Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(3): 19-29, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1791 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Selma Tabur, Nai ṁe Büyük- kaya Bayraktar, Serkan Özmen (2022). L-Ascorbic acid modulates the cyto- toxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aber- rations. Caryologia 75(3): 19-29. doi: 10.36253/caryologia-1791 Received: August 22, 2022 Accepted: November 02, 2022 Published: April 5, 2023 Copyright: © 2022 Selma Tabur, Nai ṁe Büyükkaya Bayraktar, Serkan Özmen. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations Selma Tabur1,*, Nai̇me Büyükkaya Bayraktar2, Serkan Özmen1 1 Department of Biology, Faculty of Arts and Science, Süleyman Demirel University, 32260 Isparta, Turkey 2 Süleyman Demirel Education Complex, 32260 Isparta, Turkey *Corresponding author. E-mail: taburs@gmail.com Abstract. The objective of the present study was to with all details explain of the effi- ciency of L-ascorbic acid (L-AsA) also known as vitamin C on cytotoxicity and geno- toxicity induced by salt stress in the barley apical meristems. As a result of the sta- tistical analysis salt stress caused a significant (P ≤ 0.05) decrease in mitotic index of barley seeds depending on concentration increase, while the frequency of chromosom- al aberration (CA) increased. In addition, it was determined that mitotic index value was decreased by 46% with 1 μM L-AsA supplementation as compared to control and chromosomal abnormalities were increased by 8.96% as well as. However, in the case of simultaneously application of 1 μM L-AsA and different salt concentrations, the high salt concentrations exhibited an excellent success according to low salt concentra- tions in alleviating the mitodepressive effect of salt stress. Moreover, the frequency of chromosomal aberrations in the root meristem cells of those seeds with 1 μM L-AsA supplementation germinated at different salt concentrations was substantially reduced compared to own control group (alone 1 μM L-AsA pretreatment). The 1 μM L-AsA pretreatment at the highest salt concentration (at 0.40 M) was showed an excellent suc- cess by reducing the frequency of the chromosomal aberrations by approximately 90 %. Different salt concentrations and/or 1 μM L-AsA supplementation caused micro- nuclei and granulation as well as various chromosomal aberrations in prophase, meta- phase, anaphase and telophase. Keywords: cytotoxicity, genotoxicity, Hordeum vulgare L., mitotic index, ascorbic acid, salinity. INTRODUCTION Together the global climate change, which is starting to make its pres- ence felt more and more, plants are becoming more frequently subjected to adverse abiotic stresses, such as extreme temperatures, cold, high salin- ity, and drought, which limiting plant growth and crop productivity. Salin- ity is one of the major environmental factors that reduce plant productivity 20 Selma Tabur, Nai̇me Büyükkaya Bayraktar, Serkan Özmen (Tobe et al. 2003; Sabagh et al., 2019). Nearly 20% of the world’s cultivated land and also five-hundred thou- sand hectares of irrigation area in Turkey are threat- ened by salinity (FAO 2016). Salt stress inhibits or delay growth and development of plants by negatively affects plant growth via oxidative stress, especially ion toxic- ity, nutritional and hormonal imbalance, and osmotic stress (Parida and Das 2005; Ashraf, 2009; Elsheery et al. 2020a). Moreover, the retardant effects of salin- ity stress on growth, physiological aspects, productivity and cellular activity were also recorded on other differ- ent many plants species (Bargaz et al. 2016; Nassar et al. 2016; Elsheery 2020b; Tabur et al. 2021). However, plants develop highly complex mechanisms for tolerate salin- ity. Tolerance to salt stress of plants is of three types: osmotic stress tolerance, Na+ or Cl- exclusion, and the tolerance of tissue to accumulated Na+ or Cl- (Munns and Tester 2008; Zvanarou et al. 2020). Since the mecha- nisms behind salinity are quite complex and difficult to understand, impact of salinity on plants, type and caus- es of salinity, and salt tolerance strategies of plants are still discussed in level cellular and molecular (Zhu et al. 2016). The common view of many researchers in com- bating salinity is the development of high salt-tolerant plant varieties. However, this method, which is one of the economical ways to eliminate the negative effects of salinity on plants, shows inconsistency between differ- ent crops. Therefore, there is a great scientific burden on researchers to cope with this important environmental stress that also limit crop productivity. For all these rea- sons, most of the researchers contributed to overcome the disadvantages of salt stress and to develop salt toler- ant varieties by using various hormones, plant growth regulators, leaf extracts, vitamins biofertilizer and ami- no acids (Tabur and Demir 2010 a,b; Mohsen et al. 2014; Çavuşoğlu et al. 2016 a,b; Naser et al. 2016; Mahfouz and Rayan 2017; Farheen et al. 2018; Özmen and Tabur 2020; Tabur et al. 2021). In recent studies, it has been reported that some vitamins may be effective to alleviate the negative effects of salinity by increase resistance to salt stress (Sha- lata and Neumann 2001), plant growth and yield qual- ity (El-Bassiouny et al. 2005; Bassuony et al. 2008), seed germination, seedling growth (Emam and Helal, 2008), mitotic activity (Özmen and Tabur 2020) some metabol- ic changes. Ascorbic acid (AsA) is a naturalist product that acts as an antioxidant and enzyme and also improves cofac- tor. It acts as an essential substrate in the cyclic pathway of enzymatic detoxification of hydrogen peroxide. There are various isomers of ascorbic acid or vitamin C (L-AsA, D-AsA, D-izoAsA). D-AsA and D-isoAsA do not have vitamin C function. Therefore, when ascorbic acid is mentioned, L-ascorbic acid (3-keto-L-gulofuranolaktan) comes to mind from these isomers because of the only isomer with biological activity (Dizlek and Gül, 2007). The stimulatory roles of L-AsA, a minor, water-soluble antioxidant, in plant growth and other developmental processes are well documented (Gallie 2013; Hossain et al. 2017; Gaafar et al. 2020). In plants L-AsA serves as a major redox buffer and regulates various physiological processes controlling growth, development, and stress tolerance. Being a major component of the ascorbate-glu- tathione (AsA-GSH) cycle, L-AsA helps to modulate oxi- dative stress in plants by controlling ROS detoxification alone and in co-operation with glutathione. Any fluctua- tions, increases or decreases, in cellular L-AsA levels can have profound effects on plant growth and development, as L-AsA is associated with the regulation of the cell cycle, redox signaling, enzyme function and defense gene expression (Hossain et al. 2017). The ascorbic acid concentration increases in plant cells exposed to stress conditions and plays a role in providing tolerance against oxidative stress by playing a role in the direct clearance of O-2 and OH-. As a result of enzyme and gene expression analyzes carried out under different abiotic conditions in many plants, it was deter- mined that ascorbic acid-related gene expression levels increased and these increases were given as a defense response against stress. On account of this, it is empha- sized that higher L-AsA levels are important to mini- mize oxidative stress and regulate plant metabolic pro- cesses. (Athar et al. 2008, 2009; Akram et al. 2017). The cellular AsA pool size in plants can be regulated by the coordinated action of many related enzymes. Numerous recent studies have confirmed that AsA level increases the tolerance and adaptation of crops to many abiotic stresses such as cold, drought, salinity, heavy metal tox- icity and ozone stresses (Xie et al. 2009; Çavuşoğlu and Bilir 2015; Akram et al. 2017; Xu 2017; Sabagh et al. 2019; Gaafar et al. 2020; Nunes et al. 2020; Wang et al., 2020; Chen et al. 2021). As mentioned above, there are many studies on the effects of AsA on seed germination, seedling growth, plant resistance, plant growth and yield quality, antioxi- dant enzyme activity, and some biochemical and meta- bolic changes under various abiotic stress conditions. In addition, it has been known for few decades that AsA plays an important role in plant growth and develop- ment by regulating cell division (Smirnoff 1996; Gallie 2013). However, a rather limited number of studies have been found on the response of ascorbic acid to cytotox- icity and genotoxicity caused by various abiotic stresses, including salinity (Barakat 2003; Yu et al. 2014; El-Araby 21L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells et al. 2020). For this reason, this work was designed to comprehensively test of the efficiency level of exogenous L-AsA against effects cytotoxic and genotoxic in caused by salt stress in barley meristem cells and to contribute to the gap in the literature. Namely, it is aimed at clari- fying to what extent exogenous L-AsA is able to tolerate salt stress, whether it encourages cells to enter the mito- sis division, and whether it causes any changes in the structure and behavior of chromosomes. MATERIALS AND METHODS The barley cultivar (Hordeum vulgare cv. ‘Bülbül 89’) used in this study was requested from the Field Crops Research Institute, Ankara, Turkey. NaCl and ascor- bic acid (L-AsA) used in the experiments were obtained from Merk and Sigma-Aldrich, respectively. Primarily, to prevent fungal contamination, the barley seeds were surface sterilized by immersion in 1% (w/v) NaClO solu- tion for 10 min, rinsed thoroughly five times with sterile distilled water and dried on filter papers at room tem- perature prior to experimental procedure. The sterilized seeds were divided into two groups and soaked in con- stant volumes (50 ml) of distilled water (control, C) and L-AsA (1 µM, micromolar) for 24 h at 20 ± 1oC. The solutions were filtered at the end of this pretreatment session and 20-25 barley seeds which uniform sized were placed in Petri dishes covered with two sheets filter papers moistened with 7 ml of distilled water or three different NaCl (0.32, 0.35 and 0.40 M, molar) concentra- tions. After, Petri dishes were transferred to incubators at constant temperature (20 ± 1 °C) for germination for several days. These salt levels hindering germination of seeds on a large scale and the most proper concentration of L-AsA level in alleviation of the salt inhibition at the germination were determined in a preliminary investiga- tion conducted by us. To cytogenetic analyses after 3 or 4 days, the root tips reached to 0.5-1 cm were excised, pretreated with a saturated solution of paradichlorobenzene for 4 h at 20ºC, fixed with Carnoy’s Fluid I (absolute ethanol: gla- cial acetic acid, 3:1, v/v) for 24 h, and stored in 70% eth- anol at 4ºC until required. Then, root tips were hydro- lyzed in 1 N HCl at 60ºC for 15-18 min, stained for 1-1.5 h in accordance with the standard procedure for Feul- gen staining, and squashed in 45 % acetic acid (Sharma and Gupta 1982; Elçi and Sancak 2013). After one day, microscopic slides were made permanent in by mount- ing Canada balsam by alcohol vapor exchange method. The best mitosis phases and aberrances were observed in permanent slides and photographed (100X) with a digi- tal camera (Olympus C-5060) mounted on an Olympus CX41 microscope. The prepared slides were examined under the micro- scope at 100X magnification, and mitotic index, i.e. per- centage of dividing cells were accounted by counting approximately 15000 cells (three repeat, 5000 per slide) for all per-application. The mitotic index (MI) was cal- culated using the following the equation: In addition, chromosomal aberrations (CA) occur- ring at all stages of mitosis during microscopic observa- tion of the slides were calculated according to the fol- lowing the equation for each per-application as the per- centage of 350 dividing cells counted. All