Assessment of the Effects of Newly Fabricated CaO, CuO, ZnO Nanoparticles on Callus Formation Maintainance of Alfalfa (Medicago Sativa L.) Under In Vitro Salt Stress

Nanoparticules plays an important role in plant adaptation to abiotic stress, especially in response to salt stress. In this study, two alfalfa lines (Erzurum, and Muş) were used as the material for the response NaCl to CuO, ZnO and CaO nanoparticules (NPs). CaO is evident to be higher effective than CuO, ZnO in callus induction from leaf explants. The antioxidant enzyme activities were also determined in the callus cultures. The maximum activity in MDA analysis was observed from callus treated of 50 mM NaCl with 0.8 ppm CuO NPs. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for H 2 O 2 value compared to the other NPs. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for POD value compared to the other NPs for POD activity. The best response in protein rate was obtained from callus induction stage and callus formation stage after 50 mM treatment NaCl with 0.8 ppm CuO. LSCM analysis evident that the NPs could migitate the negative effects of NaCl stress by the elimination of stress severity in callus cells. SEM analysis was supported the results obtained by LSCM analysis. Our ndings suggest that CuO, CaO and ZnO NPs can offer a simple and effective method to protect alfalfa callus from NaCl stress severity. laser scanning


Introduction
Agricultural productivity is signi cantly constrained by biotic and abiotic environmental factors (Ji et al. 2013). Salt stress is a growing issue which is becoming a serious environmental stress around the world Excess concentrations of various types of salts such as carbonates, calcium, chlorides, sulfates, magnesium, potassium and sodium, de ne various salinity growth mediums. Various control strategies have been supposed to challenge salt stress (Yazıcılar et al. 2021; Gao et al. 2016). Salt stress decreases root growth, stem expansion, leaf length, disrupts water-use capacity, and lowers plant water activity. Plants exhibit a difference of metabolic and physiological reactions at cellular and wholeorganism levels against salt stress, thus making it a confusing event (Bezirganoglu 2017). The salt resistance trait is regulated by many physiological defensive strategies through complex enzymatic controlling pathways (Parida et al. 2005;Parihar et al. 2015). Metabolic process affects growth by challenging plants with photosynthesis, protein synthesis and lipid metabolisms. Physiological strategies like osmotic, ionic, oxidative stress and hormonal imbalances are affected due to salt stress. Salinity in uences growth cope with of plants by the excess of Na and Cl ions in the medium that reduces the osmotic potential and inhibits the nutrients and water uptake (Gao et al. 2016). It is important to understand how salt stress challenges signals on the various levels of cells to activate the adaptive process in the plant (Manchanda and Garg 2008). Nanotechnology plays an important role in research tools that solutions to the multiple agriculture-related matters. Nanotechnology has a larger application than biotechnology containing gene transformation, genomics, proteomics and bioinformatics and other technologies (Vijayakumar et al. 2010; Kim et al. 2017). Nanotechnologies can provide to enhance product capacity in less yield crops which is contributing in sustainable agriculture.
Moreover, nanotechnology has helped new potential for enhancing the structure of foods, avour, higher protein content, and improved nutritional values. Nanoparticles have assisted to develop crop productivity by introducing such qualities as biotic resistance and increased abiotic stress resistance to the crops. understanding of the potential effects during in vitro culture of plant tissues. To date, microscopy techniques have been used to study the assimilation and accumulation of NPs in plants under in vitro conditions. Alfalfa is used livestock feed and is superior to other forage crops in terms of nutritional quality. It has a rich and long history and is one of the earliest crops domesticated by human (Putnam et al. 2001). Alfalfa is cultivated for its rich source of proteins, carbohydrates, vitamins, minerals and diatery bres. It also cultivated for its high yield and nutritional feeding quality, as well as its role on N xation, soil conservation (Sakiroglu and Brummer 2017). However, alfalfa production is severely reduction by salt stress. Plants thrive by maintaining cell division and proliferation. Copper is an important micronutrient with many functions including redox reactions and participating the synthesis of chlorophyll and metabolism of carbohydrate and protein. Tolerance of diseases and crop yields can be in uenced by de ciency of Cu (Dimpka et al. 2012) Zn is essential micronutrient participating directly in metabolic activities in plants such as formation of protein and carbonhydrate and synthesis of chlorophly as well as involving in the synthesis of auxin and indole acetic acid IAA from tryptophan. It was found that Zn key functions in regulation of redoxs systems and conservation of plant cells in response to oxidative stress. Zn de ciency plants have decreased crop productivity and reduced quality of crops (Lian et al. 2019). Understanding the response of alfalfa plants toward salinity stress at the nanobased level and developing salt-resistant cultivars are the vital mandates for its effective management. Till date, no previous research article is available in literature to show the effects of, CuO and ZnO NPs on callus biomass formation and production of antioxidants in callus cultures of alfalfa. Therefore, the over all objective of the current study was to evaluate the possible effects of NPs on callus induction, biomass formation and extension of desirable levels of resistance to salt stress in alfalfa. Moreover, free radical scavenging activity and the antioxidant enzyme activities were also determined in the callus cultures.

