Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 15-24, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1922 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Basoz Sadiq Muhealdin, Sahar Hussein Hamarashid, Fairuz Ibrahim Ali, Nakhshin Omer Abdulla, Syamand Ahmad Qadir (2022). Study- ing some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency. Caryologia 75(4): 15-24. doi: 10.36253/ caryologia-1922 Received: November 08, 2022 Accepted: December 14, 2022 Published: April 28, 2023 Copyright: © 2022 Basoz Sadiq Muheal- din, Sahar Hussein Hamarashid, Fairuz Ibrahim Ali, Nakhshin Omer Abdulla, Syamand Ahmad Qadir. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency Basoz Sadiq Muhealdin1, Sahar Hussein Hamarashid1,*, Fairuz Ibra- him Ali1, Nakhshin Omer Abdulla1, Syamand Ahmad Qadir2 1 Agricultural Project Management Department, Technical College of Applied Science Halabja, Sulaimani Polytechnic University, Iraq 2 Medical laboratory techniques department/Halabja Technical Institute, Research center/ Sulaimani Polytechnic University, Sulaymaniyah, Iraq *Corresponding author. E-mail: sahar.rashid@spu.edu.iq Abstract. This research evaluated the effect of foliar spraying of different elicitors on modulating the effect of water stress on the stevia. The experimental design was the split-plot based on a randomized complete block design with three repetitions. Experi- mental treatments included different irrigation regimes (90% FC, 65% FC, and 40% FC) and foliar application of different elicitors (control, chitosan, salicylic acid, and mela- tonin). In this study, the content of chlorophyll A and b was reduced by intensifying the water deficit stress. Also, the highest content of the two pigments was allocated to the treatment of melatonin application. In the present study, melatonin foliar application under 90% FC irrigation conditions had the highest plant height, leaf area index, bio- mass, and carotenoid content, Moreover, the highest content of proline, phenol, DPPH, rebaudioside A, and steviosid was assigned to melatonin foliar application treatment under 40% FC irrigation conditions. Results revealed, although water stress reduced plant height, leaf area index, and plant biomass, the application of melatonin and sali- cylic acid under different irrigation conditions moderated the effect of water stress on these traits. Application of melatonin and salicylic acid under water deficit stress condi- tions also increased the content of proline, phenol, DPPH, rebaudioside, and steviosid. Keywords: Drought stress, Enzyme activities, Foliar spraying, Pigment. INTRODUCTION About 230-220 species have been recorded for stevia, one of the most eco- nomically important species is S. rebaudian (Al Hassan et al 2017). The dis- tinguishing feature of S. rebaudiana (stevia) from other species is the relatively high production of non-toxic and non-nutritive diterpenoid anticorn glycosides in the leaves of this species, the sweetness of which is 300 times of sucrose (Aghighi Shaverdi et al., 2018). Consumers’ desire for a natural zero-calorie sweetener has made the cultivation, production, and extraction of glycosides in this plant attractive (Aghighi Shaverdi et al., 2018). Among the glycosides in 16 Basoz Sadiq Muhealdin et al. stevia leaves and tissues, the most abundant glycosides are rebaudioside A (Reb-A) and steviosid (Arnon 1949) Drought is known as one of the most destruc- tive stresses that affect the growth, development, and reproduction of plants (Bi et al 2021). A typical effect of water deficit is to cause oxidative damage through the widespread accumulation of reactive oxygen species (ROS) (Duan et al., 2022). ROS overproduction leads to lipid peroxidation, enzyme inactivation, impaired protein structure / function, and nucleic acid dam- age (Duncan 1955). Plants, in turn, use enzymatic and non-enzymatic antioxidant systems to prevent the accu- mulation of ROS (Duncan 1955). Some stimulants of biological origin or eustress or with appropriate dosage and length, not only activate chemical defense in plants, but also increase plant productivity (Elizabeth Abreu and Munné-Bosch 2008). One of the