Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 3-10, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-317 Citation: S. Tabur, M.D. Yurtlu, S. Özmen (2019) Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L.. Caryologia 72(3): 3-10. doi: 10.13128/cayologia-317 Published: December 13, 2019 Copyright: © 2019 S. Tabur, M.D. Yurtlu, S. Özmen. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. Selma Tabur*, Merve Dündar Yurtlu, Serkan Özmen Süleyman Demirel University, Arts and Sciences Faculty, Department of Biology, 32260 Isparta, Turkey *Corresponding authors: taburs@gmail.com Abstract. The effect of humic acid, which is an replace by a biostimulant on mitotic activity and chromosome behaviors in meristem cells of Hordeum vulgare L. germinat- ed under different salt concentrations were investigated. In the parallel to increasing salt concentrations, mitotic index partly decreased and observed the higher number of chromosomal abnormalities as compared to control. Also, it was determined that the mitotic index of seeds pretreated with only humic acid increased by 30% according to control and by 42% of mitotic aberrations. Whereas, humic acid along with salt signifi- cantly inhibited to mitotic index with parallel to increasing salt concentrations. Moreo- ver, the frequency of chromosomal aberrations in seeds germinated in humic acid and salty medium significantly decreased according to its own control. Humic acid revealed to a successful performance in ameliorating of the detrimental effect of salinity in the all concentrations studied. Humic acid application at 0.35 M salinity displayed perfect- ly successful by reaching to the same abnormality percentage of control. Keywords. Barley, chromosomal aberrations, cytotoxicity, humic acid, mitotic activity, salinity. INTRODUCTION It is a known fact to affect of abiotic stresses on plant growth and devel- opment. One of the most common environmental stress factors is salinity, which an increasing problem of many arid and semiarid areas of the World. Approximately 20% of the world’s cultivated land and accounts for over 6% of the world total area is threatened by salinity (FAO 2015). Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxi- dative stress are serious threats to agriculture and the natural status of the environment. Increased salinization of all arable land is expected to have devastating global effects, resulting in 30% land loss within the next 25 years, and up to 50% by the year 2050 (Wang et al. 2003). It has been known for a long time that salt adversely affects plant growth and development, hindering seed germination, seedling growth (Çavuşoğlu and Ergin 2015), enzyme activity (Dash and Panda 2001), DNA, RNA and protein synthesis (Anuradha and Rao 2001) and mitosis (Tabur and Demir 2010 a, b; Çavuşoğlu et al. 2016). However, recent investigations are focus- 4 Selma Tabur, Merve Dündar Yurtlu, Serkan Özmen ing more on the mechanisms of salt tolerance in plants (Munns and Tester 2008).The most efficient way to mini- mize the detrimental effects of salinity on plant breed- ing is the development of varieties with high salin- ity tolerance. Hence, researchers have used various plant growth regulators and leaf extracts to reduce or eradicate negative effects of salinity on seed germina- tion, seedling growth (Çavuşoğlu and Ergin 2015), and mitotic activity (Tabur and Demir 2010 a, b; Çavuşoğlu et al. 2016). However, in spite of substantial efforts, the outcome is still disappointingly poor due to the physi- ological and genetic complexity of this trait, the lack of reliable screening tools, and most importantly, the lack of a comprehensive understanding of the mechanisms behind salinity tolerance (Zhu et al. 2016). The data relating to mitotic activity are also mostly paradox. The main ingredient of organic substances in the soil is humus. The most active biochemical substance of humus is humic acid. Humic substances have been known that the germination and growth of plants has stimulated. Humic substances can pass through micropores of biological or artificial membrane systems, facilitate the transport of trace elements in plant roots and behave like growth hormones in plants (Chen et al. 2004). Therefore, humic substances are evaluated as a biostimulant by du Jardin (2012) who conducted a bib- liographic analysis of plant biostimulants. Biostimulants are derived from natural or biological sources and can i) enhance plant growth and development when applied in small quantities; ii) help improve the efficiency of plant nutrients, as measured by either improved nutrient uptake or reduced nutrient losses to the environment, or both; or act as soil amendments to help improve soil structure, function, or performance and thus enhance plant response (du Jardin 2012). Humic acid has positive effects on plant growth and nutrition (Calvo et al. 2014). In cytophotometric studies of the DNA, it was seen that humic substances increased the amount of DNA synthesis in the