Plant materialandCallus induction
In our study, two alfalfa lines (Erzurum, and Muş) were used as the material for the response to CaO, CuO and ZnO NPs nanoparticulate. The mature seeds were sterilized with 1% NaOCl for 5 min, washed several times with sterile distilled water and rinsed with several changes of sterile distilled water overnight at 4 0 C. The mature seeds were cultivated in Petri dishes containing full MS medium (Murashige and Skoog 1962) for 30 days at 25±1 and in 16 hours light / 8 hours dark photoperiod at 1500 lux illumination intensity. Leaves were removed aseptically using forceps and placed on MS medium (Murashige and

POD (Peroxidase)
The activity of POD (Peroxidase) was measured following the procedure established by Chance and Maehly (1955) by adding 100 μL of the callus extract to 3 mL of assay solution, which contained 13 mM guaiacol, 5mM H 2 O 2 and 50Mm Na-P buffer (pH 6.5). The POD (Peroxidase) activity was determined in absorbance at 470 nm of protein. The total soluble protein contents were determined by BCA (Bicinchoninic Acid) protocol (Yee et al. 2003;Erdal 2012).

Sectioning with microtonal
Having been kept in 10% formaldehyde for 3 days, callus structures were taken to the cassettes and left for an overnight wash. Then, it was kept in 70, 80, 96% ethyl alcohol, one hour apart, respectively. Absol-I, Absol-II, Xylol-I, and Xylol-II were kept for 1 hour, respectively. The calluses were embedded in para lm, and a section of 6µm was taken (Rolls et al. 2012).

Laser scanning confocal microscope (CLSM)
Callus, sectioned with microton, are kept for about 30 minutes with 1% rhodamine. Then it is passed through distilled water 3 times. Fluorescence images were obtained with a Nikon Eclipse TE2000 Confocal Laser Scanning Microscope C1si. Samples were excited with the 488 nm line of an argon laser and dye emission was collected at 520 and 610 nm. The DCF uorescence was visualized in a single optical section of the callus. All images were obtained at the same depth (Minta et al. 1989).

Scanning Electron Microscopy
Alfalfa callus tissues were pre xed in 5% buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at room temperature. After dehydration through a graded ethanol series, samples were dried with a CPD (CO2 critical-point drying) system, sputter-coated with gold (Jeol JFC-1100 E ion-sputtering system) and observed with a scanning electron microscope (HITACHI S-4700).

Statistical Analysis
Each experiment was repeated three times. Analysis of variance was conducted using a one-way ANOVA test using SPSS 13.0 and means were compared by Duncan test at the 0.05 con dence level.

