Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 21-37, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2409 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Tabur, S., Yilmaz-Ergün, Ş.B., & Özmen, S. (2024). Role of ten dif- ferent exogenous plant growth promot- ers in regulating cytotoxic and geno- toxic processes in barley exposed to high temperature stress. Caryologia 77(1): 21-37. doi: 10.36253/caryolo- gia-2409 Received: December 7, 2023 Accepted: May 26, 2024 Published: July 8, 2024 Copyright: © 2024 Tabur, S., Yilmaz- Ergün, Ş.B., & Özmen, S. This is an open access, peer-reviewed article published by Firenze University Press (ht tps://www.fupress.com/caryolo- gia) 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. Role of ten different exogenous plant growth promoters in regulating cytotoxic and genotoxic processes in barley exposed to high temperature stress Selma Tabur*, Ş. Betül Yilmaz-Ergün, Serkan Özmen Faculty of Engineering and Natural Sciences, Department of Biology, Süleyman Demirel University, Isparta 32260, Turkey *Corresponding author. Email: taburs@gmail.com Abstract. Agricultural crop affected preliminary and the most prominent by the adverse effects of global climate change have to adapt to various abiotic factors that will occur as a result of climate fluctuations in the near-future and struggle to survive. Among abiotic factors, the one of the greatest impact on plant stress is high tempera- ture. Therefore, the most important step to take action against the global threat is the development of new temperature tolerant varieties. Barley, which is the fourth most important cereal in the world after wheat, maize, and rice are affected by high ambi- ent temperatures. In this work, the effects of their alone and in double, triple combina- tions of ten various plant growth regulators (PGRs) on mitotic activity and chromo- some behaviors in root meristems of barley exposed to high temperature (30°C) were investigated. In the experiments, Hordeum vulgare L. cv. Bülbül 89 variety and GA3, KIN, BA, E, EBR, TRIA and PAs (Spm, Spd, Put, Cad) as growth regulators were used. The results obtained were compared with each other and with those of the seeds ger- minated at optimum temperature (20°C). Consequently; it has been determined that most of the PGRs studied, especially the GA3 and their combinations with GA3, exhibit a very successful performance on mitotic activity and cytogenetic aberrations in barley seeds germinated under high temperature stress- HTS conditions. The effects of these PGRs (except for EBR) and their combinations on mitotic activity and chromosome behaviors under HTS have been presented in this study for the first time. Keywords: chromosomal aberrations, heat stress, Hordeum vulgare L., mitotic index, plant growth regulators. INTRODUCTION Climate change is an inevitable phenomenon globally, which affects all aspects mankind, including agricultural production worldwide today. Accord- ing to IPCC report (2021) projects that in the coming decades climate chang- es will increase in all regions and for 1.5°C of global warming there will be increasing heat waves- longer warm seasons and shorter cold seasons. And, http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2409 https://doi.org/10.36253/caryologia-2409 https://doi.org/10.36253/caryologia-2409 https://www.fupress.com/caryologia https://www.fupress.com/caryologia mailto:taburs@gmail.com 22 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen this temperature increases is particularly predicted to increase by about 1–3°C by the mid and by about 2–5°C by the late twenty-first century. The report shows that at 2°C of global warming, heat extremes would more often reach critical tolerance thresholds for agriculture and health. Therefore, it is staminal that a lot of coun- tries especially in the south of 40° north latitude includ- ing Turkey located in the geographic region where the adverse effects of climate change are possible struggle these climatic fluctuations, take action against anticipat- ed threats, and revive strategies in this direction (Budak 2022). It have predicted to climatological extremes are caused various abiotic stresses and have a general nega- tive effect on plant growth and development as also likely all living. Thus, future agricultural crop production and thus global food security will encounter additional chal- lenges with human population increase competing for environmental resources (Bita and Gerats 2013; Pereira 2016). In this respect, performing genome-wide analyz- es of stress-resistant genotypes from agricultural crop, revealing their tolerance and selective mechanisms to against adversely conditions, and cultivating new varie- ties are of great importance to tackle all these challenges. Barley (Hordeum vulgare L.) is one of the most sig- nificant cereal crops farmed in Europe, the Middle East, North and South Africa, and Asia. This cereal, great economic value due to its use in both animal feed and the food industry are a cereal required grown in abiotic stress conditions that limit plant growth due to global climate changes in our current period. Furthermore, it is preferred as a model plant in cytogenetic researches for reasons such as its effortless supply, in vitro germination of seeds and small genome (Tabur and Demir 2010b; Özmen et al. 2022, 2023). Cytogenetic researches play an important role in understanding the chromosomal and genetic architecture of plant species. In particular, the chromosomal aberrations (CAs) have been accepted as an indicator of genetic damages and for those altera- tions which ultimately lead to mutations (Saxena 2022). Therefore, performing the CAs test has vital significant to determine whether a test substance or abiotic stress factors can cause various types of mutations over time. Among the abiotic stresses, temperature increase has major negative impact on agricultural crops susceptible to changes in temperature. Temperature stress occur result of the cumulative effect of the temperature sever- ity, the time the plant is exposed to these unfavorable condition, and the degree at which the temperature is increasing and cause significant and irreversible dam- age to plant growth and development (Hill and Li 2022). High temperatures are absolute effective as a stress fac- tor in plants during germination, and the measures tak- en by plants and their molecular responses under these stress conditions are completely different. Each plant species has a temperature range represented by a mini- mum, maximum, and optimum in which it functions optimally, and outside this range all cellular metabo- lisms and thus plant growth are adversely affected (Hat- field and Prueger 2015). High temperature stress (HTS) disrupts the vital cellular phenomena by damaging gen- erally physiological, biochemical and molecular mecha- nisms in plants (Narayanan 2018; Jacott and Boden 2020) and production of toxic metabolites and reactive oxygen species (ROS) takes place in the injured cells occurred as a result of aberrant metabolism (Wahid et al. 2007). Hence this situation causes total crop failure by decreasing in growth, product and quality (Shrestha et al. 2022; Khan et al. 2023). Increasing temperature inhibits different stages of plant development especially seriously reduces the germination and early seedling growth in a number of plant species including barley (Wahid et al. 2010). Additionally, it has been reported that HTS negatively affects cell division and micro- tubule organization in tobacco, wheat and vetch thus leading to decreased mitotic index (MI) and irregular mitotic configurations (Abou-Deif and Mohamed 2007; Öney and Tabur 2013; Öney et al. 2015). Fareghi et al. (2015) asserted that Vicia dasycarpa that are normally diploid exhibit a mixoploid state with diploid, aneuploid and tetraploid cells after temperature shock (boiling the seeds at 90°C for 3 min.). However, the ability of a geno- type to survive at high temperature depends on the type or variety of the plant, age, stage of development, the susceptibility of the cell types, the degree and duration of the elevated temperature (Wahid et al. 2007; Hasa- nuzzaman et al. 2013). Plant hormones are essential for regulating the interactions between plants and their complex biotic and abiotic environments. Most of the physiological activities occurring in the plant are under the control of these hormones. The effects