interphase nucleus of the meristematic cells in plants (Gorova et al. 2005). Furthermore, humic substances are accepted to be a plant growth promoter, particularly by changing the root structure and growth dynamics (Jindo et al. 2012; Canellas and Olivares 2014). Under both normal and salt conditions, there have been many investigations related to seed germination, seedling growth (Çavuşoğlu and Ergin 2015), root devel- opment (Sivananthi and Paul 2014), plant growth and mineral nutrient uptake (Khaled and Fawy 2011), and also some metabolic changes (El-Bassiouny et al. 2014). However, there is only limited research on the effect of humic acid on cell divison (Feretti et al. 2012) and the protective role of against effects of mutagenic and geno- toxic of various environmental conditions and chemicals (Gichner et al. 1990; Ferrara et al. 2000). In particular, no data have been recorded about effects of humic acid on mitotic activity and chromosomal aberrations in salinity conditions. In the study reported here, the influence of humic acid pretreatment on mitotic activity and chromosome behaviors in root meristems of barley seeds exposed to salinity stress were investigated. So, we have aimed to clarify to some extent to what extent humic acid can alleviate salt stress, whether it stimulates cells to enter the mitosis division or not and also whether it causes any changes in the structure and behavior of chromo- somes or not. MATERIAL AND METHODS In the present study, barley seeds (Hordeum vulgare L. cv. Bülbül 89) were used. The barley seeds were sub- jected to surface sterilization before used. For this, seeds were kept in 1% sodium hypochlorite for ten minutes, then washed with distilled water five times and dried on filter paper at room temperature. Preparation of solutions and germination of seeds NaCl and humic acid used in the experiments were obtained from Merck and Sigma-Aldrich firm respec- tively. As test solution, 28 mg/L humic acid were used due to promote germination in the best way against the inhibitory effect of salt. Concentrations of NaCl were 0.25, 0.30 and 0.35 M (molar). These salt levels and the concentration of humic acid used were determined in the result of a preliminary study. Primarily, plump-look- ing, robust and approximately equal-sized 20-25 barley seeds were selected. Then, sterilized seeds were soaked in test tubes filled with 28 mg/L humic acid and distilled water (control) at constant volume (50 ml) for 24 h at room temperature. At the end of this pretreatment ses- sion, the seeds from every application were arranged in 10 cm Petri dishes covered with two sheets of filter paper moistened with 7 ml of distilled water and different salt concentrations. Petri dishes were transferred in an incu- bator to germinate at 20 ± 1°C in continuous dark for several days. Cytogenetical analyses When the root tips were 0.5–1 cm long, they were cut off, pretreated with a saturated solution of paradi- 5Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. chlorobenzene for 4 h at 20°C, fixed with Carnoy’s Fluid I (absolute ethanol: glacial acetic acid, 3:1, v/v) for 24 h, and stored in 70% alcohol at 4°C until used. Then, the root tips were hydrolyzed in 1 N HCl at 60°C for 18 min, stained with Feulgen for 1 h, and squashed in 45% acetic acid (Sharma and Gupta 1982). After 24 h, microscopic slides were made permanent by mounting in balsam. The mitotic phases and mitotic aberrations were photo- graphed (100×) with a digital camera (Olympus C-5060) mounted on an Olympus CX41 microscope. Data analyses and statistical evaluations To determine the effect of humic acid and salt on the mitotic index, at least 3000 cells (approx. 1000 per slide) were scored in control and in treated groups. Chromosomal aberrations were calculated for each con- centration as the percentage of 300 dividing cells count- ed. Statistical analysis related to all parameters was per- formed by using SPSS program according to Duncan’s multiple range test at the level of significance p ≤ 0.05 (Duncan 1955). RESULTS Effects of humic acid on mitotic index and chromosome aberrations in normal conditions Barley seeds pretreated with 28 mg/L humic acid were germinated at 20°C in distilled water and slides were prepared with the root tips obtained. The mitotic index values calculated as a result of the cell counting procedures performed in these slides were presented in Table 1. Humic acid pretreatment caused a 30% increase in the mitotic index of barley seeds germinated in non- stress conditions as compared to those of the control group. In other words, the mitotic index of seeds treated with humic acid was even higher than seeds germinated in distilled water. As a result of cytological analyzes, chromosomal aberrations in meristem cells of barley seeds germinat- ed at 20°C in distilled water (control) were statistically insignificant. That is, all mitotic phases were normal (Fig. 1). However, the frequency of chromosomal aberra- tions in seeds