Characterization of CaO, CuO and ZnO NPs
Surface morphology examination of synthesized CaO, CuO and ZnO NPs was carried out using Zeiss brand Sigma 300 model scanning electron microscope (SEM). From the SEM images given in Figure 1A, it can be seen that CaO NPs have a particle size ranging from 35 to 160 nm. SEM images of CaO show that synthesized CaO NPs are porous, the structure is regular and has a very pleasant layered structure. When the SEM image of the obtained CuO NPs was examined ( Figure 1B), it was determined that the CuO NPs were spherical and their dimensions were distributed between 20-45 nm (Gultekin et al, 2017; Gultekin et al. 2020). When the SEM images of ZnO NPs were examined ( Figure 1C), it was determined that ZnO NPs were agglomerated and compatible with XRD results. It was observed that the structures of ZnO NPs were spherical and varying in size between 17-65 nm. In addition, when the surfaces of ZnO NPs are rough, it is clearly seen from Figure 1C. The ndings obtained are also compatible with the literature (Rajendran and Sengodan 2017; Nadaroglu and Alayli 2020).

XRD analysis
The spectra of the XRD analysis (X-ray diffraction) of the CaO NPs structure are shown in Figure 2. 28 Yazıcılar et al. 2021). The ndings obtained con rmed that the structure of CaO NPs was successfully formed. XRD and crystallographic analysis of zinc nanoparticles by green synthesis method using walnut shell extract are given in Figure 3A. Characteristic peaks of the XRD spectrum that can be indexed at 2 fas = 11.39o, 22.24o, 36.09o, 49.22o, facets (111), (200), (220) are consistent with the literature. Zn NPs structures were determined to be cubic (fcc) zinc nanocrystals (Nadaroglu and Alayli 2020). XRD and crystallographic analysis of CuO nanoparticles synthesized by green synthesis method are given in Figure 2C. 2θ = 32.2o, 39.62o, 58.9o, 70.3o of the XRD spectrum showed characteristic peaks that can be indexed at facets (110), (111) and (202) ( Figure 2C). It has been determined that CuO NPs structures have a spherical structure (Gultekin et al. 2020).

MDA (Malondialdehyde)
The callus induction stages of the Erzurum and Muş genotypes produced as a result of ZnO, CuO and CaO NPs application against NaCl were evaluated. Also, salt-free ZnO, CuO and CaO NPs applied callus formation stages were evaluated. Table 1. clearly display that MDA activities were greatly affected in callus formation stage of two alfalfa lines in presence of 0.8 ppm NPs after salt treatments. MDA values indicated a large range of variation among tested samples for salt stress treatments, ranging from 0,0168 to 0,0466 nmol g -1 FW. The maximum activity was observed from callus treated of 50 mM NaCl with 0.8 ppm CuO NPs. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for MDA value compared to the other NPs. Although the highest membrane damage was found in the treatments with 0.8 ppm CuO 50 mM NaCl in callus induction stage, the lowest membrane damage was found in 'callus formation stage' for 0.8 ppm CaO NPs 50 mM NaCl (Table 1).

H 2 O 2 (Hydrogen peroxide)
There were signi cantly differences among 1. week Muş callus and the other groups (Fig. 2). Although the highest membrane damage was found in the treatments of 0.8 ppm CuO NPs in the callus induction stage, the lowest membrane damage was found in callus formation stage for 0.8 ppm CuO NPs (Table 1).

POD (Peroxidase)
Table 1 clearly display that POD activities were signi cantly affected in callus formation stage of two alfalfa lines in presence of 0.8 ppm NPs. POD values indicated a large range of variation among tested samples for salt stress treatments, ranging from 0,1035 to 1,666 nmol g -1 FW. The maximum activity was observed from callus treated of 50 mM NaCl. The callus induction stage without salt treatments indicated a best result in 0.8 ppm CaO NPs for POD value compared to the other NPs. Although the highest membrane damage was found in the treatments of 50 mM NaCl, the lowest membrane damage was found in 'callus induction stage' for 0.8 ppm CuO NPs (Table 1).

Protein Analysis
The analysis displayed that protein levels have stronger effects in the Muş and Erzurum callus (Fig. 6). However, few accumulations of protein were detected in the control callus except 0.8 ppm CuO. CuO displayed lower protein accumulation prior to NaCl. The best response was obtained from callus induction stage and callus formation stage after 50 mM treatment NaCl with 0.8 ppm CuO. The best response was obtained from callus induction stage and callus formation callus without NaCl with 0.8 ppm ZnO (Table 1).

