, the experiment was replicated at least three times. The dry/wet weight of Erzurum's ecotype ranged from 0.114 to 0.062 g. As drought stress intensified, there was a notable decrease in dry/wet weight. Notably, the treatment of 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO (0.114 g) exhibited the most pronounced curative effect for the Erzurum ecotype. It was observed that applications of CaO NPs and GO had a beneficial impact on dry weight. Under drought stress conditions (mannitol treatment), there was an observed increase in proline concentration and a simultaneous decrease in soluble sugar concentration when compared to the control. Konya's ecotype exhibited a dry/wet weight range of 0.053 to 0.128 g (Fig. 3).

Fig. 3
figure 3

The effect of CaO NPs and GO on the percentage of dry and wet callus weight (Konya and Erzurum). The determination of significant differences was achieved using Duncan's Multiple Comparison Test at a significance level of P ≤ 0.05. *Konya 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/0.5 ppm GO, 7: 50 mM. mannitol/2 ppm CaO NPs/0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1 ppm CaO NPs, 10: 100 mM mannitol/1.5 ppm. GO, 11: 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO. **Erzurum 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/1 ppm CaO NPs, 7: 50. mM mannitol/2 ppm CaO NPs /0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1.5 ppm GO, 10: 100 mM mannitol/ 1 ppm. CaO NPs/0.5 ppm GO, 11: 100 mM mannitol/2 ppm CaO NPs /1.5 ppm GO

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In the Erzurum ecotype, drought stress conditions (mannitol treatment) led to an increase in proline concentration and a reduction in soluble sugar concentration compared to the control. Concurrent treatments with GO and mannitol, along with CaO NPs, demonstrated a beneficial impact on proline and water-soluble sugar content when compared to drought stress alone. Specifically, the application of 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO (0.968 μg/g YA) resulted in an elevated proline content, and the amounts of water-soluble sugar increased with 50 mM mannitol/1 ppm CaO NPs (0.398 mg/g YA) and 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO (0.271 mg/g YA) (Fig. 4).

Fig. 4
figure 4

The effect of basal medium on the amount of proline (A) and soluble sugars (B) in the Medicago sativa callus cultures. The determination of significant differences was achieved using Duncan's Multiple Comparison Test at a significance level of P ≤ 0.05. *Konya 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/0.5 ppm GO, 7: 50 mM. mannitol/2 ppm CaO NPs/0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1 ppm CaO NPs, 10: 100 mM mannitol/1.5 ppm. GO, 11: 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO. **Erzurum 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/1 ppm CaO NPs, 7: 50. mM mannitol/2 ppm CaO NPs /0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1.5 ppm GO, 10: 100 mM mannitol/ 1 ppm. CaO NPs/0.5 ppm GO, 11: 100 mM mannitol/2 ppm CaO NPs /1.5 ppm GO

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Similarly, in the Konya ecotype, drought stress increased proline content and decreased water-soluble sugar concentration compared to the control. However, the combined application of GO with CaO NPs exhibited a positive effect on both proline and water-soluble sugar concentrations compared to the stress factor control. Specifically, the application of 100 mM mannitol/1.5 ppm GO (0.989 μg/g FA) resulted in an elevated proline concentration, and the application of 100 mM mannitol/1 ppm CaO NPs (0.470 mg/g FA) had a positive effect on the amount of water-soluble sugar (Fig. 4).

In both Erzurum and Konya local ecotypes, drought stress increased MDA and H2O2 concentrations compared to the control, while applying CaO NPs and GO reduced them. Notably, the Erzurum variety displayed MDA levels ranging from 0.330 to 0.968 nmol/mL, with the lowest concentration observed in the treatment of 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO. H2O2 concentration varied between 0.0276 and 0.1095 μg/gr, with the lowest concentration observed in the treatment of 100 mM mannitol/1.5 ppm GO. Drought stress showed significant differences compared to the control (Fig. 5).