experiments were repeated three times. Statisti- cal evaluations of obtained data were actualized using the SPSS 14.0 program and Duncan’s multiple range test (Duncan, 1955). RESULTS The mitotic index (MI) data obtained from the cyto- logical analysis of barley root tips treated with differ- ent NaCl levels and 1 μM L-AsA (vitamin C) are sum- marized in Table 1. Based on these data, MI gradually drastic reduced with parallel to increasing NaCl levels as compared to the control group. At the highest salt level (at 0.40 M, molar), the mitotic index was reached to the lowest value by reducing from 7.0 ± 1.5 (control, in distilled water) to 1.6 ± 0.07 (77%). In the root meris- tem cells exposed to 1 μM L-AsA alone, a mitotic index reduction of about 46% was recorded according to con- trol group. When samples with L-AsA treated germinat- ed at different salt levels were compared with their selves control group (L-AsA alone), it was determined that the mitotic index increased statistically a little except for 0.32 M NaCl. 0.35 and 0.40 M NaCl levels exhibit- ed major successful compared as each other the mitotic index values of L-AsA pre-treated and untreated samples at the same salt concentrations (Table 1). Especially, it was recorded that at the highest salt concentration (0.40 M NaCl) the mitotic index value was increases approxi- mately two and a half times (from 1.6 ± 0.07 in control group to 4.0 ± 0.3 in 1 μM AsA). 22 Selma Tabur, Nai̇me Büyükkaya Bayraktar, Serkan Özmen The chromosomal aberration frequencies data obtained from barley root tips germinated both distilled water and different NaCl levels in the absence or pres- ence of 1 μM L-AsA are summarized in Table 1. In par- allel with the increasing salt concentrations, a very high rate of chromosomal aberrations observed in the root meristem cells of barley seeds. That is, while the chro- mosomal aberration frequency was 0.00±0.0 in the con- trol seeds germinated in distilled water medium, it was recorded as 2.30±1.0 at 0.32 M salinity, 8.96±2.5 at 0.35 M salinity, and 21.2±2.5 at 0.40 M salinity. On the oth- er hand, the frequency of chromosomal aberrations in seeds germinated in salt stress-free medium after 1 μM L-AsA supplementation alone was remarkably higher than that in the control group (distilled water, 0.00 M NaCl) and was also statistically significant. However, the frequency of chromosomal aberrations of seeds germi- nated at different salt concentrations after 1 μM L-AsA supplementation has exhibited a statistically significant decrease compared to the percentage of seeds treated with 1 μM L-AsA alone. When these values are com- pared with the frequencies of seeds germinated only at different salt concentrations, although1 μM L-AsA sup- plementation partially increased the chromosome aber- ration rate at the lowest salt level studied, it significantly reduced the negative effect of salt stress on this param- eter, especially at high salt levels (at 0.35 and 0.40 M salinity). In other words, while the chromosomal aber- ration rate was 8.96 % at 0.35 M salinity and 21.2 % at 0.40 M salinity, the application of 1 μM L-AsA showed an excellent success, reducing these aberration rates to 1.70% and 2.00%, respectively (Table 1). As a result of scans in mitosis slides, no abnormal- ity was found in the meristem cells of the control group barley seeds germinated in distilled water and at 20°C, and all stages of mitosis were observed normally (Figure 1). Microscopic images of a wide range of chromosome aberrances observed in the preparations prepared with Table 1. Mitotic index scores and frequency of chromosome aber- rations in meristem cells of H. vulgare L. exposed to different NaCl concentrations after 1 μM L-AsA supplementation NaCl (M, mol/L) and L-AsA (μM) Concentrations Mitotic Index (%) Chromosome Aberrations (%) Control (0.00, Distilled Water) *7.0±1.5c *0.00±0.0a 1 μM L-AsA 3.8±0.4b 8.96±2.5b 0.32 M NaCl 6.3±0.4c 2.30±1.0a 0.32 M NaCl + 1 μM L-AsA 3.3±0.7b 2.50±2.0a 0.35 M NaCl 2.8±0.1b 8.96±2.5b 0.35 M NaCl + 1 μM L-AsA 4.0±0.2b 1.70±0.6a 0.40 M NaCl 1.6±0.07a 21.2±2.5c 0.40 M NaCl + 1 μM L-AsA 4.0±0.3b 2.00±2.0a *Values with insignificant difference (P ≤ 0.05) for each column are indicated with same letters (± Standard deviation). As test solution, 1 µM ascorbic acid (L-AsA) was used. Concentrations of NaCl were 0.32, 0.35, 0.40 M (mol/L). The pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or L-AsA. Different concentrations of salt were added to germination medium. All data were evaluated as three replicates Figure 1. Normal mitosis stages in meristem cells of H. vulgare L. germinated in distilled water (Control). a- Prophase b- Metaphase (2n = 14) c- Anaphase d- Telophase. Scale bar = 10µm. Figure 2. Aberrations observed in pre-prophase and prophase stage in meristem cells of H. vulgare L. germinated at different NaCl con- centrations after 1 µM L-AsA supplementation for 24 h. a-b- micro- nuclei, c-d- chromatin granulation in interphase, e-f- disorderly prophase. Scale bar = 10µm. 23L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells root tips belonging to all other application groups are shown in Figure 2-5. The most common chromosome abnormalities observed in all application were micronu- cleus, disorderly prophase and anaphase, uncoiling chro- mosome, sticky chromosome, bridges in anaphase, and false polarization in anaphase and telophase. The abnor- malities such as alignment anaphase and vagrant chro- mosomes were observed in the minimal level. DISCUSSION In the present work, effect cytotoxic and genotoxic in the apical meristem cells of barley seeds exposed salt stress of exogenous 1 μM L-AsA supplementation were investigated comprehensive. A lt hough its mechanism has not been f u lly explained yet, the effects of salinity stress one of the most important abiotic stresses have been known for a long time by many researchers at cellular and chromo- somal levels (Lutsenko et al. 2005; Tabur and Demir 2010 a,b; Pekol