Laser Scanning Confocal Microscope (LSCM)
LSCM was used as a visual marker to verify the distribution of nanoparticles in stable callus culture. NaCl free callus and callus which applied the 50 mM NaCl alone were used as a control. The analysis displayed that the CaO, CuO and ZnO NPs are obviously traceable in the Erzurum and Muş callus tissues. However, The NaCl stress inhibited in terms of nanoparticles was at different degrees. CuO exhibited a better response than CaO and ZnO NPs in response to NaCl stress. In the rst week, accumulation of NPs inside the cell was lower activity by NaCl than in the second week in terms of Erzurum and Muş genotype. According to the result of the confocal analysis, CuO exhibited the most abundant callus induction stage, followed by ZnO exhibited at callus induction stage, 50 mM NaCl ZnO callus induction stage and nally, 50 mM NaCl CuO callus induction stage (Fig. 7). SEM analysis of callus structures SEM detection indicated that each genotype callus type had various callus structures. There was a continuous amorphous sphere, termed extracellular matrix, on the callus surface. It was also detected that cultivars belonging to the same genotypes share similar cell structure and shapes. The soft and compact character of the callus in Erzurum and Muş convert to granular-mucilage resembles, mostly likes a membranous surface and wrinkled cell mass structure under SEM detections (Fig. 8).