Fig. 5
figure 5

The effect of basal medium on the amount of H2O2 (A) and MDA (B) in the Medicago sativa callus cultures. The determination of significant differences was achieved using Duncan's Multiple Comparison Test at a significance level of P ≤ 0.05. *Konya 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/0.5 ppm GO, 7: 50 mM. mannitol/2 ppm CaO NPs/0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1 ppm CaO NPs, 10: 100 mM mannitol/1.5 ppm. GO, 11: 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO. **Erzurum 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/1 ppm CaO NPs, 7: 50. mM mannitol/2 ppm CaO NPs /0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1.5 ppm GO, 10: 100 mM mannitol/ 1 ppm. CaO NPs/0.5 ppm GO, 11: 100 mM mannitol/2 ppm CaO NPs /1.5 ppm GO

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For the Konya ecotypes, both MDA and H2O2 levels varied under drought stress, with the lowest concentrations observed in treatments involving 50 mM mannitol and 0.5 ppm GO. The most favorable results, considering both H2O2 and MDA, were found in treatments with 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO for the Erzurum ecotype and 50 mM mannitol/0.5 ppm GO for the Konya ecotype (Fig. 5).

Electron scanning microscope (SEM) and confocal laser scannİng mİcroscope (CLSM) analysis

In M. sativa callus exposed to drought stress under in vitro conditions, CaO NPs and GO were applied, and images of the callus, SEM, and CLSM were captured. Consequently, CaO NPs and GO were demonstrated to play a role in embryogenic callus formation and Ca2 + uptake by cells in alfalfa. In the Erzurum local ecotype, 1.5 ppm GO in the callus, compared to the control, reduced blackening and necrosis (Fig. 6 K1, K3). In the SEM image, membranous structures lacking Ca were found more frequently in the control (Fig. 6 S1, S2). On the other hand, 2 ppm CaO NPs exhibited dense filamentous structures, while 1.5 ppm GO showed spherical structures (Fig. 6 S3, S4).

Fig. 6
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Representative CaO accumulates images of Callus, SEM, and CLSM in Erzurum ecotype (K: callus, S: SEM, C: CLSM). K1, S1, C1: Control; K2, S2, C2: Ca-; K3, S3, C3: 2 ppm CaO NPs; K4, S4, C4: 1.5 ppm GO, K5, S5, C5: 50 mM mannitol; K6, S6, C6: 50 mM mannitol/1 ppm CaO NPs; K7, S7, C7: 50 mM mannitol/2 ppm CaO NPs/0.5 ppm, GO; K8, S8, C8: 100 mM mannitol, K9, S9, C9: 100 mM mannitol/1.5 ppm GO; K10, S10, C10: 100 mM mannitol/1 ppm CaO NPs/0.5 ppm GO; K11, S11, C11: 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO

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In the CLSM image, Ca2+ accumulation was observed to be highest at 2 ppm CaO NPs and lowest under Ca deficiency and with 1.5 ppm GO (Fig. 6 C1, C2, C3, C4). When comparing the callus images, it was evident that, under 50 and 100 mM mannitol treatments, blackening and necrosis were more pronounced in the 100 mM mannitol condition (Fig. 6 K5, K8). Notably, 50 mM mannitol with 1 ppm CaO NPs and 50 mM mannitol with 2 ppm CaO NPs and 0.5 ppm GO concentrations demonstrated a healing effect (Fig. 6 K6, K7). In the SEM image, membrane and globular structures were observed in 50 mM mannitol and 50 mM mannitol with 1 ppm CaO NPs (Fig. 6 S5, S6). For 50 mM mannitol with 2 ppm CaO NPs and 0.5 ppm GO, filamentous structures were observed, while amorphous structures were observed in 100 mM mannitol (Fig. 6 S7, S8). In CLSM, Ca2+ accumulation, from most to least, was observed in 50 mM mannitol with 2 ppm CaO NPs and 0.5 ppm GO, 50 mM mannitol with 1 ppm CaO NPs, 50 mM mannitol, and 100 mM mannitol (Fig. 6 C5, C6, C7, C8).