et al. 2016; Kiełkowska et al. 2017; El- Araby et al. 2020). All these researchers agree that salt stress causes chromotoxic actions and total inhibition of mitotic processes on meristematic cells, just as in our study. In addition to, Zvanarou et al. (2020) reported Figure 5. Aberrations observed in telophase stage in meristem cells of H. vulgare L. germinated at different NaCl concentrations after 1 µM L-AsA supplementation for 24 h. a-b- false polarization in telophase, c-d- false polarization in telophase and vagrant chromo- somes (arrows). Scale bar = 10µm. Figure 3. Aberrations observed in metaphase stage in meristem cells of H. vulgare L. germinated at different NaCl concentrations after 1 µM L-AsA supplementation for 24 h. a-b- uncoiling chro- mosomes, c-d- sticky chromosomes. Scale bar = 10µm. Figure 4. Aberrations observed in anaphase stage in meristem cells of H. vulgare L. germinated at different NaCl concentrations after 1 µM L-AsA supplementation for 24 h. a- disorderly anaphase, b-d- bridges in anaphase (arrows), e- laggard chromosome (arrow), f-g- alignment anaphase, h-ı- false polarization in anaphase. Scale bar = 10µm. 24 Selma Tabur, Nai̇me Büyükkaya Bayraktar, Serkan Özmen that dividing root meristem cells are more sensitive to NaCl than other tissues since remains in direct contact with abiotic stress factors. However, salt damage extent depends upon plant species, stages of plant development, genotype, salinity concentration, and exposure time (Vicente et al. 2004; Tabur et al. 2021). To date, many studies have been conducted on the effect of ascorbic acid on morpho-physiological, bio- chemical and metabolic changes under both normal and various stress conditions using various plant species (Khan et al. 2006; Dolatabadian ve Jouneghani 2009; Fatemi 2014; Mohsen et al. 2014; Gaafar et al., 2020; Nunes et al. 2020; Chen et al. 2021). However, studies on the protective role of exogenous L-AsA supplemen- tation against the cytotoxic effects of various abiotic stresses and its effect on mitotic activity and chromo- somal abnormalities, especially against salt stress, are quite insufficient (Barakat 2003; Yu et al. 2014; El-Araby et al. 2020). Therefore, first of all, it was found appropri- ate to compare the effects of L-AsA during germination in distilled water at 20°C before proceeding to its effects on these parameters under salt stress conditions. As mentioned in the research findings section, the mitotic index value of barley seeds that were not pretreat- ed with L-AsA (0.00 control, C) was 7.0±1.5, while this value was 3.8±0.4 in seeds that were pretreated. In other words, L-AsA supplementation alone caused a decrease of approximately 46% on the mitotic index compared to the control group (see Table 1). Mitotic index, as known is one of the most important indicators reliably identified the presence of cytotoxicity (Fiskesjö 1985). The decrease of the mitotic index value below 50% compared to the control variant leads to a sublethal effect, while below 22% it can cause lethal effects on test organisms (Mesi and Kopliku 2013). Undoubted, in this case 1µM L-AsA supplementation alone has a potential for sublethal effects. In addition, L-AsA application alone increased the rate of chromosomal aberrations by 8.96% compared to distilled water (see Table 1). As a result of this study, it was revealed that 1µM L-AsA supplementation alone reduced the mitotic index value and had a negative effect on chromosomal aberrations in barley seeds germinated in distilled water environment. Our findings regard- ing mitotic index and chromosomal abnormalities are in agreement with the study reported in Allium cepa by Asita et al, (2017). However, Cenanovic and Durakovic (2016) reported that ascorbic acid treatment at different concentrations (250, 500 and1000 μg/ml) increased the mitotic index in Allium cepa root meristems. It is thought that this difference may have occurred depending on the plant species studied and/or the dose and application time of the ascorbic acid used. As for the effect of L-AsA application on the mitotic index and chromosomal aberrations of barley seeds ger- minated in saline conditions, the data obtained from our study on the mentioned parameters will be presented for the first time for barley plant. As a result of our lit- erature research, only three previously reported studies were found that were more or less close to the subject. Firstly, Barakat (2003) reported that high salt concentra- tions significantly reduced mitotic activity and increased chromosomal aberrations in Allium cepa L. However, the researcer has determined that the ascorbic acid sup- plementation significantly increased the mitotic index and reduced chromosomal aberrations by reducing inhibitory effect of salt. Secondly, Yu et al. (2014) empha- sized that the application of the AsA (0, 0.5, 1, 2, 4 mM) decreased markedly chromosome aberrations frequency, and increased mitotic index on Vicia faba roots exposed to different concentration of Pb (NO3)2. Finally, El-Araby et al. (2020) has been researched the effects of two con- centrations of ASA (50 and 100 ppm) on the cytological parameters of pea seedlings under salinity stress. They reported that ASA (100 ppm) treatments significantly reduced the damaging effect of salinity stress on mitotic index and chromosomal abnormalities percentage. Simi- larly, also in our study, 1µM L-AsA showed an excellent performance on the mitotic index of barley seeds under high salt stress conditions. For example, 1µM L-AsA supplementation has increased mitotic index by approxi- mately two and a half times at the highest salt stress condition (at 0.40 M salinity), (see Table 1). In addi- tion, L-AsA supplementation under especially high salt stress conditions showed statistically positive effects on chromosomal aberrations in root meristems of barley seeds too. Although the application of 1µM L-AsA alone caused a significant increase of chromosomal aberra- tions in root