Discussion
Alfalfa is an important high nutritional feeding quality and N xation, which have a great potential for wildlife habitat and soil conservation. However, abiotic stress such as salinity which causes adverse effects on germination, plant vigour and crop yield, thereby affecting annual development and resulting in serious economic losses (Sakiroglu and Brummer 2017). Various cultural and chemical control strategies have been attempted to address these concerns, but the strategies were only partially powerful. In comparison, nanotechnology supplied new options to produce resistance plants against abiotic stress factors (Iqbal et al. 2020;Gohari et al. 2020). In fact, many studies have been reported on the plant species use of nanoparticles in response to stress factors. In this study, in vitro callus induction responses of alfalfa under NaCl and CaO, CuO, ZnO were examined and present study displayed signi cant differences in their responses to the NPs. The results indicated that NPs had a promoting effects callus induction, while control callus postponed the day of callus induction. It was detected that with treatment NPs the degree of callus induction also highly increased. The obtained results that compact callus with globular structure and yellowish colour callus were formed from leaf explants within 1 months, while there was callus induction in the NPs within a shorter time 1 months. Callus were induction in NPs treatments with various frequencies. The highest frequency of callus induction from leaf explant of Muş genotype was detected on the medium containing 0.8 ppm CaO. Among tested treatments, CaO is evident to be higher effective than CuO, ZnO in callus induction from leaf explants. The fresh weight in Muş callus in the presence of 0.8 ppm CaO was heavier than those of CuO and ZnO in equal concentration (data not shown). It was proved that considerable uptake and accumulation of the nutrient elements occurred. Noticeably the size all tested NPs examined in this study did identical, with a value of 20 -160 nm but varying uptake and accumulation functions of those three types of NPs were detected, and the genotype dependent uptake for NPs occured in the callus. Previous studies have shown a positive correlation between NaCl and nanoparticules accumulation in various plant species ( . This obviously con rms that CaO in the culture medium was quickly responsive and effective accumulation. In terms of confocal analysis, assessment of NPs functions in the presence of NaCl was based on the expanded coloration, tissue damage, and amount of the cell survival. In our cases, the results of laser scanning confocal analysis and SEM demonstrate the delivery of NPs alfalfa callus is supported related to the uptake of nutrient elements and translocate from culture medium (Fig. 7). CuO nanoparticle application in the Muş genotype at the 1 st week provided an expected improvement on NaCl stress. CuO application showed a dense distribution in the cell by preventing the adverse effect of 50 mM salt faster than ZnO and CaO application. The best reponse of ZnO nanoparticle application was obtained in the 2 nd week of the Muş genotype compared with the 1 st week and the 2 nd week. ZnO accumulated intensely in certain parts of the cell in 1 st week application, and as the period extended, it simultaneously distributed into the cell and prevented the negative effect of salt. These indicate that term alterations of defense responses to NaCl could be a main process. One or two weeks post NaCl application, control callus displayed salt severity on the callus tissues, resulting in a tissue damage within less than 7 days (Fig 7). By contrast, Muş and Erzurum genotypes are 1 st application CuO NPs survived longer than 2 weeks, indicating considerably improved resistance to NaCl. Our detection of NPs in callus tissues following a fairly aggressive 2 nd applications suggests that CaO, CuO and ZnO was present, not merely on the extracellular matrix, but that it penetrated inside the callus cells. SEM analysis supported with confocal analysis results in indicating that CuO was distrupted the extracellular matrix of alfalfa callus 7 days post exposure (Fig 7c). 14 days post exposure, CuO was clearly located inside callus cell tissues. It was detected that the formation of callus structure of alfalfa callus subject to media including various NPs such as CaO, CuO and ZnO can be added to the culture media. It was noticed that no harmful for cell wall in callus of the control while harmfuls were detected in cell wall of callus exposure to 50 mM NaCl. This is evident that even lower doses NaCl dramatically increased on the harmful cell wall of the callus tissues. CaO, CuO and ZnO in uence for induction NaCl stress various concentrations of NaCl. Callus produces an amorphous mass of extreme cell wall in response to exposure to different time-periods (Fig.  8). Accumulation nanoparticles and its adverse effects on applied callus tissues highly depend upon the genotype and exposure time. For example; 7 days post exposure, ZnO NPs exhibited membranous structures to be extracellular matrix and related to neighboring cells, and they were intensely ZnO that accumulated and present in the 50 mM NaCl. In contrast, CuO NPs showed partial rough and mucilagelike on the broblast in Erzurum genotypes. Although the CaO NPs exhibited wrinkle and rough structures at 1 st week of Erzurum genotype. In 1 st week of the application of 50 mM NaCl of Muş genotype mostly granular structures and nodular callus segments were showed. Interestingly the CaO NPs exhibited mucilage-like structures and differentiation of calllus primordia at 2 nd week of Muş genotype and 2 nd week of the application of 50 mM NaCl of Erzurum genotype wrinkle structures were shown (Fig. 8).