Looking at the callus images, it was observed that the highest concentration of drought stress, under 100 mM mannitol treatment, had an ameliorative effect on CaO NPs and GO treatments (Fig. 6 K9, K10, K11). Examining the SEM images, 100 mM mannitol with 1.5 ppm GO and 100 mM mannitol with 2 ppm CaO NPs and 1.5 ppm GO exhibited spherical (globular) structures, while 100 mM mannitol with 1 ppm CaO NPs and 0.5 ppm GO showed filamentous and curved structures (Fig. 6 S9, S11, S10). In CLSM, Ca2+ accumulation, from most to least, was observed in 100 mM mannitol with 2 ppm CaO NPs and 1.5 ppm GO, 100 mM mannitol with 1 ppm CaO NPs and 0.5 ppm GO, and 100 mM mannitol with 1.5 ppm GO (Fig. 6 C9, C10, C11)."

In Konya, local ecotype, CaO NPs and GO induced the initiation of embryogenic callus formation (Fig. 7 K3, 4). When the callus samples without Ca were compared with the control, necrosis and a brown structure were observed (Fig. 7 K2,1). In the SEM image, the control and Ca-free callus samples have a very compact structure. While 2 ppm CaO NPs callus had a spherical (globular) structure, 1.5 ppm GO had both a compact structure and a membranous structure (Fig. 7 S1, 2, 3, 4). In the CLSM image, compared to the control, Ca2+ accumulation was highest in 2 ppm CaO NPs and lowest in Ca-free callus samples observed (Fig. 7 C1, 2, 3, 4).

Fig. 7
figure 7figure 7figure 7

Representative CaO accumulates images of Callus, SEM, and CLSM in Konya ecotype (K: callus, S: SEM, C: CLSM). K1, S1, C1: Control; K2, S2, S2: Ca-; K3, S3, C3: 2 ppm CaO NPs; K4, S4, C4: 1.5 ppm GO; K5, S5, C5: 50 mM mannitol; K6, S6, C6: 50 mM mannitol/0.5 ppm GO; K7, S7, C7: 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO; K8, S8, C8: 100 mM mannitol; K9, S9, C9: 100 mM mannitol/ 1 ppm CaO NPs; K10, S10, C10: 100 mM mannitol/1.5 GO; K11, S11, C11: 100 mM mannitol/ 2 ppm CaO NPs/1.5 ppm GO

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In the callus images of the Konya ecotype, it was observed that necrosis and brown structures increased as drought stress increased (Fig. 7 K1, 4). The 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO and 50 mM mannitol/0.5 ppm GO treatments tolerated the drought effect (Fig. 7 K6, 7). In the SEM image, the 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO treatment exhibited an amorphous structure, and the 100 mM mannitol treatment had a very dense compact structure (Fig. 7 S7, 8). The spherical (globular) structures in control and 50 mM mannitol/0.5 ppm GO treatments were similar (Fig. 7 S5, 6). In CLSM images, the highest Ca2 + accumulation was observed in 50 mM mannitol/2 ppm CaO NPs/0.5 ppm GO and 50 mM mannitol/0.5 ppm GO, while the least accumulation was observed at 50 and 100 mM mannitol (Fig. 7 C5, 6, 7, 8)..

In callus images, it was observed that 100 mM mannitol/1 ppm CaO NPs treatment promoted somatic embryo formation (Fig. 7 K9). 100 mM mannitol/1.5 ppm GO and 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO treatments also had an ameliorative effect (Fig. 7 K10, 11). In the SEM image, spherical (globular) structures were observed, and the treatments observed from the most compact to the least compact were 100 mM mannitol/2 CaO NPs/1.5 ppm GO, 100 mM mannitol/1.5 ppm GO, 100 mM mannitol/1.5 ppm GO, 100 mM mannitol/1.5 ppm GO, and 100 mM mannitol/1.5 ppm GO, respectively, and 100 mM mannitol/1.5 ppm GO, respectively (Fig. 7 S9, 10, 11). In CLSM images, the most intense accumulation of Ca2 + was observed in 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO, while the least accumulation was at 100 mM mannitol/1.5 ppm GO (Fig. 7 C9, 10, 11).