of hormones always appear in a balance as complementary to each other (synergis- tic) or reducing the effect of each other (antagonistic) (Aerts et al. 2021). Under single or multifactorial stress combination phenomenon, fluctuations in hormonal bal- ance in plants bring about serious morphological, physi- ological, biochemical and molecular changes (Goharrizi et al. 2021; Zandalinas et al. 2022 and their cited). For example, ABA (abscisic acid) plays a major role in differ- ent stages of plant development such as stomata opening and closing, seed germination, and dormancy and trig- gers many physiological mechanisms in plants. The plant growth is severely retarded and it increases the ABA concentration in cells under drought conditions. ABA 23Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress accumulation during this period controls transpiration and inhibit stomatal disclosure (Dong et al. 2018). There are many studies that phytohormones or vari- ous PGR agents have positive effects on plant growth and development, as well as various physiological and biochemical mechanisms, and increase plant resistance against many stresses. Because the role of different indi- vidual phytohormones under abiotic stresses is too board to be covered here, we can direct readers to up-to-date research articles and reviews on the subject (Huyluoğlu et al., 2008; Moumita et al. 2019; Younis and Ismail 2019; Emamverdian et al. 2020; Islam and Mohammad 2020; Mangena 2020, 2022; Kosakivska et al. 2022; Kothari and Lachowiec 2021; Sharma 2021; El-Beltagi et al. 2022; Fat- ma et al. 2022; Sarwar et al. 2022; Shao et al. 2022; Ver- ma et al. 2022; Sultan et al. 2023). The constantly rising ambient temperature caused by rapidly changing climate warming is considered one of the most detrimental abiot- ic stresses and heat tolerance in plants can be achieved by exogenous application of various protectant substances (Rasheed et al. 2011; Qureshi et al. 2022). Therefore, since especially recent ten years, the exogenous application of protectant substances such as osmoprotectants, phyto- hormones, signal molecules, polyamines, trace elements and nutrients have studied by numberless researchers to alleviate the harmful effects of HTS on plant (Hasanuz- zaman et al. 2012; Waraich et al. 2012; Öney and Tabur 2013; Zaki et al. 2014; Öney et al. 2015; Kaur et al. 2018; Taheri and Haghighi 2018; Chen et al. 2019; Wu and Yang 2019; Alcázar et al. 2020; Jing et al. 2020; Li N. et al. 2021, Li Y. et al. 2023; Sharma et al. 2022; Wang et al. 2022; Wu et al. 2022; Huang et al. 2023; Hudelson 2023; Mei et al. 2023). As mentioned above, there are many studies that phytohormones have positive effects on plant growth, development, physiological processes, and yield and increase plant stress resistance against various stresses. On the other hand, the effects of various phytohormones or PGRs on mitotic activity and chromosomal behav- iors under normal conditions (in a stress-free environ- ment) have also investigated by many researchers since the 1970s (Powell et al. 1973; Oh and Clouse 1998; Hu et al. 2000; İsmailoğlu et al. 2004; Huyluoğlu et al. 2008; Kartal et al. 2009; Truta et al. 2011; El-Ghamrey et al. 2013; El-Ghamery and Mousa 2017; Tabur et al. 2019; Tütünoğlu et al. 2019). It is a well-known fact that the exogenous application of both natural and synthetic PGRs contributes to the increase in the relative number of embryonic cells. Therefore most of these researchers agree that the exogenous phytohormones promotes cell division and proliferation and activates DNA replication and protein synthesis, but causes chromosomal aberra- tions (CAs) by disrupting the mitotic balance. Further- more, some of these researchers argue that PGRs are more effective on cell division at high concentrations, while others assert that they are more effective at low concentrations. However, studies on PGRs effects on cell division, mitotic activity and chromosome behavior under various stress conditions (heavy metals, salinity and drought) are quite limited (Mansour and Kamel 2005; Tabur and Demir 2009; 2010a,b; Maraklı et al. 2014; Özmen et al. 2022). Moreover, a single study was found on how effec- tive only EBR are on these parameters (MI and CAs), especially under heat stress conditions as a result of our detailed literature research (Pradhan and Gupta 2013). For this reason, the effects of ten different PGRs either alone or double and triple combinations, mentioned on MI and chromosome behaviors under HTS have been comprehensively revealed in this study for the first time. As a result, aims of this work are (1) to determine the effect of HTS on MI and CAs, (2) to determine the effects of exogenous application of various PGRs alone or in double, triple combinations on the mitotic activ- ity and chromosome behaviors in barley root meristems under nonstress conditions, (3) to comparatively evalu- ate the effects of these PGRs on the mentioned param- eters in barley root meristems under HTS and to fill the gap in the literature on this subject, (4) to clarify in detail to what extent all studied PGRs and their com- binations can overcome HTS, whether they encourage cells to enter mitosis, and whether they cause any chang- es in the structure and behaviors of chromosomes. MATERIALS AND METHODS Preparation of the seeds and PGRs The barley seeds (Hordeum vulgare cv. Bülbül 89) were kindly provided from Field Crops Research Insti- tute, Ankara/Turkey. PGRs used in the experiments were obtained from Fluka and Sigma-Aldrich Firm. To forbid contamination before germination experiments, the bar- ley seeds were surface sterilized by immersion in 1% (w/v) NaClO solution for 10 min, rinsed thoroughly five times with sterile distilled water and dried on filter papers at room temperature. Ten different PGRs were used in the study: GA3 (gibberellic acid), KIN (kinetin), BA (benzy- ladenine), E (ethylene), EBR (24-epibrassinolide), TRIA (triacontanol), Spm (spermine), Spd (spermidine), Put (putrescine) and Cad (cadaverine). The concentration of each PGR (as μM, micromolar), which reduces the dam- aging effect of HTS (30°C) on germination, was deter- mined as a result of a preliminary study (Figure 1). 24 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen Germination experiments First of all, germination experiments were carried out at different temperatures between 22 and 35°C and the tolerance limit of barley seeds against heat stress was determined as 30°C. Germination processes were carried out at constant temperatures of 20°C (control) and 30°C in the dark and in an incubator. For this pro- cess, full-looking, robust and uniform sized 20 seeds were selected first. These previously sterilized seeds were pretreated in 50 mL distilled water (control), GA3, KIN, BA, E, EBR, TRIA, Spm, Spd, Put and Cad alone and in their double-triple combinations for 24 hours at room temperature. At the end of this pretreatment ses- sion, the solutions were filtered and the seeds were vac- uum-dried. Then, the seeds for each application were arranged in Petri dishes covered with two sheets of fil- ter paper moistened with 7 ml of distilled water. Imme- diately after sowing, the Petri dishes were placed in the above-mentioned 20°C and 30°C constant temperature incubators for germination. At the specified tempera- tures, they could not be studied because suitable and sufficient germination did not occur in combinations other than double combinations GA3+KIN, GA3+EBR, KIN+EBR and triple combinations GA3+KIN +EBR, GA3+KIN+E. Figure 1. Diagram showing PGRs solvents, prepared stocks and concentration of solutions used in the study. Stock solutions were prepared by dissolving each of PGRs with appropriate solvents and made up to liter (μM, micromolar) with distilled water. The stock solutions were diluted and the concentrations of solutions used in the study were obtained. For this, PGR concentrations which reduce the damaging effect of 30°C, the tolerance limit of barley seeds against heat stress were used. Seeds were pretreated in 50 mL distilled water (control), PGRs alone and their double-triple combinations for 24 hours at room temperature. Germination process carried out at constant temperatures of 20°C (control) and 30°C in an incubator. 25Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress Cytogenetic examinations For cytogenetic examinations, the root tips reached about 1 cm length after 5-7 day were excised, pretreated with a saturated solution of paradichlorobenzene for 4 h at 20 °C, fixed in solution absolute ethanol:glacial acetic acid (3:1, v/v) for overnight, and stored in 70% alcohol at refrigerator until used again. The root tips were hydro- lyzed in 1 N HCl at 60 °C for 15–18 min, stained for 1-2 h in accordance with the standard procedure for Feul- gen staining, smashed in a drop of 45% acetic acid and squashed (Elçi and Sancak 2013). After 24 h, microscopic slides were made permanent by mounting with alcohol vapor exchange method. The best mitotic phases and mitotic aberrances were photographed (100X) with digi- tal camera (Olympus C-5060) mounted on an Olympus CX41 microscope. Data analyses and statistical evaluations For detect the effect of PGRs and HTS on the MI, the prepared slides were examined under the microscope at 100X magnification, and MI, i.e. percentage of divid- ing cells were accounted by counting at least 6000 cells for per application (three repeat, 2000 per slide). The MI was calculated using the following equation: 𝑀𝑀𝑀𝑀 (%) = total number of dividing cells total number of analyzed cells × 100 At the same time, CAs occurring at all stages of mitosis during microscopic observation of the slides were calculated according to the following the equation for each per-application as the percentage of 350 divid- ing cells counted. 𝐶𝐶𝐶𝐶 (%) = total number of aberrant cells total number of dividing cells × 100 All experiments were repeated three times. Statisti- cal evaluation concerning all obtained parameters was realized by using SPSS 14.0 program according to Dun- can’s multiple range test, at p ≤ 0.05 level of significance (Duncan 1955). RESULTS As explained in detail in the Introduction, it is known that under normal conditions, GA3, cytokinins (CKs= BA and KIN) and E generally promote cell divi- sion and cell elongation, thereby increasing growth. However, the effects of EBR, TRIA and PAs (Spm, Spd, Put and Cad) on cell division have not yet been fully elu- cidated. Therefore, the present study aimed to clarify the counterchecks of these chemicals alone or in combina- tion on MI and CAs, under both optimum (20˚C) and HTS (30˚C). Counterchecks of PGRs against the adverse effects of HTS on MI The MI values calculated as a result of cell count- ing of barley meristems after pre-application of various PGRs alone or in combinations at 20°C and 30°C are presented in Figure 2. At optimum temperature (20°C), the application of GA3, BA and Put, respectively, from the PGRs studied here showed statistically quite a lot successful effect on the MI of barley root meristems compared to the con- trol group. Especially, at GA3 the MI were reached to the highest value by increasing from 6,2±0,3 (at 20°C, in distilled water) to 15,1±0,8 (approx. 2.5 fold). While E, Spm and Cad applications were partially successful on this parameter KIN, EBR, TRIA and Spd applications exhibited an inhibitory effect on the MI. Considering the alone PGRs pre-applications, GA3 treatment on the MI was more successful than all other treatments, while TRIA had the most negative effect. Among the double combinations of PGRs, GA3+KIN showed a more suc- cessful effect on the MI than the control group and oth- er double combinations. In addition, both of the triple combinations studied (GA3+KIN+EBR and GA3+KIN+E) displayed an excellent performance by showing a very successful effect on the MI compared to all other treat- ments except GA3 alone. Considering all the PGRs appli- cations both alone and in double/triple combinations, the most positive effect on the MI was obtained with the application of GA3 alone and GA3+KIN+E from the tri- ple combinations. But, at the KIN+EBR, one of the dou- ble-combinations, the MI were recorded as the lowest value by decreasing from 6,2±0,3 to 1,5±0,5 (Figure 2). At HTS (30°C), the MI in barley root meristem cells germinated in distilled water medium decreased by 35% compared to the control (at 20°C). When applied alone, it was determined that the PGRs, which showed a very successful performance on the MI of meristem cells at HTS compared to own control group (in distilled water/at 30°C), were GA3, BA, E, Put and Spm, respectively. How- ever, KIN, EBR, TRIA, Spd and Cad alone were not suc- cessful in alleviating the negative effect of HTS on the MI. Considering the pre-applications of all PGRs alone, GA3 treatment increased from 5,7±0,3 (at 30°C, in distilled water) to 15,3±0,7 was more successful than the others in 26 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen mitigating the negative effect of HTS on the MI, but Spd was extremely unsuccessful on this parameter. Among the double combinations of PGRs, again GA3+KIN was quite a lot successful in mitigating the negative effect of HTS on the MI compared to its control group and other double combinations. However, the KIN+EBR double combina- tion had the lowest MI value together with Spd from the single combinations. Similarly, both of the triple combina- tions studied resulted in a statistically significant increase in the MI under HTS (Figure 2). Among all studied applications, GA3, E, GA3+KIN and KIN+EBR showed a more positive effect on the MI compared to those at optimum temperature in barley meristem cells exposed to HTS. None of the other appli- cations under HTS could reach MI values under own self optimum conditions. Under these conditions, double combinations (except GA3+EBR) were more successful than single PGRs. Especially, while KIN+EBR had the lowest MI value (1,5±0,5) under optimum conditions, this value increased approximately twice and reached 2,9±1,3 at HTS (Figure 2). Counterchecks of PGRs against the adverse effects of HTS on CAs The percentages of CAs into barley meristem cells germinated in distilled water and at 20°C (control) and 30°C after pretreatment of various PGRs alone or in double/triple combinations were summarized in Fig- ure 3. Representative images of CAs for all applica- tions were given in Figure 4. As a result of cytological examinations, no aberration was found in the chromo- some structures of barley meristem cells germinated in Figure 2. Mitotic index scores in meristem cells of barley exposed to high temperature stress after plant growth regulators supplementation.*Values with insignificant difference (P ≤ 0.05) for each column are indicated with same letters (± Standard deviation). Seeds were germinated at constant temperatures of 20°C (control) and 30°C in the dark and in an incubator. As test solution, 900 μM GA3, 100 μM KIN, 100 μM BA, 400 μM E, 3μM EBR, 10 μM TRIA and 10 μM PAs (Spm, Spd, Put and Cad) were used. The pretreatment process of seeds was performed by soaking 24 h in constant volumes (50 mL) of distilled water (control) or each PGR. All data were evaluated as three replicates. 27Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress distilled water and at 20°C and all of the mitotic stages were observed normally (Fig. 4 A-D). Whereas, the rate of CA in samples subjected to HTS (30°C) was deter- mined to be 20,0±2,2%. Also, in terms of genotoxic- ity, these aberrations were observed to increase signifi- cantly (p≤0.05) with all PGR treatments compared to the control, as evidenced by the CA percentages. Under optimum conditions, the highest CA frequency was detected in KIN+EBR, one of the double combinations by increasing from 0,00 ±0,0% to 22,3±9,3%, followed by KIN (13,2±2,3%), TRIA (12,2±2,4%), Cad (11,8±3,4%) and GA3+KIN+E (11,4±2,7%). The applications with the lowest CA frequency are also GA3 alone (5,5±1,3%) and triple combination GA3+KIN+EBR (5,6±1,2%) (Figure 3). Under HTS, most of the PGR pretreatments studied greatly attenuated the negative effect on CA percent- ages in barley meristem cells. At the temperature level in mentioned, however, the percentage of CAs respec- tively, at BA (27,1±7,0%), KIN+EBR (23,6±7,8%) and TRIA (23,1±7,2%), increased even more compared to own control group (20,0±2,2%). It was determined that the GA3 application alone was the most successful appli- cation (almost the same as optimum conditions) com- pared to all other combinations studied by reducing the detrimental effect of HTS on the percentage of CA from 20,0±2,2% to 5,9±2,0% (Figure 3). In all PGR applications studied, HTS significantly increased CAs compared to own optimum conditions. Especially with BA, GA3+KIN+EBR and TRIA applica- tions respectively, CA rates increased by 2 times or more compared to own optimum conditions in HTS. Microscopic images of a wide range of CAs observed in the preparations prepared with root tips belonging to all application groups are shown in Figure 4. Generally, the most extensive aberrations observed in all applica- tion were micronucleus (Figure 4 a, b), disorderly pro- Figure 3. Frequency of chromosome aberrations in meristem cells of barley exposed to high temperature stress after plant growth regulators supplementation. *Values with insignificant difference (P ≤ 0.05) for each column are indicated with same letters (± Standard deviation). Seeds were germinated at constant temperatures of 20°C (control) and 30°C in the dark and in an incubator. As test solution, 900 μM GA3, 100 μM KIN, 100 μM BA, 400 μM E, 3μM EBR, 10 μM TRIA and 10 μM PAs (Spm, Spd, Put and Cad) were used. The pretreatment process of seeds was performed by soaking 24 h in constant volumes (50 mL) of distilled water (control) or each PGR. All data were evaluated as three replicates. 