purposes of plant physiologists is to identify and introduce substances that improve the resistance of plants to biotic and abiotic stresses. Salicylic acid is one of these substances. Sali- cylic acid treatment with appropriate dose has increased resistance to environmental stresses in different species. Regulation of tougher pressure and activation of the antioxidant system are the mechanisms of salicylic acid in improving water shortage stress resistance (Eraslan et al., 2007). Another substance that plays a role in modu- lating environmental stresses is chitosan. It is a natural polymer derived from chitin, which is used as a biologi- cal elicitor in agriculture (EFSA 2010). Chitosan treatment enhances photosynthesis and stomatal closure by ABA synthesis (Guo et al., 2022; 2021). This substance increases antioxidant enzymes through the H2O2 and NO signaling pathways and induces the produces sugars, amino acids, organic acids, and other metabolites needed to signal stress, osmotic balance, and energy metabolism under unfavorable envi- ronmental conditions (Gao et al., 2016). Another compound that increases plant resistance to biological and non-biological stresses is melatonin (N-acetyl-5-methoxytryptamine) (Janda et al., 1999; Meng et al., 2014; Ma et al., 2018; Porra et al., 1989; Peng et al., 2021; Su et al., 2019; Sun et al., 2021). A wide range of physiological reactions has been attributed to mela- tonin, which can moderate the adverse effects of environ- mental stresses such as drought and salinity on plants. Melatonin has an antioxidant activity and can detoxify ROS in cells (Karimi et al., 2015). Melatonin can increase photosynthetic capacity by increasing the absorption of water and nutrients and increasing the expression of genes associated with mitogen-activated protein kinases (LI et al., 2021). It has been reported that the use of mela- tonin under drought stress conditions prevents the accu- mulation of abscisic acid in the cell and has a synergistic effect with cytokinin (Lehmann et al., 2010; Liang et al., 2019; Mahajanab et al., 2021 ). Therefore, in the present study, we investigate the role of chitosan, salicylic acid, and melatonin on the physiological and biochemical responses of stevia under water deficit stress conditions. MATERIALS AND METHODS This experiment was carried out in the two cropping years of 2019 and 2020 as a split-plot based on a rand- omized complete block design with three replications in a research farm in the city of Sulaymaniyah located in the north of Iraq. The experimental area was located at 45°11’ N; 45°58’ E longitude and altitude, respectively, 690 m above sea level. The average annual rainfall in the experimental area was about 250 mm and the aver- age maximum and minimum temperatures were 22 and 9 degrees, respectively. The soil properties of the test site are listed in Table 1. Experimental treatments included different irriga- tion regimes (90% FC, 65% FC, and 40% FC) and foliar application of different elicitor (control, chitosan (2 gL-1), salicylic acid (100 mg L-1), and melatonin (100 μM), which were assigned to main and sub-plots, respectively. Table 1. Soil physical and chemical properties of the experimental site. Physical Properties Value Unit Chemical properties Value Unit sand 133.6 g kg-1 Organic mater 20 g Kg-1 silt 244.3 g kg-1 pH 7.57 clay 622.1 g kg-1 ECe 1.4 dS m-1 soil texture Clay Total nitrogen 20 mg Kg-1 Bulk density 1200 Kg m-3 Phosphors (soluble) 19 mg Kg-1 Field capacity (33 k Pa) 320 g kg-1 Potassium (soluble) 13.7 Meq Kg-1 Wilting point (1500 k Pa) 188 g kg-1 Calcium (soluble) 7.5 Meq Kg-1 17Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency The seedlings obtained by tissue culture were ini- tially cultivated in peat moss medium to select the well- established plantlets. After three weeks, the uniform seedlings were trans-planted into soil in May each year. In this experiment, the dimensions of each plot were considered 2×2.5 m. Each plot also included four rows of crops. In this study, the distance between the rows was 50 and the distance between the plants was 30 cm. The first two weeks the soil moisture was maintained within the