Evaluation of altered mtr-miR159 and mtr-miR393 gene expression by RT-qpcr

The fold change in mtr-miR159 and mtr-miR393 gene levels was calculated according to 2−ΔΔCT. In the Konya ecotype, mtr-miR159 increased in the 50 mM/2 ppm CaO NPs/0.5 ppm GO treatment (35.405) compared to the control (0.381). The expression of mtr-miR393 in the Konya ecotype was highest in the 100 mM/2 ppm CaO NPs/1.5 ppm GO treatment (43.630), followed by the 50 mM/2 ppm CaO NPs/0.5 ppm GO treatment (20.284), and the 100 mM/1 ppm CaO NPs treatment (12.102) (Fig. 8).

Fig. 8
figure 8

Heat map diagram of mtr-miR159 and mtr-miR393 gene expression levels for drought stress genes analyzed by Real-Time Quantitative PCR. The heat map was drawn according to fold change value. Columns and rows in the heat map represent treatment and genes, respectively. Medicago sativa (A Konya, B Erzurum) ecotype names are displayed upper the heat map. Color scale indicates fold changes in gene expression (used heatmapgenerator5 tool). *Konya 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/0.5 ppm GO, 7: 50 mM. mannitol/2 ppm CaO NPs/0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1 ppm CaO NPs, 10: 100 mM mannitol/1.5 ppm. GO, 11: 100 mM mannitol/2 ppm CaO NPs/1.5 ppm GO. **Erzurum 1: Control, 2: Ca-, 3: 2 ppm CaO NPs, 4: 1.5 ppm GO, 5: 50 mM mannitol, 6: 50 mM mannitol/1 ppm CaO NPs, 7: 50. mM mannitol/2 ppm CaO NPs /0.5 ppm GO, 8: 100 mM mannitol, 9: 100 mM mannitol/1.5 ppm GO, 10: 100 mM mannitol/ 1 ppm. CaO NPs/0.5 ppm GO, 11: 100 mM mannitol/2 ppm CaO NPs /1.5 ppm GO

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In the Erzurum ecotype, mtr-miR159 increased with the 100 mM/1 ppm CaO NPs/0.5 ppm GO treatment (4.605) compared to the control (4.208). For mtr-miR393 in the Erzurum ecotype, the highest expression was observed in the 100 mM treatment (9.397), followed by the 100 mM/2 ppm CaO NPs/1.5 ppm GO treatment (4.104), and the 100 mM/1.5 ppm GO treatment (2.5) (Fig. 8).

Dıscussıon

Drought stress encompasses a wide array of cellular events, including morphological traits in plants such as leaf area, stem and root length, as well as physiological, biochemical, and gene-level changes (Gopal et al. 2008; Redmond and Tseng 1979). The adverse impacts of drought stress on plants can be characterized by various parameters, including morphological, physiological, biochemical, and gene-level changes, along with alterations in nutrient content that are utilized to monitor plant growth. In recent years, there has been an increasing focus on studying the drought tolerance of crops to address current and future risks associated with climate change (Chaves et al. 2002; Gray et al. 2012).

Previous research has demonstrated the importance of nanoparticles in seed germination and development (Salama 2012; Zheng et al. 2005). However, it has been observed that plants require calcium for their growth and development. Recent studies indicate that CaO NPs have garnered global attention due to their promising agricultural applications. In this study, the assessment of stress tolerance in the presence of CaO NPs, graphene oxide (GO), and mannitol-induced drought was based on the determination of dry weight, proline, soluble sugars, malondialdehyde (MDA), and hydrogen peroxide (H2O2) content, in addition to changes at the gene level, and analysis using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).