meristem cells of seeds germinated in dis- tilled water, in parallel with the increasing of the salt concentrations, the detrimental effect of supplementa- tion 1µM L-AsA has seriously reduced, from 8.96 ± 2.5% abnormal cells (at distilled water, control) to 2.00 ± 2.0% (at 0.40 M). Moreover, while ratio of the chromosomal aberrations in the highest salt level studied (at 0.40 M) was 21.2 ± 2.5%, it was reduced to 2.00 ± 2.0% with the application of 1µM L-AsA. In other words, 1µM AsA application at 0.40 M salinity has shown an excellent success by almost zeroing the detrimental effect of salt stress (see Table 1). That is, we can say that L-AsA appli- cation may be more successful in high salt levels than in low salt levels in alleviating the detrimental effect of salt stress on chromosome structure and behaviors. From here, it can be concluded that effective in including the adaptive response to genotoxic stress since L-AsA at 25L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells high salt concentrations significantly reduces the clas- togenic effects induced by salinity. Also, it is important to point out that L-AsA, known as vitamin C, have the ability to reduce the toxic effect of various genotropic toxicants if used in appropriate doses and at the con- venient stage of growth and development. In unstressed conditions, administration of L-AsA alone might have been function as a stimulator, slowing down the mitotic cycle by suppressing the synthesis of proteins required for normal cell division (Tabur et al. 2021). The slow- down in the mitotic cycle might have triggered mitode- pressive effects during cell division, thus causing a sig- nificant increase (8.96%) of chromosomal aberrations. It has been known for a long time that external stimula- tory growth regulator applications are useless and even harmful under normal conditions without stress (Tabur ve Demir 2010a). Therefore, it is not surprising that L-AsA application alone in distilled water reduces the mitotic index and increases chromosome aberrations. Then, we can say that L-AsA supplementation under stress conditions, especially at high salt concentrations (at 0.35 M and 0.40 M salinity), may have accelerated mitotic activity and consequently reduced chromosomal aberrations caused by stress. Undoubtedly, these results supported that exogenous L-AsA may play a protective role against the harmful effect of salt stress on chro- mosomes by eliminating the mitodepressive effects that occur under stress conditions. Chromosomal abnormalities that occur spontane- ously or as a result of exposure to environmental stresses are indicate the harmful effect of a toxic agent on plant cells (Nag et al. 2013). Many biotic and abiotic toxic agents can promote the occurrence of chromosome aber- rations by different mechanisms, including aneugenic (changes in total chromosome number) and clastogenic (changes in chromosome structure) actions. Feretti et al. (2007) sugessted that if toxic ajans cause damage to plant cell chromosomes, they may also be potentially harmful for mammalian cell chromosomes. Micronucle- us (MN) assay is accepted as the most effective endpoint to analyze the mutagenic effect of the toxic agents. The large MN in the cell indicates aneugenic effect result- ing from chromosome loss while small MN indicates clastogenic effect due to chromosome breaks (Kontek et al. 2007). Briand and Kapoor (1989) have reported that the micronuclei (Figure 2 a, b) are probably the result of vagrant chromosomes and fragments. Dane and Dalgıç (2005) reported that chromatin granulation is related to the inhibition of enzymes and histone proteins. It emphasized by many researchers that several chroma- tin regulation-related factors, such as histone modifica- tion enzymes, linker histone H1, HMG proteins and ATP-dependent chromatin remodeling factors have been functioned in plant abiotic stress responses (Kim et al. 2010; Asensi-Fabado et al. 2017). Chromatin granula- tion at interphase (Figure 2 c, d), most likely caused to deformation of the nuclear material by toxic agents, might be a consequence of all these reasons and abnor- mal chromatin condensation and indicative of many abnormalities that may occur in future mitosis phases. Uncoiling chromosomes (Figure 3 a, b) and disorder- ly prophase (Figure 2 e, f ) may be the result of a weak mitotic effect and irregular chromosome contractions (Tabur et al. 2021). Sticky chromosomes (Figure 3 c, d) could be originated from abnormal DNA condensation, abnormal chromosomal wrapping and inactivation of the axes (Asita and Mokhobo 2013). At the same time it has been asserted that such aberrations may be a result of improper folding of the chromatin fibers (Klášterská et al. 1976). According to some researchers, sticky chro- mosomes are a marker of high toxic effect on chroma- tin and irreversibility of the change (Fiskesjö and Levan 1993; Türkoğlu 2007). In the current study, all of mito- classic impacts in anaphase and telophase (Figure 4-5) that form an important portion of chromosomal abnor- malities might have been largely resulted from spindle dysfunction. Fiskesjö (1997) have informed that bridges (Figure 4 b-d) are clastogenic effects, both resulting from chromosome and chromatid breaks. According to Tabur and Demir (2010 b) the bridges in anaphase and telophase might have been the result of inversions. Moreover, Bonciu et al. (2018) have asserted that nucleo- plasmic bridges originate from dicentric chromosomes or occur as a result of as faulty longitudinal break of sis- ter chromatids during anaphase. The disorganizations in mitosis such as disorderly anaphase (Figure 4 a), fault polarization at ana-telophases (Figure 4 h, ı; Figure 5 a-d), alignment anaphase (Figure 4 f, g) and bridges may be mainly the result of faulty kinetochore attachment or of spindle dysfunction (Rieder and Salmon 1998). Such irregularities constitute a significant portion of chro- mosomal aberrations. Vagrant (Figure 5 c, d) and