range of field capacity and, then, the irrigation treatments were applied as 90, 65, and 40% of field capac- ity (FC). The soil moisture content was measured using the gravimetric method. Irrigations for each plat were conducted to replenish 100% of soil field capacity. Chitosan, salicylic acid, and melatonin were sprayed three times after 30, 40, and 50 days from transplanting in both years. Harvesting was done 62 days after transplanting the seedlings to the field. In the first stage, the leaves and stems were separated and then weighed. Evaluating morphological characteristics At the time of harvest, the plants in each plot were carefully removed from the soil and the roots of each sample were washed to remove soil residues. Stem length, leaf area index, and biological yield of each sam- ple were measured. Leaf area index was calculated according to Formula (1), in which LA leaf area and LG land area were occu- pied LAI = LA/LG photosynthetic pigments 80% of acetone extract with adsorption readings at 663, 646, and 440 nm was used to measure the concentra- tion of photosynthetic pigments according to Arnon’s (18) method. In addition to quantify the measurements, Porra et al. (19) method was used Chlorophyll a = 12.25×A663–2.55×A646 Chlorophyll b = 20.31×A646–4.91×A663 Carotenoids = 4.69×A440–0.267× (A663×A646) Pigment content was expressed as mg/gDW. Proline content To determined the proline content. 50 mg of leaf dry matter was homogenized in 5 ml of ethanol: Water mix- ture (60:40) and the homogenized solution was incubated for 24 h at +4 °C. After centrifugation at 10,000 rpm, the supernatant was collected. In the next step, 100 mL of nin- hydrin acid 1% was mixed with 500 μL of supernatant of the extracted solution. The samples (reaction mixture) were placed in a water bath at 95 °C for 20 min and, then, cooled to the room temperature. After cooling, the reaction absorbance was measured at 520 nm and using proline as standard, the proline content of the samples was measured. The proline content of the samples was expressed based on mg proline / g fresh weight Zheng et al. (2018). Secondary metabolite analysis To extract phenol, plant samples were dried and, then, macerated in 80% methanol ((HPLC grade). In the next step, the samples were incubated for 24 h at 4 °C. The incubated samples were centrifuged at 2000 rpm for 15 min and the supernatants of the extracts were collected for further analysis. Finally, Wolf et al.’s (20) method was used to analyze the phenol content. In this method, folin-ciocalteu reagent and gallic acid were used as the standard. The total antioxidant capacity was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay according to Karimi et al. (21). Analysis of steviol glycosides To estimate the glycoside content of the leaves, at the time of harvest, the leaves in each plot were randomly selected and dried at 40 °C in an air oven. Then, 100 g of the powdered leaf sample was placed in a 25 ml flask containing 10 ml of methanol and filtered for 24 hours. The reduced pressure filter was vacuum dried and, then, defatted with n-hexane. Afterwards, the moving phase was used to solve the samples (HPLC grade-acetonitrile: water in the ratio 1:1). Filters with pores of 0.22 μm were used to re-filter the samples. Filtered samples were used to determine the content of stevoside, Reb-A by liquid chromatography-mass spectrometry (LCMS-Shimadzu, 2020 system) (22). Standard curves with the samples were used to quantify glycoside content; these curves are used for standard stevoside and Reb-A samples (22). Statistical analysis Analysis of variance was performed using SAS soft- ware, also the comparison of the mean of treatments with the Duncan (1955) method was performed at a level of 5% probability. RESULTS Results showed the effect of irrigation regimes on all the traits was significant at the level of 1% probability. 