germinated in distilled water after humic acid pretreatment was remarkably higher than that in the control (Table 2). For example, while the rate of chromosomal aberrations in control seeds was 0.04%, it was 0.42% in seeds pretreatmented with humic acid. A resulting of the application of humic acid alone, chromosomal aberrations such as fragment formation, lagging chromosome, anaphase and telophase bridges, fault polarization in telophase and anaphase were fre- quently observed (Fig. 2). Effects of humic acid on mitotic index and chromosome aberrations in salinity conditions Mitotic index scores obtained from this study made to determine the activity degree of humic acid on the Table 1. Mitotic index of barley seeds germinated in distilled water and different NaCl concentrations after humic acid pretreatment. Mitotic index (%) NaCl (M, molar) Control Humic acid (28 mg/L) 0.0 (distilled water) *0.19 ± 0.13bcd 0.27 ± 0.04e 0.25 0.19 ± 0.03bcd 0.17 ± 0.03abcd 0.30 0.18 ± 0.01abc 0.18 ± 0.02bcd 0.35 0.18 ± 0.03abc 0.12 ± 0.02ab The pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or humic acid. As test solution, 28 mg/L humic acid was used. Different salt con- centrations (0.25, 0.30, 0.35 M NaCl) were exogenously applied to germination medium. Data are the means of three replications ± standard deviation. * Shows values with insignificant difference (p ≤ 0.05) for each col- umn shown with same letters Fig. 1. Normal mitosis phases in root tips meristems of barley ger- minated in distilled water (control). (a) Prophase; (b) metaphase (2n = 14); (c) anaphase; (d) telophase. Scale bar = 10μm. 6 Selma Tabur, Merve Dündar Yurtlu, Serkan Özmen mitotic index of barley seeds germinated under salt stress was summarized in Table 1. The mitotic index value of barley seeds was statis- tically decreased at especially high concentrations as parallel to the increase of salt concentration as com- pared with control. It was found that the seeds applied alone humic acid shows a considerable increase on the mitotic index as compared with seeds germinated in dis- tilled water. That is, the addition of 28 mg/L humic acid increased the mitotic index by 30%. However, humic acid pretreatment caused a significant decrease in the mitotic index with increasing salt concentrations. The mitotic index value (0.12) in root meristems of the seeds germinated at the highest salt concentration (0.35 M) after treated with humic acid reduced to a large extent (Table 1). Considering all of the application groups, it was determined that humic acid pretreatment together with salinity decreased the mitotic index at 0.25 M and 0.35 M salinity, while it was at the same level with its own control at 0.30 M salinity (Table 1). In addition to, chromosomal aberration scores were presented in Table 2. While there is a chromosomal aberration that can be ignored statistically in control seeds germinated in distilled water, screening mitotic divisions revealed the many numbers of chromosomal aberrations as parallel to increasing salt concentrations. It was determined that chromosomal aberrations at the highest salt concentration (0.35 M) were higher approximately 60% according to those in distilled water. At the same time, alone humic acid pretreatment caused a 42% increase in chromosomal aberrations as com- pared to control. However, the frequency of chromo- somal aberrations of seeds germinated at different salt concentrations after treatment with humic acid showed a decrease which can be considered statistically significant from those of the seeds germinated in distilled water after treatment with humic acid. Namely, humic acid has been quite successful in mitigating the detrimental effect of salt stress on chromosomal aberrations at all the salt concentrations studied here. Moreover, humic acid appli- cation at the highest salt concentration (0.35 M) signifi- cantly reduced the detrimental effect of salt stress and reached the percentage of the same abnormality (0.04) as the seeds germinated in distilled water (Table 2). The most prominent chromosomal aberrations in seeds belonging to all application groups were the disor- ganizations in anaphase and telophase such as bridges, Table 2. Frequency of chromosomal aberrations of barley seeds ger- minated in distilled water and different NaCl concentrations after humic acid pretreatment Chromosomal aberrations (%) NaCl (M, molar) Control Humic acid (28 mg/L) 0.0 (distilled water) 0.04 ± 0.18a 0.42 ± 0.05cd 0.25 0.25 ± 0.27bc 0.22 ± 0.01bc 0.30 0.35 ± 0.07c 0.30 ± 0.15bc 0.35 0.58 ± 0.05d 0.04 ± 0.04a The pretreatment process of seeds was performed by soaking 24 h in constant volumes of distilled water (control) or humic acid. As test solution, 28 mg/L humic acid was used. Different salt con- centrations (0.25, 0.30, 0.35 M NaCl) were exogenously applied to germination medium. Data are the means of three replications ± standard deviation. * Shows values with insignificant difference (p ≤ 0.05) for each col- umn shown with same letters