28 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen Figure 4. Representative images of microphotographs of normal mitotic stages (A-D) and aberrations (a-t) observed in all application groups studied in barley plant. A prophase, B metaphase (2n=14), C anaphase, D telophase. a, b micronucleus (arrows) c disorderly pro- phase with micronucleus d uncoiled chromosomes e chromosomal ringing (arrow) f vacuolated sequencing at metaphase g sticky chro- mosomes h chromosomal irregularity in the equatorial plane i stellar anaphase j disorderly anaphase k, l anaphase with multiple bridges m alignment anaphase with vagrant chromosome (arrow) n laggards in anaphase (arrows) o multipolar anaphase p polar slip in anaphase q bridges in telophase r laggard in telophase (arrow) s vagrant chromosome in telophase (arrow) t polar slip in telophase. Scale bar = 10 µm. 29Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress anaphase (Figure 4 c, j), uncoiled chromosomes (Figure 4 d), sticky chromosomes (Figure 4 g), chromosomal irregularity in the equatorial plane (Figure 4 h), align- ment anaphase (Figure 4 m), multipolar anaphase (Fig- ure 4 o), laggard and vagrant chromosomes (Figure 4 m, n, r, s), bridges (Figure 4 k, l, q) and polar slip (Figure 4 p, t) in ana-telophase. The minimal level aberrations recorded were chromosome ringing (Figure 4 e), vacu- olated sequencing at metaphase (Figure 4 f) and stellar anaphase (Figure 4 i). DISCUSSION As a result of the extensive literature review, ade- quate study were not found on the effects of some of the PGRs studied here (especially EBR, TRIA and PAs) on cell division and chromosomal behaviors. Moreover, it was seen that there is still no consensus among research- ers about the role of also GA3, KIN, BA and E on these parameters. For this reason, it was found appropriate to compare the effects of these parameters under optimum conditions before moving on to the effects of the above mentioned PGRs under HTS conditions. Effects of exogenous PGRs on cytotoxicity and genotoxicity at optimum conditions In this part of the study, the effects cytotoxic and genotoxic the effects of hormone/with hormone-like activity shown chemicals such as exogenously GA3, KIN, BA, E, EBR, TRIA and PAs in the barley meristem cells of at optimum conditions were investigated. The results were compared with the relevant literature and among themselves. According to our findings, while the MI value of barley seeds in the control group (in distilled water, at 20°C) was 6,2±0,3%; KIN, EBR, TRIA, Spd, GA3+EBR and KIN+EBR treatments could not reach this (see Fig- ure 2). Similarly, some researchers suggested that exter- nally applied GA3, KIN, BA (Tabur and Demir 2010a; Tütünoğlu et al. 2019) and TRIA application (Tabur and Demir 2008a) under stress-free conditions reduced mitotic activity in barley root meristems. In that case, it can be said that exogenously application of some stimulatory growth regulators under normal condi- tions without stress may be useless. On the other hand, it has been reported that exogenous GA3 (Mansour and Kamel 2005; MacDonald and Little 2006), low concen- tration BA (Huyluoğlu et al. 2008; Truta et al. 2011; El- Ghamrey et al. 2013) and TRIA (Hangarter and Ries 1978) applications promote cell division and thus MI during germination under normal conditions. How- ever, the effects of E, BRs and PAs on cell division have not been fully elucidated. It has been stated that these PGRs may have positive or negative effects on cell divi- sion. Some researchers asserted that these PGRs pro- mote cell division and MI at low concentrations (Kartal et al. 2009; Maraklı et al., 2014), while at high concen- trations reported that they had an inhibitory effect (Hu et al., 2000; İsmailoğlu et al., 2004; Tabur and Demir, 2009, 2010 a,b; Özmen et al. 2022). Our findings reveal that the application of GA3, BA and Put alone had sta- tistically a very successful effect on the MI of barley seeds compared to the control group and also E, Spm and Cad applications were had partially successful. But, TRIA, EBR, Spd and KIN applications had an inhibitory effect on this parameter (see Figure 2). Considering the PGRs studied, either alone or in double-triple combina- tions, it is seen that the most positive effect on the MI is obtained with GA3 pre-application alone (15,1±0,8%) and generally the combinations with GA3 are statisti- cally more significant than the control group (6,2±0,3%). For example, the GA3+KIN+EBR triple combination cre- ated with the addition of GA3 to the KIN+EBR double application, where the most negative effect on the MI was observed, increased statistically significantly the MI compared to the control group. Moreover, considering all the PGR applications studied, the most positive effect on the MI was obtained with the application of GA3 alone and GA3+KIN+E from the triple combinations (see Figure 2). This indicates that GA3 has an indispensable place in cell division. Data on double and triple com- binations of PGRs studied here on MI under optimum conditions are presented for the first time in this study. No any chromosomal abnormalities (CA) were came across in barley root meristems germinated under optimum conditions. However, as a result of the PGRs pretreatment studied here, either alone or in double/ triple combinations, various types and percentages of CAs were generally observed (see Figure 3-4). This is due to the fact that even any externally applied stimu- lator under optimum conditions is perceived as a stress factor by the plant. The least percentage of CA was obtained with GA3 pretreatment alone. In particular, it was determined that the percentage of CA in seeds with KIN pretreatment was higher than other PGRs applied alone. Among all the combinations studied, the most CA again was observed in the KIN+EBR application, which is one of the double combinations by increasing from 0,00±0,0% abnormal cells (at distilled water, control) to 22,3±9,3%. Moreover, the CA ratio was reduced in the GA3+KIN+EBR triple combination (5,6±1,2%) formed by adding GA3 to this double combination until to the 30 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen level in the GA3 application alone (5,5±1,3%) (see Figure 3). In this case, as mentioned above, we can say that the negative effect of KIN+EBR double application on the MI also is due to these CAs caused by the mitotic irreg- ularities during cell division. Again, data on double and triple combinations of PGRs studied under optimum conditions on CAs also are presented for the first time in this study. In our study, it was observed that CKs and E caused the formation of CAs such as micronucleus, disorder- ly pro-anaphase, chromosome ringing, chromosomal irregularity in the equatorial plane, multipolar anaphase, sticky and uncoiled chromosomes, especially bridges in ana-telophase. In addition, CAs such as sticky chromo- somes, chromosome ringing, laggard and vagrant chro- mosomes in ana-telophase, and alignment anaphase were frequently encountered in GA3, EBR and TRIA applications. On the other hand, it has been determined that PAs cause CAs in the form of sticky chromosomes, disorderly anaphase, chromosome bridges in ana-telo- phase, and polar slip in ana-telophase (see Figure 4 a-t). Information on the effects of various PGRs on chro- mosome behavior under optimum conditions is limited to only a few studies conducted in the last 20 years. It has been reported