The application of CaO NPs, GO, and mannitol-induced drought to the callus of local alfalfa ecotypes subjected to drought stress resulted in the mitigation of drought damage across all developmental stages of alfalfa compared to control callus. Calcium ion (Ca2+) accumulation increased in Erzurum and Konya callus treated with CaO NPs and GO compared to the control callus (Fig. 6 C3,7 and Fig. 7 C3,7).

The response to CaO NPs and GO can be influenced by factors such as plant developmental stages, characteristics of tissue culture media, explant sources, genotype, and media temperature. Our results demonstrate a significant genotype and mannitol level-dependent variation in the impact of CaO NPs and GO, as depicted in Figs. 3, 4, and 5. A similar observation of Onobrychis viciifolia regeneration capacity was noted exclusively under CaO NPs treatment during drought stress (Ertuş and Yazıcılar 2023). Positive effects of CaO NPs on chickpea (Cicer arietinum L.) growth were also reported (Gandhi et al. 2021).

Calcium (Ca2+) faces challenges due to its immobility in the phloem, restricting the redistribution of Ca2+ from old tissues to young tissues in plants (Hamza et al. 2021). Given the shortcomings of traditional calcium application methods, using nano-sized calcium particles appears to be more efficient in supplying calcium to alfalfa. Furthermore, employing low levels of sulfonated graphene can remove reactive oxygen species (ROS) in maize roots, leading to changes in root structure and fostering better seedling growth (Liu et al. 2020).

Adding CaO NPs and GO to the medium altered the structure of the callus. The health of the callus is associated with its tendency to become glassy in texture (vitrification); therefore, changes in the amount of ions or water in the callus can be important. High doses of Ca2+ NPs in M. sativa callus suggest such a correlation. Ali et al. (2016) demonstrated the importance of Ag NPs applications in adjusting the micronutrient content of the medium for Caralluma tuberculata callus structures under in vitro conditions.

Confocal laser scanning microscopy (CLSM) analysis is crucial for understanding the localization of Ca2+ NPs, stress response, and tolerance of plant varieties to drought stress. It has been widely used to observe Ca2+ accumulation in stressed cells and rapidly verify Ca2+ localization in living cells (Sun et al. 2014). The application of CLSM in M. sativa callus serves as an essential strategy for understanding tolerance in response to drought stress, as evidenced by the observed Ca2+ accumulation in Erzurum and Konya callus.

Nanoparticles, either alone or in combination with drought stress, have been applied to some plant species to control both biotic and abiotic stress in plant tissue culture. In our study, we evaluated the response to treatments with CaO NPs and GO, including mannitol, in Erzurum and Konya's ecotypes under a confocal laser scanning microscope. Drought stress development was assessed over a 30-day period. In the 2 ppm CaO NP treatment, callus cells showed a strong red color, indicating significant absorption of calcium ions (Ca2+) (Fig. 6 C1 and Fig. 7 C1). Likewise, there was a high accumulation of Ca2+ in the 2 ppm CaO NP treatment under drought conditions. However, callus treated with 50 and 100 mM mannitol without added calcium showed almost no presence of Ca2+.In other treatments, Ca2+ accumulation was observed at a moderate level. Both callus and SEM images indicated that CaO NPs and GO application significantly increased the somatic embryonic rate and reduced blackening and necrosis structures. Various structures, including amorphous, compact, filamentous, membranous, and spherical (globular), were observed in SEM images (Fig. 6 and Fig. 7). Ca accumulation rates, based on SEM and EDS analysis, increased with 2 ppm and increasing CaO NPs and decreased with the severity of drought stress. Ertuş and Yazıcılar (2023) obtained similar results in Onobrychis viciifolia exposed to drought stress through CaO NPs application, aligning with our findings.