Fig. 2. Chromosomal aberrations in root meristems of barley seeds germinated in distilled water and different NaCl concentra- tions after treatment with 28 mg/L humic acid: a) micronucleus; b) granulation c) disorderly prophase; d) disrupted equatorial plate; e) uncoiling chromosomes f ) fragments g) sticky chromosomes; h) bridges in anaphase and lagging chromosome; ı) fault polarization in anaphase; j) vagrant chromosome in anaphase (arrow); k) align- ment anaphase; l) bridges in telophase; m) fault polarization in telo- phase n) distant poles in telophase; o) vagrant chromosome in telo- phase. Scale bar = 10 μm. 7Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. lagging chromosome, fault polarization, distant poles, vagrant chromosomes and alignment anaphase (Fig. 2h–o). In addition, other chromosomal aberrations observed were the presence of micronucleus, granula- tion, disorderly prophase, disrupted equatorial plate, uncoiling chromosomes, fragments, sticky chromo- somes (Fig. 2a–g). The minimal common aberrations were micronucleus, granulation and disrupted equato- rial plate. DISCUSSION It is well known that humic substances stimulate germination in seeds of various species by increasing enzymatic activities in seed tissues during germination and decreasing mitotic activity under normal conditions (Feretti et al. 2012). However, no information has been found on effects of humic acid on mitotic activity and chromosomal aberrations especially under saline condi- tions. All the data on the effect of humic acid on these parameters in saline conditions are presented for the first time in this study. In the present work, it was determined that humic acid pretreatment alone in non-stress conditions sig- nificantly increase the mitotic index of barley seeds as compared to distilled water (Table 1). Whereas, Feretti et al. (2012) have suggested in their study using vari- ous plant tests (Allium cepa test, Tradescantia and Vicia faba micronucleus test) that alone humic acid pretreat- ment decreases the mitotic index in studied two solu- tions. Because of the differences in the findings of these researchers may be due to the species of plant studied or the concentration of the humic acid used. However, our findings have been endorsed by the data expressed that the humic substances increase the amount of DNA synthesis in meristematic cells in plants (Gorova et al. 2005). In addition, we found that the application of alone humic acid increased the chromosome abnormal- ity rate by 42% under normal conditions (Table 2). Our this finding is consistent with Ferretti et al. (2012)’s find- ings. For this reason, we can reach the result that alone humic acid pretreatment increases the mitotic index val- ue in barley seeds germinated in distilled water but may have a genotoxic effect because of the negativity that it has shown on chromosome behavior. This reveals the fact that alone humic acid application can create muta- tions in various types over time. As is known, plant growth and development are adversely affected by salinity. High salinity is an impor- tant factor negatively inf luencing plant growth and development even in most halophy tes. At present, approximately 20% of cultivated lands in the World are affected by salinity (FAO 2015). Generally, it is suggest- ed that salinity impairs seed germination, retards plant development and reduces productivity. In some cases, the plant dies before completing the life cycle. There have been numerous investigations conducted to explore the effects of salt on plant growth, but mechanisms of salt stress have not yet been explained precisely (Munns and Tester 2008; Zhu et al. 2016). We determined that mitotic index in root meris- tems of barley seeds significantly decreased with increas- ing salt concentrations (Table 1). The inhibitory effects of salt stress on mitotic activity are known for a long time (Lutsenko et al. 2005). Salt induced-inhibition of cell division may relate to osmotic effect and ion uptake (Munns and Tester 2008 ), inhibition of DNA, RNA and protein synthesis (Anuradha and Rao 2001), distrup- tion the activity of enzymes required for cells (Miller et al. 2010) and hinderance of mitosis division (Tabur and Demir 2010 a, b; Çavuşoğlu et al. 2016). It is worth mentioning again that the relation between salinity and mitotic activity was confirmed by the present work. In our study, it was also detected that there was a remark- able increase in all kinds of chromosomal aberrations at the root meristems of barley parallel to the rise of salt concentrations (Table 2). The detrimental effects of salt stress on chromosomal aberrations in plants have been studied for over the past decade. These recent studies have shown that the higher concentrations of NaCl has chromotoxic effects and increases the percentage of total aberrations (Tabur and Demir 2010 a, b). Furthermore, it was reported that these high salt concentrations delayed mitosis and caused various anaphase aberrations