Obviously, CaO, ZnO and CuO are greatly reactive and are able to pass through the cell membrane in both cases and recover the callus from NaCl stress. It is evident that callus exposed to NaCl in the 2 nd week will provide higher physiological properties regardless of nanoparticule types which are linked to the regeneration capacity in callus cells. This veri es us that chemical composition and structural regulating on the callus tissues might play important functions in morphological formation. Medium conditions in tissue culture induced activation of various cellular defense strategies, adjusting cell adaptation under new environmental conditions. These ndings are consistent with one of the rst reports on formation of the ECM mediated on the outer surface layer (Zari et al. 2015). They suggested that ECM formation might be a stress response of explants, revealed by speci c tissue culture conditions. NaCl severity resulted in an obvious increase in MDA values in callus. A reduction of MDA was also observed in callus formation stage subject to 0.8 ppm CaO 50 mM NaCl period. Based on genotype, the NPs applied callus had considerably higher MDA values compared with 1 st and 2 nd weeks. This indicated that NaCl-activated stress severity was alleviated by ZnO, CuO and CaO (Fig. 3). These results indicated that the effect of NPs was linked to the callus structure and genotypes. These ndings are in agreement with one of the reports in absence of NPs mediated tolerance to NaCl stress potato callus (Rizwan et al. 2015). Levels of MDA in callus induction stages higher activity than callus formation stage in callus cells of both genotypes. The obtained results suggested that MDA activity from callus induction stage was higher promoting NPs uptake and binding capacity in the cytosol even without NaCl. This verifying that the NaCl induced stress was mitigated by nano-Cao, CuO and ZnO. Under stress conditions, H 2 O 2 is generated and mediates crosstalk among metabolic process. Therefore, the H 2 O 2 is a likely signaling molecule that participates to the events (Soleymanzadeh et al. 2020). In our cases, the results veri ed that the production of H 2 O 2 is dependent on genotype and nanoparticules types and this was con rmed by the statistical test. However, application of Muş genotype with 1 st week was signi cantly different from 2 nd week as well as Erzurum genotype. and there was higher H 2 O 2 production at the callus formation stage in the presence of CuO NPs. Additionally, Application of long period callus cells with CuO considerably recovered stress severity at callus formation stage (Fig. 4). These results are contributed with the outcomes obtained on callus cells tissues in different Triticale genotypes (Yazıcılar et al. 2021). The POD activity with NPs in callus tissues was considerably decreased exposed control and in presence of 50 mM NaCl. Moreover; The results of this study displayed that calli decreased callus formation stages that the POD decreased; and these decreases were seen to have changed depending on the genotypes, type and period of the nanoparticules applied. This decrease was detected changeable depending on the applied NPs period. Although the maximum POD activity was recorded at in presence of NaCl, the minimum POD activity was recorded at 0.8 ppm CuO callus formation stage (Fig. 5). Based on our ndings, decreases in POD activity in NPs-treated callus could be linked to promotion of callus development parameters and protective role of NP as direct or indirect. Additionally; Induction in POD activity may be explained that Cu +2 ions led to higher ROS generation, and initially promoted their antioxidant system to challenge the ROS but then lost capacity to regulate antioxidant enzyme activity. This is consistent with the earlier report indicating that POD activity is greatly linked to NPs ions. (Liang et al. 2018;Castiglione et al. 2014). It is evident that organs and tissues of plants exposed to environmental stress in long-term will gradually lose their biochemical and enzymatical structures. 1 st week Muş applications exhibited an enough activity of POD that mainly due to rapid and e cient inducer of CuO NPs, whereas ZnO was slowly effective, which could probably be an outcome of stronger stability of ZnO. Stability is a main strategy determining the transformation, transport, fate, and toxicity of ZnO NPs in various growth media. This can be explained that the behavior of NPs depends on the intrinsic physiochemical properties and the chemistry of the surrounding different environment media. The nanoparticules uptake mechanisms can be in uenced by several factors including salinity, total organic carbon, pH, redox potential, water properties like ionic strength, natural organic material (NOM), redox potential, and other chemical components in uence the short-and long-term behavior of CuO NPs (Sousa and Texeira 2013; Conway et al. 2015). Applied longterm cultured callus cells with 0.8 ppm NPs exhibited the best response and considerably recovered ZnO NPs as compared to control callus in terms of protein content. After NaCl exposure, ZnO was found to be hardly recovered at callus induction stage and callus formation stage in response to NaCl stress as compared to 50 mM NaCl and control callus (Fig. 6). This explains that ZnOuptake, translocation and accumulation The uptake and biotransformation of ZnO NPs in plants are not only related concentrations but also particle adhesion onto cell surface, therefore ZnO uptake may also arise due to particle dissolution in the culture medium. This suggested that the callus cells were slightly effective at NaCl severity in 0.8 ppm ZnO applied. As explained above, CuO was more effective than ZnO on callus cells in response to NaCl severity. It is well-known that callus cells within explant resources demonstrate intensive cell division and hence there is necessary for active synthesis of nucleic acids. Need for DNA production improves the synthesis of NTP. That is an early substrate for nucleic acid synthesis. Increased NTP synthesis increases pH level within cells. This statement con rms that protein synthesis is signi cant for plant growth and development, which are highly sensitive to the NaCl stress. The result may have veri ed by the microscopy studies with a SEM in which display a reduction in the formation of continous surface in presence of of ZnO NPs. In fact, by extending the period ZnO, the formation of membranous structures as well as some wrinkle and amorphous compounds were detected. These effects are likely due to synthesis protein and inducing structural changes in protein and thus the formation of various conformational changes in the callus surfaces.

Conclusions
It was observed that NaCl resulted in altered pattern of CuO and ZnO in the callus tissues of the two alfalfa genotypes. NaCl tolerance in alfalfa can be improve by increase in NPs uptake e ciency. Moreover; callus induction stage in presence of CaO were affected but did not appear to have expected positive relationship with antioxidant enzyme activities under NaCl. It is inferred from the outcomes of the present study that decreased the adverse affects of NaCl on alfalfa callus tissues through improving MDA, H 2 O 2 and protein rates and lowering the activity of POD in presence of NPs. In the near future; these  Tables   Due to technical limitations, table 1,2 is only available