Proline is recognized as one of the osmoprotectants synthesized by plants in response to stress conditions (Grzesiak et al. 2013; Kumar et al. 2008; Nagesh Babu and Devaraj 2008; Saradhi et al. 1995). Despite its primary osmoprotective role, some studies have indicated that proline plays a crucial role in maintaining redox balance within cells (Mohammadkhani and Heidari 2008). In the current study, the concentration of proline increased with the intensity of drought stress, with the highest proline content observed in the Erzurum ecotype (Fig. 4). Cano et al. (1998) noted that stress-sensitive genotypes exhibited higher proline accumulation than stress-resistant genotypes in tomatoes grown under in vitro conditions. Additionally, proline has been widely employed as a marker for stress resistance (Alvarez et al. 2003). However, the relationship between proline accumulation and abiotic stress tolerance in plants is not universally clear. For instance, while Arabidopsis mutants with elevated proline content are hypersensitive to salt and cold (Liu et al. 2017, 2018), the proline content of drought-tolerant rice cultivars is not necessarily associated with salt tolerance in barley (Hordeum vulgare) (Zhang et al. 2015; Allen et al. 2007).

Drought stress poses a threat to the integrity of plant cell membranes. The membrane system in plants serves as a crucial barrier between cells and the external environment, playing a pivotal role in maintaining the microenvironment and supporting normal cellular metabolism. ROS (reactive oxygen species) can inflict damage on membrane phospholipids, leading to peroxidation of membrane lipids and subsequent generation of MDA (malondialdehyde) (Møller et al. 2007). The accumulation of MDA is commonly utilized as an indicator of electrolyte conductance (EC) through cells, cell membrane damage, and drought resistance in plants. Additionally, Moore (2006) observed that nanoparticles (NPs) interact with the lipid bilayer of the plasma membrane, inducing alterations in ROS levels and metabolic processes. Both MDA and H2O2 (hydrogen peroxide) content serve as crucial markers of oxidative stress in plants (Vosough et al. 2015). Under stress conditions, lower levels of these compounds correlate with reduced damage to plants (Pirasteh-Anosheh and Emam 2018). Genotypes and cultivars exhibiting lower MDA and H2O2 level under stress conditions have been associated with higher stress tolerance.

In the context of this thesis, both MDA and H2O2 content exhibited an increase with the severity of drought stress; however, the response varied among the different ecotypes (Fig. 5). Additionally, the impact of stress was evident in the reduction of dry/wet weight and soluble sugars. Intriguingly, the application of CaO NPs and GO treatments resulted in an increase in dry/wet weight and soluble sugars, suggesting a potential ameliorative effect under stress conditions (Fig. 3, 4).

miRNAs play a crucial role in negatively regulating the expression of target genes by either stopping or inhibiting translation, thereby serving as key regulators at various stages of plant growth (Bartel 2004; Achkar et al. 2016; Jones-Rhoades et al. 2006; Wani et al. 2020). They are known to play a regulatory role in enhancing the adaptability of plants to drought stress (Liu et al. 2020; Fan et al. 2017). Graphene oxide (GO) has been shown to up-regulate the expression of aquaporins and phosphate transporter-related genes (Cao et al. 2020), and graphene material, in general, can up-regulate genes associated with root development and auxin content, promoting morphological development and biomass accumulation in tomato seedlings (Guo et al. 2021). GO has also been found to directly enhance plant defense enzymes, hormone content, and the expression of drought-related genes, thus improving drought resistance in soybeans. Furthermore, it has been reported to increase drought tolerance in Zea mays L., Paeonia ostii, and M. sativa (Lopes et al. 2022; Chen et al. 2021).