in bar- ley (Tajbakhsh et al. 2006) and in onion (Çavuşoğlu et al. 2016). There is no relevant literature data relating to effects of humic acid on either mitotic activity or/and chromo- somal abnormalities in saline conditions. The present study is the first one revealing the cytogenetic respons- es to the salt stress of humic acid. However, there are a few studies about the effect of humic acid application against the genotoxic effects of various chemicals such as N-nitrous compounds, maleic hydrolase and some disinfectants (Gichner et al. 1990; Ferrara et al. 2000). These studies have argumented that humic acid exhibits an anticlastogenic or antimutagenic activity in differ- ent plants. Ferretti et al. (2012) determined that humic acid alone reduces the mitotic index and has genotoxic effects. However, there could not be made explanation for the effect of humic acid on these disinfectants since these investigators have not determined any evidence of the genotoxic effect of disinfectants alone. 8 Selma Tabur, Merve Dündar Yurtlu, Serkan Özmen In the present work, we analyzed that humic acid pretreatment in salt stress conditions was not suffi- ciently successful on the mitotic index of barley seeds, but exhibit a performance very successfully statistically on chromosome behaviors. Although humic acid appli- cation alone was caused a significant increase (42%) of chromosomal aberrations in root meristems of bar- ley seeds germinated in distilled water, it has shown remarkable success in alleviating a large majority of these abnormalities caused by salinity in salt stress con- ditions. In parallel to salt concentrations rise, humic acid was reduced the detrimental effects of salinity and caused complete elimination of chromosome abnormali- ties at the highest salt concentration studied. That is, the application of 28 mg/L humic acid at 0.35 M salin- ity achieved an excellent success on the negative effect of salt stress, reaching to the same percentage (0.04) as the seeds germinated in distilled water. The important point here is that humic acid should be used at the appropri- ate concentrations, considering the negative effects on chromosome behaviors when it was used in non-stressed conditions. Humic acid application alone under non- stressed conditions may have functioned as a stimula- tor by triggering the synthesis of proteins necessary for normal cell division and by accelerating the mitotic cycle. The acceleration of the mitotic cycle may have led to a number of disruptions during the cell division and a significant increase of chromosomal abnormalities. As is known, external stimulator growth regulator applica- tions are useless under normal conditions where there is no stress (Tabur and Demir 2010 a). Therefore, it is not surprising that the application of humic acid under stress conditions slows down mitotic activity in parallel with salt concentrations rise and eventually alleviating the negative effects of stress by regulating chromosome behaviors, and even removing (at 0.35 M salinity). In addition, we can explain as follows the reason why various chromosomal aberrations observed during the microscopic examination of the root meristem cells of seeds belonging to all the applications: In general, accurate chromosome segregation in mitosis requires that sister kinetochores attach to microtubules emanat- ing from opposite spindle poles. Because kinetochore attachment is a stochastic process, it is error prone and can result in chromosome malorientation (Rieder and Salmon 1998). Mitotic irregularities such as disorderly prophase, fault or distant polarization, alignment ana- phase, vagrant chromosome and bridges may be main- ly the result of the above mentioned reasons or spindle dysfunction. Generally, such abnormalities constitute a significant portion of chromosomal aberrations. The formations of micronuclei are likely the consequence of vagrant chromosomes and fragments. Also, some researchers reported that MNs, indicators of chromo- somal genotoxicity and instability, are formed from one or more chromosomes (Bonciu et al. 2018). It is known that fragments are considered as structural changes in chromosomes and that chromosomes are affected by physical or chemical agents outside normal condi- tions (El-Ghamery et al. 2000). It has been reported that certain regions of chromosomes are broken by reacting with chemical substances and these regions are particularly heterochromatic regions (Rieger et al. 1973). Abnormal chromatin condensation expressed as chromatin granulation is concerned with the inhibi- tion of enzymes and histone proteins. While laggard chromosomes could be the result of the failure of spin- dle apparatus to organize in normal way, sticky chro- mosomes may result from the improper folding of the chromatin fibres (Klášterská et al. 1976). According to some researchers, chromosomal stickiness is a marker of the toxic effect on chromatin (Fiskesjö and Levan 1993). The prophase and metaphase cells