Mitigation of Drought Stress Effects on Alfalfa (Medicago sativa L.) Callus through CaO Nanoparticles and Graphene Oxide in Tissue Culture Conditions

Drought stress poses a signi�cant threat to fertile soils worldwide, triggering profound physiological, biochemical, and molecular changes in plants that adversely impact agricultural productivity. This study explores the potential of nanotechnology, speci�cally Calcium Oxide Nanoparticles (CaO NPs) and Graphene Oxide (GO), to ameliorate the negative effects of drought stress on two distinct alfalfa ecotypes. Seeds from Erzurum and Konya regions were regenerated in the Murashige and Skoog (MS) medium, and ensuing callus formation was induced through 2,4-D and Kinetin. The callus samples underwent a one-month treatment with varying concentrations of mannitol (50 and 100 mM), CaO NPs, and GO (0.5 and 1.5 ppm). Results revealed a decrease in dry/wet weight with increasing mannitol concentration, contrasting with an increase in weight under CaO NPs and GO treatment. Proline, DNSA, MDA, and H 2 O 2 exhibited proportional increases under drought stress, while CaO NPs and GO treatments mitigated these effects. Physiological and biochemical analyses identi�ed optimal conditions for Erzurum as 50 mM mannitol/2 CaO NPs/0.5 ppm GO, and for Konya as 50 mM mannitol/0.5 ppm GO. Gene expression analysis indicated up-regulation of mtr-miR159 and mtr-miR393 with heightened drought stress, with down-regulation observed in CaO NPs and GO treatments. Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) con�rmed Ca 2+ accumulation in alfalfa tissues. In conclusion, CaO NPs and GO treatments exhibited a signi�cant reduction in the adverse effects of drought stress on alfalfa callus under tissue culture conditions. This research sheds light on the potential of nanotechnological interventions to alleviate the impact of environmental stressors on crop plants, opening avenues for sustainable agriculture in the face of changing climatic conditions. Further investigations are warranted to elucidate the underlying mechanisms and scalability of these �ndings for �eld applications.


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 xation 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 noncoding 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.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 scienti c 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.
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.However, the complexity of nanomaterials, in uenced by factors such as class, concentration, characteristics, plant species, and seed size, has become evident in recent studies (Wu et al. 2012).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. 2011; Khan et al. 2019).Supported transition metal oxide in reduced graphene oxide catalyzed reactions offers advantages such as high atomic e ciency, simpli ed 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.Insu cient Ca availability results in symptoms such as tip blight in lettuce (Lactuca sativa L.) and ower 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 speci cally 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 modi cations of CaO NPs and GO, in comparison to a control group.As drought stress poses a signi cant challenge to agricultural productivity, understanding the speci c 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-speci c responses that may emerge.

Hydrothermal Synthesis of CaO NPs
CaO NPs were synthesized through a hydrothermal process.In a Te on-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 CaCl 2 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 hours.The resultant precipitate was isolated through centrifugation at 15,000 xg for 30 minutes 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 NaNO 3 , and 115 mL of 96.4% sulfuric acid were combined in an ice bath and placed on a magnetic stirrer for 1 hour.Subsequently, 15 g of KMnO 4 was cautiously introduced into the mixture.
After removing the ice bath, the mixture was stirred on a magnetic stirrer for an additional 2 hours.Following this, 500 mL of deionized water was added, and the mixture continued to be stirred on the magnetic stirrer for another 1 hour.To this, 8.4 mL of H 2 O 2 was incorporated into the mixture and stirred for an additional 2 hours.The resulting mixture underwent ltration, with deionized water being added until reaching a pH of 7. The ltrate 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 minutes, followed by three washes in distilled water.Leaves were excised from three-week-old plants and placed on a hormone-free MS medium (Murashige & Skoog, 1962) in vitro.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.The samples were maintained in an acclimatization chamber for a total duration of 1 month, under conditions of 16 hours of light and 8 hours of darkness, at a temperature of 25°C, and with a humidity level of 50%.

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 minutes, 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 hour, 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 minutes.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 minutes.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.
Hydrogen peroxide (H 2 O 2 ) To quantify H 2 O 2 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 x g for 10 minutes, and the pellet was discarded.A 1.5 mL aliquot of the supernatant was retrieved and combined with 150 µL of 5% Ti(SO 4 ) 2 (titanium disulphate) and 0.3 mL of 19% NH 4 OH (ammonium hydroxide).The resulting mixture underwent centrifugation again at 10,000 x g for 10 minutes.The pellet was dissolved in 3 mL of 2 M H 2 SO 4 and thoroughly mixed.The absorbance of the nal mixture was measured at 415 nm.The results are expressed as the quantity of H 2 O 2 per gram of callus (µg/g of callus) (Hao et al. 2014).

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 minutes, 100 µL of chloroform was added and slowly inverted.The samples were then left at room temperature for 3 minutes and centrifuged at 12,000 rpm at 4°C for 15 minutes.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 minutes, they were centrifuged at 12,000 rpm at 4°C for 10 minutes.
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 minutes.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-speci c 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 5x HOT FIREPol EvaGreen® qPCR Supermix commercial kit was employed.
The RT-qPCR conditions were completed over 40 cycles, comprising 15 seconds at 95°C, 20 seconds at 58°C, and 20 seconds at 72°C, with a total duration of 92 minutes.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 signi cant differences was achieved using Duncan's Multiple Comparison Test at a signi cance level of P ≤ 0.05.