that high concentrations of CKs nega- tively affect chromosomal behaviors with a clastogenic effect and cause different types of genetic and chro- mosomal variations (Huyluoğlu et al. 2008; Truta et al. 2011; El-Ghamrey et al. 2013; El-Ghamery and Mousa 2017). However, Tabur and Demir (2010a) reported in their study that BA and GA3 application did not cause any chromosomal abnormality, but KIN and E applica- tion increased CAs significantly compared to the con- trol. According to Tütünoğlu et al. (2019) argue that increasing GA3 concentrations depending on time and dose show cytotoxic and genotoxic effects and the dif- ference between control and treatment groups is statisti- cally significant, while Mansour and Kamel (2005) argue that there is statistically an insignificant increase in CAs. Again, some researchers reported that exogenous applied TRIA, EBR and HBRs under optimum conditions nega- tively affect chromosomal behavior in barley meristems (Tabur and Demir, 2008a, 2009; Kartal et al., 2009). Similarly, Ünal et al. (2002) on barley seeds, İsmailoğlu et al. (2004) on diploid, tetraploid and hexaploid wheat seeds in their studies stated that also PAs cause mitotic irregularities. Tabur and Demir (2010b) reported that PAs inhibited the MI in barley meristems, significant- ly increased the CAs of other PAs except Spd and Put had the highest abnormality rate in total. Özmen et al. (2022) also stated that PAs significantly increased the CA rate by causing various mitotic abnormalities, and the PA with the highest abnormality percentage was Spm. If a comparison is made in the light of all these studies; it can be said that the effects of PGRs on MI and CAs under optimum conditions may differ depending on the plant species studied, plant development stages, genotype, used concentration, exposure time and pre- application method. Effects of exogenous PGRs on cytotoxicity and genotoxicity at HTS conditions HTS may inhibit seed germination and mitosis, thereby reducing germination rate (Çavuşoğlu and Kabar, 2007; Sharma et al. 2022) and mitotic activ- ity (Öney and Tabur, 2013; Öney et al. 2015). It may be cause a decrease in the amount of protein and stop the synthesis of proteins that act as osmoprotectants that play a role in temperature tolerance (Xu et al. 2021). According to the results obtained from our study, it has been confirmed once again that HTS reduces the MI also in barley plant, and limited literature information on this subject has been contributed. The reason for the decrease in mitotic activity at high temperature may be directly or may be related to the loss of enzyme activa- tion, which is responsible for mitosis, and also proteins denaturation and lipid peroxidation (Sheikhi et al. 2023). At the same time, HTS showed quite unfavorable effects on the chromosome behavior of barley seeds. In our study, it was determined very high rate and various types of chromosome aberrations in barley root mer- istems germinated at 30°C (see Figure 3-4). These aber- rations may be due to the damaging effects of HTS on microtubule organization (Wahid et al. 2007), which may have led to irregular mitotic configurations and CAs, mainly involving spindle fibers and metaphase (Abou-Deif and Mohamed, 2007). In addition, it has been reported that reactive oxygen species (ROS) such as hydroxyl (OH), superoxide (O2-), hydrogen peroxide (H2O2) and single oxygen (1O2), which occur due to HTS may be cause deaggregation of DNA, RNA and nucleic acids (Liu and Huang, 2000). Faraghi et al. (2015) sug- gest that Vicia dasycarpa, which is normally diploid, exhibits a mixoploid state with aneuploid and tetraploid cells after temperature shock. However, Öney and Tabur (2013) reported in their study that high temperature (30°C) did not cause any CA in Vicia faba root meristem cells. In the light of all these studies, we can emphasize again that heat stress may have different effects depend- ing on the type of plant used, the severity and duration of the application of stress. Because the upper and lower threshold limits of abiotic stresses can show different effects in different species, sometimes even in different 31Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress varieties of the same species (Wahid et al. 2007; Heman- taranjan et al. 2014). On the other hand, it was determined that PGRs were effective at different degrees in mitigating the nega- tive effects of HTS on the MI, and this difference was sta- tistically significant (see Figure 2). Especially KIN, EBR, TRIA, Spd, Cad, GA3+EBR and KIN+EBR applications have not been successful in alleviating the negative effects of HTS on the MI. It was observed that the most negative effect on the MI occurred with Spd application alone and KIN+EBR application from double combinations. In this case, it would not be right to expect every stimulator to be successful in overcoming the heat stress on this param- eter. Indeed, it has been emphasized by many researchers that the type(s) and concentrations of stimulators may vary from species to species in overcoming various abi- otic stresses (Mirza and Bagni 1991; Tabur and Demir 2010a,b; Korek and Marzec 2023). However, considering all PGR pre-applications, either alone or in double/triple combinations, in our study, more than half of the tested applications showed a successful performance in alleviat- ing the unfavorable effect of HTS on the MI. In particu- lar, the most positive effect was obtained with the applica- tion of GA3 alone, GA3+KIN from double combinations, and GA3+KIN+E from triple combinations (see Figure 2). In addition, in the case of HTS alone GA3 and GA3+KIN double combinations reached a higher value than they have shown success under optimum conditions. Moreo- ver, considering the success of other combinations with GA3 on the MI, it is seen that again GA3 has an indis- pensable place in alleviating the negative effects of stress compared to their own control groups (at 30°C in distilled water). Similarly, GA3+KIN (8,2±1,3%) combination was more successful than KIN alone (4,0±0,4%) and GA3+EBR (4,5±0,8%) combination was more successful than EBR alone (3,1±0,9%) in overcoming HTS on the MI. Also, the excellent success of the GA3+KIN+EBR (8,0±1,4%) triple combination on the MI compared to the KIN+EBR (2,9±1,3%) double combination indicates that GA3 cre- ates a noticeable synergism with these PGRs. It has also been emphasized in previous reports that combinations with GA3 against abiotic stresses are more effective role on seed germination and MI (Çavuşoğlu and Kabar 2007; Tabur and Demir 2008b). With this together, consider- ing that the internal amount of stimulators such as CKs (El-Mashad and Kamel, 2001) and GAs (Prakash and Prathapasenan, 1990) decreases in seeds under stress con- ditions, these externally applied promoters is not surpris- ing that they increase mitotic activity; it can be expect. Although the information about the response of PGRs to stress factors during cell division has not been sufficiently clarified, it is known that various priming applications increase resistance to stress factors by pro- moting cell division, DNA replication (Giri and Schil- linger 2003) and antioxidative defense (Afzal et al. 2006). The most common response under stress conditions is the acceleration of synthesis of protective compo- nents, especially osmoprotectants. Based on general lit- erature information, it would be correct to say that the PGRs may have been successful in alleviating the dam- aging effect of HTS on the MI by increasing the activ- ity of enzymes involved in cell division or by accelerat- ing the synthesis of proteins that act as osmoprotectants that play a role in temperature tolerance. However, as mentioned in the Introduction, there is only one study (Pradhan and Gupta 2013) on the effects of these PGRs on mitotic activity, especially under high temperature conditions. In this previous study, it was reported that only EBR application was studied and increased the MI in Brassica oleracea var. botrytis root meristems ger- minated under low (4°C) and high (44°C) temperature stress. Contrary to our findings, these researchers sug- gested that increasing concentrations of EBR under high and low temperature stress increased MI. This paradox may be due to the type of plant, the concentration of EBR used and the applied temperature degree. As for CAs, so far no studies have been conducted on the effects of all the