lag- ging chromosomes (Figure 4 e) occurs during the ana- phase where one or more chromatids gets detached from the rest of the chromatids and is incapable of moving towards the poles. Patil and Bhat (1992) have suggested that laggard chromosomes could be originate from the failure of spindle apparatus to organize in normal way. Also, the laggard of chromosomes may have occurred due to a weak mitotic impress. It known that salt stress, particularly NaCl caused too many c-mitotic reactions (Fiskesjö 1997). Therefore, increasing salt concentrations may have been reason to the formation of laggard chro- mosomes at high rates. Briefly, L-AsA alone and/or dif- 26 Selma Tabur, Nai̇me Büyükkaya Bayraktar, Serkan Özmen ferent salt levels used in our study may have been caused to all these abnormalities mentioned above by trigger- ing the stimulation/ inhibition of enzymes and proteins necessary for the normal cell division, by disturbing the spindle mechanism. CONCLUSION In the present work, it has been compared the inter- actions between the mitotic index and chromosome behaviors of L-AsA under normal and salt stress using barley seeds. As known, the mechanisms by which salin- ity affections plant growth and development are rather complex and also controversial since a long time. Unfor- tunately, although the causes of salinity have been char- acterized, our understanding of the mechanisms by which salinity prevents plant growth is still rather poor. In summary, it was determined that L-AsA supplemen- tation alone significantly reduced mitotic activity (46%) and caused a very high (8.96%) abnormality on chromo- some behaviors in this study. In this case, L-AsA supple- mentation alone can create various types of mutations over time. However, this study supports that exogenous L-AsA pretreatment, especially at high salt concentra- tions, can eliminate the negative effects of salinity on the mentioned parameters in barley plant. The obtained results in our work may provide new conceptual tools for designing the hypotheses of different salt tolerance in plants and to brighten many contradictions particu- larly in relation to effects of L-AsA and high salt stress on mitotic activity and chromosomal abnormalities. Surely! Further investigation is needed to confirm these findings. Consequently, surveying the effects of L-AsA on princi- pal metabolic events, which can be directly or indirectly effective on cell division and chromosome configuration will contribute to clarify of this mechanism. REFERENCES Akram NA, Shafiq F, Ashraf M. 2017. Ascorbic acid- a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front Plant Sci. 8:613. Asensi-Fabado MA, Amtmann A, Perrella G. 2017. Plant responses to abiotic stress: The chromatin context of transcriptional regulation. Biochim Biophys Acta. 1860:106-122. Ashraf M. 2009. Biotechnological approach of improv- ing plant salt tolerance using antioxidants as markers. Biotechnol Adv. 27:84-93. Asita AO, Mokhobo MM. 2013. Clastogenic and cyto- toxic effects of four pesticides used to control insect pests of stored products on root meristems of Allium cepa. Environ Nat Resour Res. 3(2):133–145. Asita AO, Moramang S, Rantśo T, Magama S. 2017. Mod- ulation of mutagen-induced genotoxicity by vitamin C and medicinal plants in Allium cepa L. Caryologia. 70:151-165. Athar HR, Khan A, Ashraf M. 2008. Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat. Environ Exp Bot. 63:224-231. Athar HR, Khan A, Ashraf M. 2009. Inducing salt toler- ance in wheat by exogenously applied ascorbic acid through different modes. J Plant Nutr. 32:1799-1817. Barakat H. 2003. Interactive effects of salinity and certain vitamins on gene expression and cell division. Int J Agric Biol. 3:219-225. Bargaz A, Nassar RMA, Rady MM, Gaballah MS, Thompson SM, Brestic M, Schmidhalter U, Abdelha- mid MT. 2016. Improved salinity tolerance by phos- phorus fertilizer in two Phaseolus vulgaris recombi- nant inbred lines contrasting in their P-efficiency. J Agron Crop Sci. 202:497-507. Bassuony FM, Hassanein RA, Baraka DM, Khalil RR. 2008. Physiological effects of nicotinamide and ascorbic acid on Zea mays plant grown under salin- ity stress II-changes in nitrogen constituents, protein profiles, protease enzyme and certain inorganic cati- ons. Aust J Basic & Appl Sci. 2(3):350-359. Bonciu E, Firbas P, Fontanetti CS, Wusheng J, Karaismailoğlu MC, Liu D, Menicucci F, Pesnya DS, Popescu A, Romanovsky AV, Schiff S, Ślusarczyk J, Souza CP, Srivastava A, Sutan A, Papini A. 2018. An evaluation for the standardization of the Allium cepa test as cytotoxicity and genotoxicity assay. Caryolo- gia. 71(3):191-209. Briand CH, Kapoor BM. 1989 The cytogenetic effects of sodium salicylate on the root meristem cells of Alli- um sativum L. Cytologia 54:203–209. Cenanovic M, Durakovic C. 2016. In vivo genotoxic- ity testing of vitamin C and naproxen sodium using plant bioassay. Southeast Europe J Soft Comput 4(2):66-71. Chen X, Zhou Y, Cong Y, Zhu P, Xing J, Cui J, Xu W, Shi Q, Diao M, Liu HY. 2021. Ascorbic acid-induced photosynthetic adaptability of processing tomatoes to salt stress probed by fast OJIP fluorescence rise. Front Plant Sci. 12:594400. Çavuşoğlu K, Bilir G. 2015. Effects of ascorbic acid on the seed germination, seedling growth and leaf anatomy of barley under salt stress. ARPN J Agric Biol Sci. 10(4):124-129. 27L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells Çavuşoğlu D, Tabur S, Çavuşoğlu K. 2016a. The effects of Aloe vera L. leaf extract on some physiological and cytogenetical parameters in Allium cepa L. seeds ger- minated under salt stress. Cytologia. 81(1):103-110. Çavuşoğlu D, Tabur S, Çavuşoğlu K. 2016b. Role of Ginkgo biloba L. leaf extract on some physiological and cytogenetical parameters in Allium cepa L. seeds exposed to salt stress. Cytologia. 81(2):207-213. Dane F, Dalgıç Ö. 2005. The Effect of fungicide benom- yl (benlate) on growth and mitosis in onion (Allium cepa L.) root apical meristem. Acta Biol Hung. 56 (1-2):119-128. Dizlek H, Gül H. 2007. L-ascorbic acid and its func- tions at bread making. Süleyman Demirel Univ J Fac Agric. 