The miR159 family is highly conserved among monocot and dicot plants. However, under drought conditions, miR159 exhibits tissue- and species-specific variations. For instance, miR159 is up-regulated by drought stress in Arabidopsis and maize, while it is down-regulated in cotton and potato. In barley and alfalfa, it is down-regulated in the root but up-regulated in the leaf (Liu et al. 2008; Wei et al. 2009; Xie et al. 2015; Hackenberg et al. 2015). LjmiR156 increased soluble sugar content in alfalfa, contrasting with the decrease observed when MsmiR156d was overexpressed (Aung et al. 2015). miR159 is induced by ABA (abscisic acid) and drought treatments in regenerating Arabidopsis seeds (Reyes and Chua 2007). In Arabidopsis, miR159a mediates the cleavage of MYB33 and MYB101 transcripts (Reyes and Chua 2007; Allen et al. 2007). Overexpression of miR159a suppresses MYB33 and MYB101 mRNA levels, rendering plants hypersensitive to ABA; transgenic plants overexpressing cleavage-resistant forms of MYB33 and MYB101 also show hypersensitivity to ABA treatment. MYB transcription factors bind to cis-elements in the dehydration-responsive Gene Response to Dehydration 22 (RD22) promoter and co-activate RD22. Overexpression of both MYC2 and MYB2 improves the osmotic stress tolerance of transgenic plants (Abe et al. 2003). A study on Moringa oleifera showed that identifying microRNAs and their target genes in callus led to enhanced production of medicinal compounds under cold stress conditions by increasing the levels of miR159 and miR393 (Pirrò et al. 2019). Overexpression of miR397 in Liriodendron callus promotes callus formation and somatic embryo induction whereas retard callus proliferation (Wang et al. 2021). In Rice embryogenic callus, expression levels of miR319 and miR156 exhibit distinct alters during the transition from callus phase to embryogenic callus phase when somatic embryogenesis occurs, suggesting regulatory roles of microRNAs in vitro regeneration development of somatic embryos (Chen et al. 2011).

In our study, mtr-miR159 was found to be down-regulated in the Konya and Erzurum ecotypes, resulting in decreased MYB transcription levels for MYB33 and MYB10 in these ecotypes (Fig. 8).

miR393 has been demonstrated to play a pivotal role in regulating auxin signaling, thereby influencing plant growth and development under drought stress conditions. It is well-established that miR393 is widely up-regulated during drought stress in Arabidopsis, rice, and sugarcane (Saccharum spp.) (Sunkar and Zhu 2004; Ferreira et al. 2012; Li et al. 2017). In Arabidopsis, the target of miR393 encodes TIR1 (transport inhibitor response 1), an auxin receptor. The TIR1 enzyme acts as a positive regulator of auxin signaling by facilitating the degradation of Aux/IAA proteins through ubiquitination (Dharmasiri and Estelle 2002). Xia et al. (2012) reported that rice seedlings overexpressing miR393 exhibited suppressed growth after a 1-day drought treatment compared to control plants. Therefore, elevated miR393 levels down-regulate auxin signaling, leading to reduced plant growth under drought stress. In this study, mtrmiR393 was found to be down-regulated in the Konya ecotype and up-regulated in the Erzurum ecotype. Down-regulation was associated with a negative impact on plant growth by suppressing mRNA. With the application of CaO NPs and GO, down-regulation was observed in the Konya ecotype, accompanied by increased plant growth (Fig. 8).

In this investigation, CaO NPs and GO were applied to drought-exposed Erzurum and Konya callus to explore changes at the biological, biochemical, and gene levels. An unorganized cell community was preferred, and CaO NPs and GO were employed to mitigate the effects of drought stress under in vitro conditions. Upon evaluating the results, a reduction in blackening and necrosis in the callus structures was noted, accompanied by an increase in embryogenic callus formation. Moreover, mtr-miR393 and mtr-miR159 were up-regulated under drought stress, while they were down-regulated following treatment with CaO NPs and GO. SEM and CLSM results indicated a decrease in Ca content under drought stress, whereas an increase was observed under CaO NPs and GO treatment. Taken together, positive outcomes were observed in the callus. The subsequent phase of this study aims to regenerate callus structures for the formation of complete plants. Regenerated plants provide an opportunity for detailed observation of all morphological, physiological, and biochemical changes, as all tissue and organ structures develop in these plants.