above-mentioned PGRs on this parameter under HTS. Therefore, our study includes the first findings describing the data obtained on this parameter in detail. Accordingly, it was determined that the studied PGRs also showed statistically signifi- cant effects on the percentages of CAs in barley seeds germinated under HTS (see Figure 3). Although most of the PGRs applied alone or in double/triple combina- tions were successful in improving the CAs caused by HTS, only BA, TRIA and KIN+EBR applications could not show sufficient success on this parameter. Especially among all applications, the most positive effect on CAs was obtained again with the GA3 application alone, while the most damaging effect was in BA application. Thus, the GA3 application demonstrated once again on CAs its successful performance on the MI under HTS. For example; GA3+KIN double application (10,0±2,5%) was more successful than KIN application alone (13,5±3,3%) in ameliorating the damaging effects of HTS by reducing the percentage of CAs. Various mitotic aberrations were observed during microscopic scans of root meristem cells of barley seeds belonging to all application groups (see Figure 4 a-t). Aneugenic and clastogenic impacts that form an impor- tant portion of CAs might have been largely resulted from spindle dysfunction and chromosomal breaks respectively. The CAs, such as bridges and break, are 32 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen indicators of a clastogenic action, whereas chromosome losses, laggards, sticky, multipolarity and C-metaphase originate from aneugenic effects (Silveira et al. 2017). As known, accurate chromosome segregation in mitosis requires that sister kinetochores attach to microtubules emanating from opposite spindle poles (biorientation). Because kinetochore attachment is a stochastic process, it is error prone and can result in chromosome malori- entation (Banerjee et al. 2020). Mitodepressive actions such as disorderly pro-anaphase, alignment anaphase, multipolar anaphase, stellar anaphase, bridges and polar slip in ana-telophase may be mainly the result of the above reasons. Moreover, Tabur and Demir (2010b) asserted that the nucleoplasmic bridges in ana-telophase might have been occurs as a consequence of inversions while Bonciu et al. (2018) have asserted originate from dicentric chromosomes or occur as a result of as faulty longitudinal break of sister chromatids during anaphase. Fiskesjö (1997) have claimed also that bridges are clasto- genic effects, both resulting from chromosome and chro- matid breaks. The large micronucleus (MN) in the cell indicates aneugenic effect resulting 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 MNs are likely the consequence of vagrant chromosomes and fragments. Uncoiled chromosomes and chromosome ringing’s may be the result of a weak mitotic effect and irregular chro- mosome contractions (Tabur and Demir 2010b). Asita and Mokhobo (2013) asserted that sticky chromosomes could be originated from abnormal DNA condensa- tion, irregular chromosomal wrapping and inactiva- tion of the axes. At the same time, such aberrations may be a result of improper folding of the chromatin fibers (Klášterská et al. 1976). According to some researchers, sticky chromosomes are a marker of high toxic effect on chromatin and irreversibility of the change (Fiskesjö and Levan 1993; Türkoğlu 2007). Chromosomal irregu- larity in the equatorial plane and vacuolated sequencing at metaphase may originate from unequal distribution of chromosome and spindle dysfunction. Laggard and vagrant chromosomes occurs during the anaphase where one or more chromatids gets detached from the rest of the chromatids and is incapable of moving towards the poles. Aberrations of these kinds may have occurred due to a weak mitotic impresses a consequence of failures in chromosomal attachment to the mitotic spindle (Patil and Bhat 1992). Generally, it was concluded that BA, E, Put and Spm, respectively, among the PGRs alone studied, including at first GA3, showed a very successful performance statisti- cally in mitigating the negative effect of HTS on the MI. In addition, when the effects of the double/triple com- binations of these PGRs on this parameter were evalu- ated, it was determined that all the studied combina- tions, except the KIN+EBR application from the double combinations, showed a superior success in overcoming the negative effect of HTS on the MI. In fact, this suc- cess was higher than most of the PGRs applied alone (see Figure 2). On the other hand, as a result of the statistical evaluations, it was proved that all PGRs studied, except for KIN+EBR, BA and TRIA applications, both alone and in double/triple combinations, showed an important suc- cessfully in the improvement of CAs (see Figure 3 ). CONCLUSION Various growth agents can be effective in differ- ent events in different species, even in individuals of the same species, and can be found in different amounts. Accordingly, which hormone is in effective concentration in any event in a plant this hormone would be respon- sible for growth and development events by perform- ing its function. Indeed, as Khan (1971) points out, any event is unlikely to be governed by the absolute presence or absence of a hormone. In response to environmental conditions, some hormones in the plant may be more effective, some may be less effective or not effective at all. Therefore, it seems more plausible that whichever hor- mone is most effective, it functions in the relevant case. In our study, the interactions between mitotic activ- ity and mitotic irregularities and various stimulating growth agents, which can be counted as possible mech- anisms of tolerance to increased heat stress as a result of global climate changes, were examined in barley, an important cereal crop. Thus, it has been tried to serve to fill a gap in the literature regarding these parameters. It is thought that the use of suitable PGRs for plants that will be grown in regions exposed to high tempera- tures will provide very beneficial results economically. However, a detailed investigation of the effects of these chemicals on basic metabolic events such as hydrolase synthesis and activity, nucleic acid metabolism, protein and enzyme synthesis, which can be directly or indirect- ly effective on mitotic activity, will help to elucidate the mechanism in question. Consequently, thanks to these and similar studies, it can be contribute to the development of genetically tem- perature-tolerant products by changing the plant’s sens- ing, signaling and regulatory pathways without disturb- ing other vital processes. In addition, a comprehensive explanation of the response of plants to high tempera- ture tolerance and temperature tolerance mechanisms 33Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress and the development of possible strategies in this regard are mandatory. Therefore, it is necessary to map gene loci related to thermotolerance and to elucidate different genetic approaches that provide tolerance to heat stress (Asthir, 2015). ACKNOWLEDGEMENTS The authors would like to thank Ayşegül Özmen (SDU Faculty of Fine Arts Department of Graphic Design) for her help in the arrangement of figures. FUNDING This study was funded by The Department of Scien- tific Research Project Management of Süleyman Demirel University (SDUBAP 2276–YL-10). AUTHOR CONTRIBUTIONS ST and ŞBYE designed and performed the experi- ments. SÖ helped to conduct the experiments. ST wrote the manuscript. All authors read and approved the man- uscript and have equal contribution. REFERENCES Abou-Deif MH, Mohamed FI. 2007. Effect of heat stress on chromosomes and protein patterns in six hexa- ploid wheat varieties. Res J Cell Mol Biol. 1(1): 42- 49. Aerts N, Mendes MP, Van Wees SCM. 2021. Multiple lev- els of crosstalk in hormone networks regulating plant defense. Plant J. 105: 489-504. Afzal I, Basara SMA, Faooq M, Nawaz A. 2006. Allevia- tion of salinity stress in spring wheat by hormonal priming with ABA, salicylic acid and ascorbic acid. Int J Agric Biol. 8: 23-28. Alcázar R, Bueno M, Tiburcio AF. 2020. Polyamines: Small amines with large effects on plant abiotic stress tolerance. Cells (MDPI). 9: 2373. 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. Banerjee A, Adames N, Peccoud J, Tyson JJ. 2020. A sto- chastic model for error correction of kinetochore- microtubule attachments in budding yeast. PLo- SONE. 15(8): e0236293. Bita CE, Gerats T. 2013. Plant tolerance to high tempera- ture in a changing environment: scientific funda- mentals and production of heat stress-tolerant crops. Front Plant Sci. 4: 273. 