2(1):26-34. Dolatabadian A, Jouneghani RS. 2009. Impact of exog- enous ascorbic acid on antioxidant activity and some physiological traits of common bean subjected to salinity stress. Not Bot Horti Agrobot Cluj-Napoca. 37(2):165-172. Duncan DB. 1955. Multiple range and multiple F tests. Biometrics 11:1-42. El-Araby HG, El Hefnawy SFM, Nassar MA, Elsheery NI. 2020. Comparative studies between growth regu- lators and nanoparticles on growth and mitotic index of pea plants under salinity. Afr J Biotech 19(8):564- 575. El-Bassiouny MSH, Gobarah ME, Ramadan AA. 2005. Effect of antioxidants on growth, yield and favism causative agents in seeds of Vicia faba L. plants grown under reclaimed sandy soil. J Agron. 4:281- 287. Elçi Ş, Sancak C. 2013. Research methods and observa- tions in cytogenetics. Ankara Univ Publish House. Beşevler/ANKARA, 227p. Elsheery NI, Helaly MN, Omar SA, John SVS, Zaboch- nicka-Swiatek M, Kalaji HM, Rastogi A. 2020a. Phys- iological and molecular mechanisms of salinity tol- erance in grafted cucumber. South Afric J Bot. 130: 90-102. Elsheery NI, Helaly MN, El-Hoseiny HM, Alam-Eldein SM. 2020b. Zinc oxide and silicone nanoparticles to improve the resistance mechanism and annual pro- ductivity of salt-stressed mango trees. Agronomy. 10:953-975. Emam MM, Helal NM. 2008. Vitamins minimize the salt-induced oxidative stress hazards. Aust J Basic & Appl Sci. 2(4):1110- 1119 FAO 2016. FAOSTAT. Food and Agriculture Organiza- tion of the United Nations, Rome, Italy. Web. http:// faostat.fao.org/default. aspx. Accessed: 12 February 2019. Farheen J, Mansoor S, Abideen Z. 2018. Exogenously applied salicylic acid improved growth, photosyn- thetic pigments and oxidative stability in mungbean seedlings (Vigna radiata) at salt stress. Pak J Bot. 50:901-912. Fatemi SN. 2014. Ascorbic acid and its effects on alle- viation of salt stress in sunflower. Ann Res Rev Biol. 4(24):3656-3665. Feretti D, Zerbini I, Zani C, Ceretti, Moretti M, Monar- ca S. 2007. Allium cepa chromosome aberration and micronucleus tests applied to study genotoxicity of extracts from pesticide-treated vegetables and grapes. Food Addit Contam. 24(6):561-572. Fiskesjö G. 1985. The Allium test as a standard in envi- ronmental monitoring. Hereditas. 102:99-112. Fiskesjö G. 1997. Allium test for screening chemicals; evaluation of cytological parameters. In: Wang W, Lower WR, Gorsuch JW, Hughes JS (eds) Plant for Environmental Studies. Lewis Publishers, New York, pp 308-333. Fiskesjö G, Levan A. 1993. Evaluation of the first ten MEIC chemicals in the Allium test. Altern Lab Anim. 21:139–149. Gaafar AA, Ali SI, El-Shawadfy A, Salama ZA, Sekara A, Ulrichs C, Abdelhamid MT. 2020. Ascorbic acid induces the increase of secondary metabolites, anti- oxidant activity, growth, and productivity of the common bean under water stress conditions. Plants. 9:627. Gallie DR. 2013. Ascorbic acid: A multifunctional mol- ecule supporting plant growth and development. Sci- entifca. 795964:1-24. Hossain MA, Munne-Bosch S, Burritt DJ, Diaz-Vivancos P, Fujita M, Lorence A. 2017. Ascorbic acid in plant growth, development and stress tolerance. Springer, Cham 514p. Khan MA, Ahmed MZ, Hameed A. 2006. Effect of sea salt and L-ascorbic acid on the seed germination of halophytes. J Arid Environ. 67(3):535-540. Kiełkowska A. 2017. Cytogenetic effect of prolonged in vitro exposure of Allium cepa L. root meristem cells to salt stress. Cytol Genet. 51(6):478-484. Kim JM, To TK, Nishioka T, Seki M. 2010. Chromatin regulation functions in plant abiotic stress responses. Plant Cell Environ. 33:604-611. Klášterská I, Natarajan AT, Ramel C. 1976. An interpre- tation of the origin of subchromatid aberrations and chromosome stickiness as a category of chromatid aberrations. Hereditas. 83:153-162. Kontek R, Osiecka R, Kontek B. 2007. Clastogenic and mitodepressive effects of the insecticide dichlor- vos on root meristems of Vicia faba. J Appl Genet. 28 Selma Tabur, Nai̇me Büyükkaya Bayraktar, Serkan Özmen 48(5):359-361. Lutsenko EK, Marushko EA, Kononenko NV, Leonova TG. 2005. Effects of fusicoccin on the early stages of sorghum growth at high NaCl concentrations. Russ J Plant Physiol. 52:332–337. Mahfouz H, Rayan WA. 2017. Antimutagenics effects of stigmasterol on two salt stressed Lupinus termis culti- vars. Egypt J Genet Cytol. 46:253-272. Mesi A, Kopliku D. 2013. Cytotoxic and genotoxic potency screeningof two pesticides on Allium cepa L. Proc Tech. 8:19–26. Mohsen AA, Ebrahim MKH, Ghoraba WFS. 2014. Role of ascorbic acid on germination indexes and enzyme activity of Vicia faba seeds grown under salinity stress. J Stress Physiol Biochem. 10(3):62-77. Munns R, Tester M. 2008. Mechanisms of salinity toler- ance. Annu Rev Plant Biol. 59:651-681. Nag S, Dutta R, Pal KK. 2013. Chromosomal aberrations induced by acetamiprid in Allium cepa L. root meris- tem cells. Ind J Fund Appl Life Sci. 3(2):1-5. Naser HM, El-Hosieny H, Elsheery NI, Kalaji HM. 2016. Effect of biofertilizers and putrescine amine on the physiological features and productivity of date palm (Phoenix dactylifera L.) grown on reclaimed salinized soil. Trees. 30:1149-1161. Nassar R, Nermeen T, Reda F. 2016. Active yeast extract counteracts the harmful effects of salinity stress on the growth of leucaena plant. Scienta Hortic. 201:61-67. Nunes LRL, Pinheiro PR, Silva JB, Dutra AS. 2020. Effects of ascorbic acid on the germination and vig- our of cowpea seeds under water stress. Rev Ciênc Agron. 51(2):e20196629. Özmen S, Tabur S. 2020. Functions of folic acid (vitamin B9) against cytotoxic effects of salt stress in Hordeum vulgare L. Pak J Bot. 52(1):17-22. Parida AK, Das AB. 2005. Salt tolerance and salinity effects on plants: A review. Ecotoxicol Environ Saf. 60:324-349. Patil BC, Bhat GI. 1992. A comparative study of MH and EMS in the induction of chromosomal aberrations on lateral root meristem in Clitoria ternetea L. Cyto- logia. 