with uncoiled chromosomes may be due to disorderly chromosome contractions. The disrupted equatorial plate may result from unequal distribution of chromosomes and spindle dysfunction. Bonciu et al. (2018) asserted that nucleo- plasmic bridges originate from dicentric chromosomes or occur as a result of a faulty longitudinal breakdown of sister chromatids during anaphase. It has also been reported that anaphase and telophase bridges may have been the result of inversions (Tabur and Demir 2010 b). It is thought that humic acid alone or salt concentrations used in our study may have been caused to all these abnormalities mentioned above by triggering the stimu- lation/ inhibition of enzymes and proteins necessary for the normal cell division, by disturbing the spindle mech- anism and by accelerating mitotic cycle. CONCLUSION The mechanisms by which salinity inhibits growth are complex and controversial. Moreover, these mecha- nisms may vary substantially according to factors, such as plant species, the developmental stage of the plant, the strength of the stress and duration of the treatment. Unfortunately, a universal mechanism about this con- tradiction has not been established yet. Although the causes of salinity have been characterized, our under- standing of the mechanisms by which salinity prevents plant growth is still rather poor. This work may pro- vide new conceptual tools for designing hypotheses of salt tolerance in plants. As a result, we have attempted 9Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. to serve the filling of a gap in the literature by compar- ing their interactions between the mitotic activity and chromosome behaviors of humic acid under normal and salt stress conditions using barley seeds, an important model plant for molecular studies. In future studies, the investigation of the effects of humic acid on fundamen- tal metabolic events such as nucleic acid metabolism, protein synthesis, and enzyme synthesis, which may be directly or indirectly effective on mitotic activity and chromosomal abnormalities will contribute to the clari- fication of mentioned mechanism. ACKNOWLEDGEMENTS We would like to thank Dr. Dilek Çavuşoğlu (SDU Atabey Vocational School, Food Business Adminis- tration) for their help in shaping. In addition, we are grateful to the Süleyman Demirel University Scientific Research Projects Management Unit for the financial support of this work. FUNDING This project is supported as financial by Süleyman Demirel University Scientific Research Projects Manage- ment Unit under Grant Project No. 3275-YL1-12. REFERENCES Anuradha S, Rao SSR. 2001. Effect of brassinosteroids on salinity stress induced inhibition of seed germination and seedling growth of rice (Oryza sativa L.). Plant Growth Regul. 33(2): 151–153. 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Evaluating predictive values of various physiological indices for salinity stress tolerance in wheat. J Agron Crop Sci. 202: 115–124. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 3 - 2019 Firenze University Press Role of humic acid against salt-induced cytotoxicity in Hordeum vulgare L. Selma Tabur*, Merve Dündar Yurtlu, Serkan Özmen Characterization of intraspecific hybrid in Clitoria ternatea (L.) using morpho-physiological, cytogenetic, metabolic and molecular markers Aparupa Naik1, Amiya K. Patel1, Sujit K. Mishra1, Atul Nag2, Jogeswar Panigrahi1,2,* Floral architecture, breeding system, seed biology and chromosomal studies in endangered Himalayan Angelica glauca Edgew. (Apiaceae) Kamini Gautam1,2,*, Ravinder Raina1,3 Cytotoxic and genotoxic activity of Plantago major L. extracts Amira Ždralović, Aner Mesic, Izet Eminović, Adisa Parić* Genetic diversity of Rhododendron simsii Planch. natural populations at different altitudes in Wujiashan Mountain (central China) Shuzhen Wang, Yanyan Luo, Tao Yang, Yujia Zhang, Zhiliang Li, Weibin Jin, Yuanping Fang* Nonreduction via meiotic restitution and pollen heterogeneity may explain residual male fertility in triploid marine halophyte Limonium algarvense (Plumbaginaceae) Sofia I. R. Conceição1, Ana Sofia Róis1,2, Ana D. Caperta1,* Effects of zinc on pollen gamete penetration to pistils in some apple crosses assessed by fluorescence microscopy Yavar Sharafi Active chemical constituents of Cynanchum viminale and its cytotoxic effects via apoptotic signs on Allium cepa root meristematic cells Neethu Kannan Bhagyanathan*, John Ernest Thoppil Clastogenic and cytotoxic effects of aerial parts’ aqueous extract of Synedrella nodiflora (L.) Gaertn. on Wistar rat bone marrow cells Saumabha Chatterjee1,2, Sanjib Ray2* Cytogenetics of Accanthopus velikensis (Piller et Mitterpacher, 1783) (Tenebrionidae: Helopini) Dirim Şendoğan1, Beril Gündoğan1, Maxim V. Nabozhenko2,3, Bekir Keskin1, Nurşen Alpagut Keskin1,* Chromosome number and genome size diversity in five Solanaceae genera Amanda Teixeira Mesquita1,*, Marìa Victoria Romero-da Cruz1, Ana Luisa Sousa Azevedo2, Eliana Regina Forni-Martins1