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. Budak Ü. 2022. Possible effects of global climate change on Turkey’s plant diversity. In: Doğan H, Fidan H, editors. Global Climate Change: Agriculture and Food Science Perspective. Iksad Publishing House, Ankara- Turkey, pp. 149-165. Chen D, Shao Q, Yin L, Younis A, Zheng B. 2019. Poly- amine function in plants: Metabolism, regulation on development, and roles in abiotic stress responses. Front Plant Sci. 9: 1945. Çavuşoğlu K, Kabar K. 2007. Comparative effects of some plant growth regulators on the germination of barley and radish seeds under high temperature stress. Eur- Asian J BioSci. 1: 1-10. Dong H, Bai L, Chang J, Song CP. 2018. Chloroplast pro- tein PLGG1 is involved in abscisic acid-regulated later- al root development and stomatal movement in Arabi- dopsis. Biochem Biophys Res Commun. 495: 280-285. Duncan DB. 1955. Multiple range and multiple F tests. Biometrics. 11: 1-42. El-Beltagi HS, Ismail SA, Ibrahim NM, ShehataWF, Alkhateeb AA, Ghazzawy HS, El-Mogy MM, Sayed EG. 2022. Unravelling the effect of triacontanol in combating drought stress by ımproving growth, pro- ductivity, and physiological performance in strawber- ry plants. Plants. 11: 1913. Elçi Ş, Sancak C. 2013. SİTOGENETIKTE ARAŞTIRMA YÖNTEMLERI VE GÖZLEMLER [Research meth- ods and observations in cytogenetics]. Ankara Univ Pub House. Beşevler/ANKARA, 227 p. Turkish. El-Ghamrey AA, Mahgoub HAM, Mousa MA. 2013. Effect of plant growth regulators on protein banding profile of some higher plants in relation to mitotic activity and the total abnormalities. Int J Adv Res. 1(10): 980-993. El-Ghamery AA, Mousa MA. 2017. Investigation on the effect of benzyladenine on the germination, radicle growth and meristematic cells of Nigella sativa L. and Allium cepa L. Ann Agric Sci. 62: 11–21. 34 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen El-Mashad AAA, Kamel EA. 2001. Amelioration of NaCl stress in Pisum sativum. Indian J Exp Biol. 39(5): 469-475. Emamverdian A, Ding Y, Mokhberdoran F (2020) The role of salicylic acid and gibberellin signaling in plant responses to abiotic stress with an emphasis on heavy metals. Plant Signal Behav 15 :7, 1777372. Fareghi S, Rashidi V, Haghighi AR. 2015. Investigating the effect of heat shock on chromosomal aberration of Vicia dasycarpa. Cumhuriyet Sci J. 36(3): 657-663. Fatma M, Asgher M, Iqbal N, Rasheed F, Sehar Z, Sofo A, Khan NA. 2022. Ethylene signaling under stress- ful environments: analyzing collaborative knowledge. Plants. 11: 2211. 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. Boca Raton, New York: CRC Lewis Publishers pp. 308-333. Fiskesjö G, Levan A. 1993. Evaluation of the first ten MEIC chemicals in the Allium test. Altern Lab Anim (ATLA). 21: 139-149. Giri GS, Schillinger WF. 2003. Seed priming winter wheat for germination, emergence and yield. Crop Sci. 43(6): 2135-2141. Goharrizi KJ, Hamblin MR, Karami S, Nazari M. 2021. Physiological, biochemical, and metabolic responses of abiotic plant stress: salinity and drought. Turk J Bot. 45: 623-642. Hangarter R, Ries SK. 1978. Effect of triacontanol on plant cell cultures in vitro. Plant Physiol. 61: 855-857. Hasanuzzaman M, Nahar K, Alam MM, Fujita M. 2012. Exogenous nitric oxide alleviates high temperature induced oxidative stress in wheat (Triticum aestivum) seedlings by modulating the antioxidant defense and glyoxalase system. Aust J Crop Sci. 6(8): 1314-1323. Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M. 2013. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci. 14: 9643-9684. Hatfield JL, Prueger JH. 2015. Temperature extremes: Effect on plant growth and development. Weather Clim Extrem. 10: 4-10. Hemantaranjan A, Bhanu AN, Singh MN, Yadav DK, Patel PK, Singh R, Katiyar D. 2014. Heat stress responses and thermotolerance. Adv Plants Agric Res. 1(3): 62-70. Hill CB, Li C. 2022. Genetic improvement of heat stress tolerance in cereal crops. Agronomy. 12(5): 1205. Hu Y, Bao F, Li J. 2000. Promotive effect of brassinoster- oids on cell division involves a distinct CycD3-induc- tion pathway in Arabidopsis. Plant J. 24(5): 693-701. Huang J, Zhao X, Bürger M, Chory J, Wang X. 2023. The role of ethylene in plant temperature stress response. Trends Plant Sci. 28(7): 808-824. Hudelson TJ. 2023. Elevated atmospheric ethylene and high temperature independently inhibit fruit set but not vegetative growth in tomato. Hortsci. 58(3): 247- 253. Huyluoğlu Z, Ünal M, Palavan-Ünsal N. 2008. Cytologi- cal evidences of the role of meta-topolin and benzy- ladenin in barley root tips. Adv Mol Biol. 1: 31-37. IPCC 2021 Climate change widespread, rapid, and inten- sifying-IPCC. Available from: https://www.ipcc. ch/2021/08/09/ar6-wg1-20210809-pr/ [Accessed: May 05, 2023] Islam S, Mohammad F. 2020. Triacontanol as a dynamic growth regulator for plants under diverse environmen- tal conditions. Physiol Mol Biol Plants. 26(5): 871-883. İsmailoğlu I, Ünal M, Palavan-Ünsal N. 2004. Effects of spermidine, spermine and cyclohexylamine on mitot- ic activity of 2X, 4X and 6X wheats. J Cell Mol Bio. 3: 83-88. Jacott CN, Boden SA. 2020. Feeling the heat: develop- mental and molecular responses of wheat and bar- ley to high ambient temperatures. J Exp Bot. 71 (19): 5740-5751. Jing J, Guo S, Li Y, Li W. 2020. The alleviating effect of exogenous polyamines on heat stress susceptibility of different heat resistant wheat (Triticum aestivum L.) varieties. Sci Rep. 10: 7467. Kartal G, Temel A, Arican E, Gözükırmızı N. 2009. Effects of brassinosteroids on barley root growth, antioxidant system and cell division. Plant Growth Regul. 58: 261-267. Kaur H, Sirhindi G, Bhardwaj R, Alyemeni MN, Siddique KH, Ahmad P. 2018. 28-homobrassinolide regulates antioxidant enzyme activities and gene expression in response to salt-and temperature-induced oxidative stress in Brassica juncea. Sci Rep. 8: 8735. Khan AA. 1971. Cytokinin: Permissive role in seed ger- mination. Science. 171: 853-859. Khan AH, Min L, Ma Y, Zeeshan M, Jin S, Zhang X. 2023. High-temperature stress in crops: male steril- ity, yield loss and potential remedy approaches. Plant Biotechnol J. 21: 680-697. 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 dichlorvos on root meristems of Vicia faba. J Appl Genet. 48(5): 359-361. https://www.ipcc.ch/2021/08/09/ar6-wg1-20210809-pr/ https://www.ipcc.ch/2021/08/09/ar6-wg1-20210809-pr/ 35Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress Korek M, Marzec M. 2023. Strigolactones and absci- sic acid interactions affect plant development and response to abiotic stresses. BMC Plant Biol. 23: 314. Kosakivska IV, Vedenicheva NP, Babenko LM, Voytenko LV, Romanenko KO, Vasyuk VA. 2022. Exogenous phytohormones in the regulation of growth and development of cereals under abiotic stresses. Mol Biol Rep. 49(1): 617-628. Kothari A, Lachowiec J. 2021. Roles of brassinosteroids inmitigating heat stress damage in cereal crops. Int J Mol Sci. 22: 2706. Li N, Euring D, Cha J., Lin Z, Lu M, Huang LJ, Kim WY. 2021. Plant hormone-mediated regulation of heat tol- erance in response to global climate change. Front Plant Sci. 11: 627969. Li Y, Han X, Ren H, Zhao B, Zhang J, Ren B, Gao H, Liu P. 2023. Exogenous SA or 6-BA maintains photosyn- thetic activity in maize leaves under high tempera- ture stress. Crop J. 11(2): 605-617. Liu X, Huang B. 2000. Heat stress injury in relation to membrane lipid peroxidation in creeping bent grass. Crop Sci. 40(2): 503-510. MacDonald JE, Little CH. 2006. Foliar application of GA3 during terminal long-shoot bud development stimu- lates shoot apical meristem activity in Pinus sylvestris seedling. Tree Physiol. 26(10): 1271-1276. Mangena P. 2020. Role of benzyladenine seed priming on growth and physiological and biochemical response of soybean plants grown under high salinity stress condition. Int J Agron. 8847098. Mangena P. 2022. Evolving role of synthetic cytokinin 6-benzyl adenine for drought stress tolerance in soy- bean (Glycine max L. Merr.). Front Sustain Food Syst. 6: 992581. Mansour MM, Kamel EAR. 2005. Interactive effect of heavy metals and gibberellic acid on mitotic activity and some metabolic changes of Vicia faba L. plants. Cytologia. 