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 speci c 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.
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.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 con gurations.
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 re ects 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 1.200-1.060cm 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 intensi ed, 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 bene cial 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).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 bene cial impact on proline and water-soluble sugar content when compared to drought stress alone.Speci cally, 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).
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.Speci cally, 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).Drought stress showed signi cant differences compared to the control (Fig. 5).
For the Konya ecotypes, MDA levels under drought stress varied between 0.3357 and 0.640 nmol/mL.The treatment with the lowest MDA concentration was 50 mM mannitol/0.5 ppm GO (0.335 nmol/mL).The H 2 O 2 concentration ranged between 0.014 and 0.096 µg/gr, with the lowest concentration observed in the treatment of 50 mM mannitol/0.5 ppm GO (0.014 µg/gr).Upon analyzing H2O2 and MDA, the treatments of 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 yielded the most favorable results (Fig. 5).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. 6K1, K3).In the SEM image, membranous structures lacking Ca were found more frequently in the control (Fig. 6S1, S2).On the other hand, 2 ppm CaO NPs exhibited dense lamentous structures, while 1.5 ppm GO showed spherical structures (Fig. 6S3, S4).
In the CLSM image, Ca 2+ accumulation was observed to be highest at 2 ppm CaO NPs and lowest under Ca de ciency and with 1.5 ppm GO (Fig. 6C1, 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. 6K5, 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. 6K6, 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. 6S5, S6).For 50 mM mannitol with 2 ppm CaO NPs and 0.5 ppm GO, lamentous structures were observed, while amorphous structures were observed in 100 mM mannitol (Fig. 6S7, S8).In CLSM, Ca 2+ 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. 6C5, C6, C7, C8).
In Konya, local ecotype, CaO NPs and GO induced the initiation of embryogenic callus formation (Fig. 7K3, 4).When the callus samples without Ca were compared with the control, necrosis and a brown structure were observed (Fig. 7K2,1).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. 7S1, 2, 3, 4).In the CLSM image, compared to the control, Ca 2+ accumulation was highest in 2 ppm CaO NPs and lowest in Ca-free callus samples.observed (Fig. 7C1, 2, 3, 4).
In the local ecotype of Konya, CaO NPs and GO induced the initiation of embryogenic callus formation (Fig. 7K3, 4).When comparing callus samples without Ca with the control, necrosis and a brown structure were observed (Fig. 7K2,1).In the SEM image, the control and Ca-free callus samples exhibited a very compact structure.While the 2 ppm CaO NPs callus had a spherical (globular) structure, the 1.5 ppm GO had both a compact structure and a membranous structure (Fig. 7S1, 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. 7C1, 2, 3, 4).

DISCUSSION
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 (H 2 O 2 ) 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 (Ca 2+) accumulation increased in Erzurum and Konya callus treated with CaO NPs and GO compared to the control callus (Fig. 6C3,7 and Fig. 7C3,7).
The response to CaO NPs and GO can be in uenced by factors such as plant developmental stages, characteristics of tissue culture media, explant sources, genotype, and media temperature.Our results demonstrate a signi cant 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 (Ca 2+ ) faces challenges due to its immobility in the phloem, restricting the redistribution of Ca 2+ from old tissues to young tissues in plants (Hamza et al. 2021).In light of this limitation in conventional Ca 2+ application, nano-sized Ca particles prove more effective in providing Ca nutrition in alfalfa.Additionally, low concentrations of sulfonated graphene scavenge reactive oxygen species (ROS) in maize roots, altering root morphology and promoting healthier seedling formation (Liu et al. 2020).
The introduction of CaO NPs and GO into the medium induced changes in the callus structure.Callus viability is linked to vitri cation in the tissue; thus, alterations in callus ion strength or water status can be signi cant.High doses of Ca 2+ 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 Ca 2+ NPs, stress response, and tolerance of plant varieties to drought stress.It has been widely used to observe Ca 2+ accumulation in stressed cells and rapidly verify Ca 2+ localization in living cells (Hussain et al. 2016;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 Ca 2+ 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.Callus cells exhibited intense red coloration due to Ca 2+ uptake in the 2 ppm CaO NP treatment (Fig. 6C1 and Fig. 7C1).Similarly, Ca 2+ accumulation was high in the 2 ppm CaO NPs treatment with the drought factor, whereas Ca 2+ was almost absent in callus with 50 and 100 mM mannitol and no Ca.In other treatments, Ca 2+ accumulation was observed at a moderate level.Both callus and SEM images indicated that CaO NPs and GO application signi cantly increased the somatic embryonic rate and reduced blackening and necrosis structures.Various structures, including amorphous, compact, lamentous, 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 ndings.
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 Devraj 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(Liu et al. , 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 in ict damage on membrane phospholipids, leading to peroxidation of membrane lipids and subsequent generation of MDA (malondialdehyde) (Møller 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 H 2 O 2 (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 content under stress conditions have been associated with higher stress tolerance.
In the context of this thesis, both MDA and H 2 O 2 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 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 droughtrelated 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-speci c 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  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 in uencing 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;Zhao et al. 2007).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.

Declarations
Con ict of interest authors have declared that no con ict of interests exists.

Figure 1 .
Figure 1.SEM (a), FT-IR spectra (b) and X-ray diffraction pattern (c) of CaO NPs SEM, FT-IR, and XRD Analyze Results of GO

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