57:259–264. Pekol S, Baloğlu MC, Altınoğlu YÇ. 2016. Evaluation of genotoxic and cytologic effects of environmental stress in wheat species with different ploidy levels. Turk J Biol. 40:580-588. Rieder CL, Salmon ED. 1998. The vertebrate cell kine- tochore and its roles during mitosis. Trends Cell Biol. 8:310–318. Sabagh AE, Hossain A, Barutçular C, Islam MS, Rat- nasekera D, Kumar N, Meena RS, Gharib HS, Saneo- ka H, Silva JAT. 2019. Drought and salinity stress management for higher and sustainable canola (Bras- sica napus L.) production: A critical review. Aust J Crop Sci. 13(1):88-97. Shalata A, Neumann PM. 2001. Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. J Exp Bot. 364:2207- 2211. Sharma PC, Gupta PK. 1982. Karyotypes in some pulse crops. Nucleus, 25: 181-185 Smirnoff N. 1996. The function and metabolism of ascor- bic acid in plants. Ann Bot. 78(6):661-669. Tabur S, Demir K. 2010a. Role of some growth regula- tors on cytogenetic activity of barley under salt stres. Plant Growth Regul. 60:99-104. Tabur S, Demir K. 2010b. Protective roles of exogenous polyamines on chromosomal aberrations in Hordeum vulgare exposed to salinity. Biologia. 65:947-953. Tabur S, Avcı ZD, Özmen S. 2021. Exogenous salicylic acid application against mitodepressive and clasto- genic effects induced by salt stress in barley apical meristems. Biologia. 76:341–350. Tobe K, Zhang L, Omasa K. 2003. Alleviatory effects of calcium on the toxicity of sodium, potassium and magnesium chlorides to seed germination in three nonhalophytes. Seed Sci Res. 13:47-54. Türkoğlu S. 2007. Genotoxicity of five food preservatives tested on root tips of Allium cepa L., Mut Res. 626:4- 14. Wang M, Ding F, Zhang S. 2020. Mutation of SlSB- PASE aggravates chilling-induced oxidative stress by impairing glutathione biosynthesis and suppress- ing ascorbate-glutathione recycling in tomato plants. Front Plant Sci 11:565701. Xie JQ, Li GX, Wang XK, Zheng QW, Feng ZZ. 2009. Effect of exogenous ascorbic acid on photosynthesis and growth of rice under O3 stress. Chin J Eco-Agric. 17:1176-1181. Xu W. 2017. Alleviative effects and mechanism of exog- enous ascorbic acid on chromium (Cr6+) toxicity in wheat. Nanjing: phd, Nanjing Agricultural Univ. Vicente O, Boscaiu M, Naranjo MA, Estrelles E, Belle’s JM, Soriano P. 2004. Responses to salt stress in the halophyte Plantago crassifolia (Plantaginaceae). J Arid Environ. 58:463-481. Yu CM, Xie FD, Ma LJ. 2014. Effects of exogenous appli- cation of ascorbic acid on genotoxicity of Pb in Vicia faba roots. Int J Agric Biol. 16(4):831-835. Zhu M, Shabala S, Shabala L, Fan Y, Zhou MX. 2016. Evaluating predictive values of various physiological indices for salinity stress tolerance in wheat. J Agro Crop Sci. 202:115-124. Zvanarou S, Vágnerová R, Mackievic V, Usnich S, 29L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells Smolich I, Sokolik A, Yu M, Huang X, Angelis KJ, Demidchik V. 2020. Salt stress triggers generation of oxygen free radicals and DNA breaks in Phy- scomitrella patens protonema. Environ Exp Bot. 180:104236. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 3 - 2022 Firenze University Press Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques Kamika Sribenja1, Alongklod Tanomtong1, Nuntaporn Getlekha2,* Chromosome number of some Satureja species from Turkey Esra Kavcı1, Esra Martin1, Halil Erhan Eroğlu2,*, Fatih Serdar Yıldırım3 L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations Selma Tabur1,*, Nai̇me Büyükkaya Bayraktar2, Serkan Özmen1 Characterization of the chromosomes of sotol (Dasylirion cedrosanum Trel.) using cytogenetic banding techniques Kristel Ramírez-Matadamas1, Elva Irene Cortés-Gutiérrez2, Sergio Moreno-Limón2, Catalina García-Vielma1,* Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context A Cius¹, CA Lorscheider2, LM Barbosa¹, AC Prizon¹, CH Zawadzki3, LA Borin-Carvalho¹, FE Porto4, ALB Portela-Castro1,4 Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Durre Shahwar1,2,*, Zeba Khan3, Mohammad Yunus Khalil Ansari1 Biogenic synthesis of noble metal nanoparticles using Melissa officinalis L. and Salvia officinalis L. extracts and evaluation of their biosafety potential Denisa Manolescu1,2, Georgiana Uță1,2,*, Anca Șuțan3, Cătălin Ducu1, Alin Din1, Sorin Moga1, Denis Negrea1, Andrei Biță4, Ludovic Bejenaru4, Cornelia Bejenaru5, Speranța Avram2 Polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (Foeniculum vulgare Mill.) Egizia Falistocco Methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in Pisum sativum L. Sazada Siddiqui*, Sulaiman A. Alrumman Comparative study and genetic diversity in Malva using srap molecular markers Syamand Ahmed Qadir1, Chnar Hama Noori Meerza2, Aryan Mahmood Faraj3, Kawa Khwarahm Hamafaraj4, Sherzad Rasul Abdalla Tobakari5, sahar hussein hamarashid6,* Nuclear DNA 2C-values for 16 species from Timor-Leste increases taxonomical representation in tropical ferns and lycophytes Inês da Fonseca Simão1, Hermenegildo Ribeiro da Costa1,2,3, Helena Cristina Correia de Oliveira1,2, Maria Helena Abreu Silva1,2, Paulo Cardoso da Silveira1,2,* Nuclear DNA content and comparative FISH mapping of the 5s and 45s rDNA in wild and cultivated populations of Physalis peruviana L. Marlon Garcia Paitan*, Maricielo Postillos-Flores, Luis Rojas Vasquez, Maria Siles Vallejos, Alberto López Sotomayor Identification of genetic regions associated with sex determination in date palm: A computational approach Zahra Noormohammadi1,*, Masoud Sheidai2, Seyyed-Samih Marashi3, Somayeh Saboori1, Neda Moradi1, Samaneh Naftchi1, Faezeh Rostami1 Comparative karyological analysis of some Turkish Cuscuta L. (Convolvulaceae) Neslihan Taşar¹, İlhan Kaya Tekbudak2, İbrahim Demir3, Mikail Açar1,*, Murat Kürşat3 Identifying potential adaptive SNPs within combined DNA sequences in Genus Crocus L. (Iridaceae family): A multiple analytical approach Masoud Sheidai1,*, Mohammad Mohebi Anabat1, Fahimeh Koohdar1, Zahra Noormohammadi2