70(3): 275-282. Maraklı S, Temel A, Gözükırmızı N. 2014. Salt stress and homobrassinosteroid interactions during germination in barley roots. Not Bot Horti Agrobot Cluj-Nap. 42(2): 446-452. Mei W, Chen W, Wang Y, Liu Z, Dong Y, Zhang G, Deng H, Liu X, Lu X, Wang F, Chen G, Tang W, Xiao Y. 2023. Exogenous kinetin modulates ROS homeostasis to affect heat tolerance in rice seedlings. Int J Mol Sci. 24: 6252. Mirza JI, Bagni N. 1991. Effects of exogenous polyam- ines and difluoromethylornithine on seed germina- tion and root growth of Arabidopsis thaliana. Plant Growth Regul. 10: 163-168. Moumita, Mahmud JA, Biswas PK, Nahar K, Fujita M, Hasanuzzaman M. 2019. Exogenous application of gibberellic acid mitigates drought-induced damage in spring wheat. Acta Agrobot. 72(2): 1776. Narayanan S . 2018. Effects of high temperature stress and traits associated with tolerance in wheat. Open Access J Sci. 2(3): 177‒186. Oh MH, Clouse SD. 1998. Brassinosteroid affects the rate of cell division in isolated leaf protoplasts of Petunia hybrida. Plant Cell Rep. 17: 921-924. Öney S, Tabur S. 2013. Cytogenetical and molecular responses of exogenous potassium sulphate for toler- ance to extreme temperatures in Vicia faba L. J Pure Appl Microbiol. 7: 663-670. Öney S, Tabur S, Tuna M. 2015. Cytogenetic and molecu- lar responses of ammonium sulphate application for tolerance to extreme temperatures in Vicia faba L. Cytol Genet. 49(5): 328-337. Özmen S, Tabur S, Öney-Birol S, Özmen S. 2022. Molecu- lar responses of exogenous polyamines under drought stress in the barley plants. Cytologia. 87(1): 7-15. Özmen S, Tabur S, Öney-Birol S. 2023. Alleviation role of exogenous cadaverine on cell cycle, endogenous pol- yamines amounts and biochemical enzyme changes in barley seedlings under drought stress. Sci Rep. 13: 17488. 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. Pereira A. 2016. Plant abiotic stress challenges from the changing environment. Front Plant Sci. 7: 1123. Powell JN, Grant CJ, Robinson SM, Radford SG. 1973. A comparison with halothane of the hormonal and anaesthetic properties of ethylene in plants. Br J Anaesth. 45(7): 682-690. Pradhan SK, Gupta RC. 2013. Cytogenetic response of 24-epibrassinolide in Brassica oleracea var. botrytis under temperature stress. Crucif Newsl. 32: 11-13. Prakash L, Prathapasenan G. 1990. Interactive effect of NaCl salinity and gibberellic acid and gibberellin like substances and yield of rice (Oryza sativa L. var. GR.3). Proc Indian Acad Sci. 100: 173-181. Qureshi H, Abbas MH, Jan T, Mumtaz K, Mukhtar H, Khan U. 2022. Plant responses to heat stress. J Adv Nutr Sci Technol. 2(2): 40-53. Rasheed R, Wahid A, Farooq M, Hussain I, Basra SMA. 2011. Role of proline and glycine betaine pretreat- ments in improving heat tolerance of sprouting sug- arcane (Saccharum sp.) buds. Plant Growth Regul. 65: 35-45. Sarwar M, Anjum S, Alam MW, Ali Q, Ayyub CM, Haid- er MS, Ashraf MI, Mahboob W. 2022. Triacontanol regulates morphological traits and enzymatic activi- 36 Selma Tabur, Ş. Betül Yilmaz-Ergün, Serkan Özmen ties of salinity affected hot pepper plants. Sci Rep. 12: 3736. Saxena S. 2022. Chromosomal aberrations in plant bioas- says: A review. Plant Arch. 22(2): 327-330. Shao J, Huang K, Batool M, Idrees F, Afzal R, Haroon M, Noushahi HA, Wu W, Hu Q, Lu X, Huang G, Aamer M, Hassan MU, El Sabagh A. 2022. Versatile roles of polyamines in improving abiotic stress tolerance of plants. Front Plant Sci. 13: 1003155. Sharma SK. 2021. Brassinosteroids application responses in fruit crops – A review. Int J Agric Environ Bio- technol. 14(2): 123-140. Sharma S, Singh V, Tanwar H, Mor VS, Kumar M, Punia RC, Dalal MS, Khan M, Sangwan S, Bhuker A, Dagar CS, Yashveer S, Singh J. 2022. Impact of high temper- ature on germination, seedling growth and enzymatic activity of wheat. Agriculture MDPI. 12: 1500. Sheikhi S, Ebrahimi A, Heidari P, Amerian MR, Rashi- di-Monfared S, Alipour H. 2023. Exogenous 24-epi- brassinolide ameliorates tolerance to high-tempera- ture by adjusting the biosynthesis of pigments, enzy- matic, non-enzymatic antioxidants, and diosgenin content in fenugreek. Sci Rep. 13: 6661. Shrestha S, Mahat J, Shrestha J, Madhav KC, Paude K. 2022. Influence of high temperature stress on rice growth and development. A review. Heliyon. 8: e12651. Silveira GL, Lima MGF, dos Reis GB, Palmieri MJ, Andrade-Vieria LF. 2017. Toxic effects of environ- mental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L. Chemosphere. 178: 359-367. Sultan K, Perveen S, Parveen A, Atif M, Zafar S. 2023. Benzyl amino purine (BAP), moringa leaf extract and ascorbic acid induced drought stress tolerance in pea (Pisum sativum L.). Gesunde Pflanz. 75 (6): 2423- 2436. Tabur S, Demir K. 2008a. Effects of triacontanol pretreat- ment on mitotic index and chromosome abnormali- ties under salt stress. J Biol Sci Res. 1(1): 11-15. Tabur S, Demir K. 2008b. Effects of combinations of some plant growth regulators on mitotic index and chromosomal aberrations of barley seeds germinated under saline (NaCl) conditions. Süleyman Demirel Univ Art Sci J Sci. 3(2): 162-173. Tabur S, Demir K. 2009. Cytogenetic response of 24-epi- brassinolide on the root meristem cells of barley seeds under salinity. Plant Growth Regul. 58: 119- 123. Tabur S, Demir K. 2010a. Role of some growth regula- tors on cytogenetic activity of barley under salt stress. 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, Yurtlu MD, Özmen S. 2019. Role of humic acid against salt induced cytotoxicity in Hordeum vulgare L. Caryologia. 72(3): 3-10. Taheri M, Haghighi M. 2018. Benzyl adenine is more effective than potassium silicate on decreasing the detrimental effects of heat stress in pepper (Capsicum annum cv. PS301). Iran Agric Res. 37(1): 89-98. Truta E, Zamfirache MM, Rosu C, Olteanu Z, Mihai C, Gherghel D. 2011. Cytogenetic effects induced by 2,4-D and kinetin in radish and common bean root meristems. Rom Agric Res. 28: 207-215. Türkoğlu S. 2007. Genotoxicity of five food preservatives tested on root tips of Allium cepa L. Mut Res. 626: 4-14. Tütünoğlu B, Aksoy Ö, Özbek R, Uçkan F. 2019. The effects of gibberellic acid on Allium cepa root tip meristematic cells. Biol Plant. 63: 365-370. Ünal M, Palavan-Ünsal N, Tüfekçi MA. 2002. Role of putrescine and its biosynthetic inhibitor on seed ger- mination root elongation and mitosis in Hordeum vulgare L. Bull Pure Appl Sci Sec B-Bot. 21: 33-38. Verma T, Bhardwaj S, Singh J, Kapoor D, Prasad R. 2022. Triacontanol as a versatile plant growth regulator in overcoming negative effects of salt stress. J Agric Food Res. 10: 100351. Wahid A, Farooq M, Rasheed R, Gelani S, Rasul E. 2010. Sugarcane under thermal stress: some biotechnologi- cal considerations. In: Kumar A (eds) Plant genetic transformation and molecular markers. Pointer Pub- lishers, Jaipur, pp. 109-123. Wahid A, Gelani S, Ashraf M, Foolad MR. 2007. Heat tolerance in plants: an overview. Environ Exp Bot. 61: 199-223. Wang Y, Zhou Y, Wang R, Xu F, Tong S, Song C, Shao Y, Yi M, He J. 2022. Ethylene response factor LlERF110 mediates heat stress response via regulation of LlHs- fA3A expression and interaction with LlHsfA2 in Lil- ies (Lilium longiflorum). Int J Mol Sci. 23: 16135. Waraich EA, Ahmad R, Halim A, Aziz T. 2012. Alleviation of temperature stress by nutrient management in crop plants: A review. J Soil Sci Plant Nutr. 12: 221-244. Wu YS, Yang CY. 2019. Ethylene-mediated signaling con- fers thermotolerance and regulates transcript levels of heat shock factors in rice seedlings under heat stress. Bot Stud. 60: 23. Wu J, Lu Y, Zhou B, Hu Z. 2022. Effects of cytokinin and abscisic acid on heat resistance of Vetiveria zizanioides. Not Bot Horti Agrobot Cluj-Nap. 50(3): 12755. 37Role of exogenous plant growth promoters in regulating cytotoxic and genotoxic of high temperature stress Xu Y, Chu C, Yao S. 2021. The impact of high-tempera- ture stress on rice: Challenges and solutions. Crop J. 9: 963-976. Younis AA, Ismail HA. 2019. Triacontanol alleviated nickel toxicity in maize seedling by controlling its uptake and enhancing antioxidant system. J Adv Plant Biol. 1(3): 1-14. Zaki MES, Shalaby MAF, Khalil IS, Abou-Sedera FA, Abd Allah MSA. 2014. Protective role of benzyladenine and putrescine on snap beans (Phaseolus vulgaris L.) productivity grown under high temperature. Middle East J Appl Sci. 4(4): 905-910. Zandalinas SI, Balfagón D, Gómez-Cadenas A, Mittler R. 2022. Plant responses to climate change: metabolic changes under combined abiotic stresses. J Exp Bot. 73(11): 3339–3354.