18 Basoz Sadiq Muhealdin et al. A significant difference was detected among the differ- ent treatments in terms of the effect on plant height, leaf area index, biological yield, proline, chlorophyll a, chlo- rophyll b, phenol, rebaudioside A, and steviosid at the probability level of 1% and in terms of carotenoid con- tent at the level of 5% probability. There was a significant interaction effect of irrigation and allicator treatments on plant height, biological yield, proline, phenol content, DPPH, rebaudioside A, and steviosid at 1% probabil- ity and on leaf area index and carotenoid content at 5% probability level was observed (Table 2). Plant Height In this study, the highest plant height with the average of 37.17 cm was allocated to normal irrigation conditions (90% FC) and the foliar application of sali- cylic acid. With the intensification of water stress, the plant height significantly decreased; but, in treatment of the 65% FC, application of salicylic acid could sig- nificantly increase plant height compared to the control treatment. There was no significant difference between the control treatment and other treatments using growth stimulants (Table 4). The results also showed that under irrigation conditions of the 40% FC, there was no considerable difference among the control treat- ment and the application of elicitors. Results revealed, the lowest plant height was allocated to irrigation treat- ment of the 40% FC in all the four growth elicitor treatments (Table 4). Leaf Area Index The results of mean comparisons showed that mela- tonin application under normal irrigation conditions (90% FC) had the highest leaf area index, while the con- trol and chitosan treatment in the treatment of 40% FC showed the lowest amount. In this study, although in the irrigation treatment of 65% FC, there was no consider- able difference between the control treatment and the other elicitor treatments, in the irrigation treatment of 40% FC, the use of salicylic acid increased the leaf area index significantly compared with the control and other treatments (Table 4). Biological Yield Among the interactions of irrigation and plant growth stimulant treatments, the use of melatonin in 90% FC treatment with the average of 49.34 g/plant showed the maximum biological yield. The lowest bio- logical yield with the average of 12.14 and 10.97 g/plant was allocated to the control treatment and application of chitosan under irrigation conditions of 40% FC. Results showed that although biological yield decreased with exacerbation of water deficit, the melatonin spraying was able to improve this trait in the treatment of the 65% FC by 45.61% compared with the control treatment, In addition there was no remarkable difference among con- trol treatment and elicitors application treatments in the irrigation treatment of the 40% FC (Table 4). Table 2. Combine analysis of variance of irrigation and spraying elicitors on studied traits in stevia (Stevia rebaudiana Bertoni). SOV Df Mean square Plant Height LAI Biomass Proline Chloro- phyll a Chloro- phyll b Carot- enoid Phenol DPPH Rebaudio- side A Steviosid Year (Y) 1 39.01ns 1.25ns 0.60ns 0.0004ns 0.0004ns 0.008ns 0.00021ns 0.027ns 0.056ns 0.50ns 2.72ns Year (Replication) 4 13.08 0.85 0.21 0.0005 0.0002 0.004 0.0006 0.018 0.021 0.64 1.24 Irrigation levels (I) 2 1606.57** 305.92** 4182.82** 0.0029** 0.0057** 28.32** 0.0031** 0.065** 42.81** 27.73** 98.38** Y× I 2 7.76ns 17.36ns 10.06ns 0.0001ns 0.0004ns 0.0024ns 0.00007ns 0.017ns 0.18ns 5.50ns 4.05ns Error 1 8 13.56 5.88 60.04 0.0006 0.0007 4.33 0.000012 0.011 3.71 5.91 11.73 Elicitors (E) 3 37.29** 31.62** 289.38** 0.0075** 0.0147** 29.94** 0.0013* 0.040** 1.95ns 13.47** 216.91** Y×E 3 19.94ns 0.45ns 0.06ns 0.0074ns 0.0004ns 0.052ns 0.000025ns 0.012ns 0.092ns 0.47ns 7.16ns I × E 6 41.33** 8.88* 189.68** 0.0011** 0.0005ns 5.54ns 0.00040* 0.332** 3.98** 8.39** 46.95* Y×E×I 6 10.36ns 1.54ns 11.16ns 0.0004ns 0.0004ns 0.0011ns 0.000032ns 0.031** 0.05ns 0.46ns 6.27ns E2 36 11.12 3.41 48.21 0.0003 0.0006 2.12 0.00013 0.011 1.05 0.98 14.34 CV% - 12.56 14.20 24.78 15.16 19.01 16.96 20.57 10.52 9.67 13.51 15.55 ns, *, and ** were significant at levels 1 and 5% respectively. 19Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency Proline The results revealed that the leaf proline content increased with exacerbation of drought, so that the irri- gation conditions of the 40% FC with spraying of mela- tonin with the average of 0.173 (Mg/gFW) had the maxi- mum proline content. Results demonstrated that spray- ing of melatonin in both 65% FC and 40% FC conditions was able to improve the proline content compared with the control by 41.62 and 55.85%, respectively. The lowest proline content was assigned to 90% FC treatment with all four eliminator treatments (Table 4). Photosynthetic Pigments The results showed that irrigation conditions of 40% FC and 65% FC reduced the chlorophyll content by 10.85 and 20.91 percent, respectively, compared to 90% FC irrigation conditions (Table 3). In the present study, the use of melatonin with the average of 0.163 (Mg/gFW) had the highest leaf chloro- phyll a content and increased the amount of this trait compared to the use of chitosan, salicylic acid, and control by 15.60, 15.60, and 72.30 percent, respectively. In this study, the control treatment with the average of 0.094 (Mg/gFW) had the lowest chlorophyll a content (Table 3). Table 3. Mean comparison of main effects of irrigation and spraying elicitors treatments of on studied traits in Stevia (Stevia rebaudiana Bertoni). Irrigation Plant Height (cm) LAI Biomass (g plant-1) Proline (Mg gFW- 1) Chloro- phyll a (Mg gFW-1) Chloro- phyll b (Mg gFW-1) Carotenoid (Mg gFW-1) phenol mgGAE gDW-1 DPPH (%) Rebaudio- side A (%) Steviosid (%) 90% FC 34.50a 16.71a 41.13a 0.108b 0.149a 0.096a 0.069a 0.961b 9.36c 6.48b 22.34b 65% FC 26.99b 12.75b 28.19b 0.120a 0.133b 0.086b 0.052b 1.026a 10.45b 7.03b 24.36ab 40% FC 18.16c 9.58c 14.73c 0.130a 0.118c 0.074c 0.047b 1.067a 12.00a 8.55a 26.39a Elicitors Control 24.41b 11.49c 23.08b 0.148a 0.094c 0.068c 0.046c 0.822d 10.49a 6.63b 21.63b Chitosan 27.42a 12.64bc 27.56ab 0.119b 0.141b 0.087b 0.056b 1.174a 10.52a 6.43b 21.09b Salicylic acid 26.99ab 14.68a 28.57ab 0.108bc 0.136b 0.089ab 0.055b 0.993c 10.31a 8.11a 27.33a Melatonin 27.38ab 13.23ab 32.85a 0.102c 0.163a 0.098a 0.067a 1.087b 11.07a 8.24a 27.39a Data in columns followed by different letters are significantly different (p ≤ 0.01) by Duncan’s multiple range test. Table 4. Mean comparison of irrigation and spraying elicitors interaction treatments of on studied traits in Stevia (Stevia rebaudiana Ber- toni). Irrigation Elicitors Plant Height (cm) LAI Biomass (g plant-1) Proline (Mg gFW-1) Carot- enoid (Mg gFW-1) Chlo- rophyll a (Mg gFW-1) Chlo- rophyll b (Mg gFW-1) Phenol mgGAE gDW-1 DPPH (%) Rebau- dioside A (%) Steviosid (%) 90% FC Control 30.35bcd 14.73bcd 32.88bcd 0.0937d 0.062bc 0.109a 0.081a 1.1197bc 8.11f 5.887ghi 18.76f Chitosan 33.95abc 15.94abc 38.84abc 0.0957cd 0.058b-e 0.147a 0.091a 1.1017bc 9.29ef 6.65f-h 20.81ef Salicylic acid 37.17a 17.65ab 43.45ab 0.1207bcd 0.07b 0.164a 0.102a 0.6317d 10.03de 5.302i 26.65bc Melatonin 36.53ab 18.51a 49.34a 0.123bcd 0.087a 0.176a 0.068a 0.674d 9.94de 8.088cd 23.15c-f 65% FC Control 23.08ef 11.36def 20.61def 0.1033cd 0.047de 0.086a 0.111a 0.6747d 11.12bcd 6.328f-i 22.49c-f Chitosan 26.57de 13.69cd 28.26cde 0.1237bcd 0.056cde 0.149a 0.082a 1.1567ab 10.37de 5.649h-i 20.40ef Salicylic acid 30.02cd 14.03bcd 26.82cde 0.1063cd 0.045e 0.134a 0.089a 1.1683ab 9.92de 8.883bc 29.10b Melatonin 28.89cde 11.9def 37.07abc 0.1498ab 0.059bcd 0.164a 0.105a 1.108bc 10.4de 7.277def 25.43bcd 40% FC Control 19.8f 8.39f 12.14f 0.1113cd 0.031f 0.088a 0.055a 1.0033c 12.24ab 7.7de 23.66cde Chitosan 18.9f 8.3f 10.97f 0.1057cd 0.055cde 0.126a 0.094a 1.2647a 11.91abc 7.018d-g 22.06def Salicylic acid 17.22f 12.36cde 20.05def 0.1313bc 0.048cde 0.11a 0.07a 1.18ab 10.98cd 10.15a 26.25bcd Melatonin 16.71f 9.28ef 15.75ef 0.1733a 0.055cde 0.148a 0.079a 1.151ab 12.88a 9.362ab 33.60a Data in columns followed by different letters are significantly different (p ≤ 0.01) by Duncan’s multiple range test. 20 Basoz Sadiq Muhealdin et al. Chlorophyll b content decreased due to water short- age so that the supply of the 90% FC and 40% FC had the maximum and minimum chlorophyll b contents, respectively (Table 3). Results revealed that melatonin application had the highest chlorophyll b content. The difference between melatonin and salicylic acid was not significant. The lowest chlorophyll content was recorded for control treatment of foliar application (Table 3). In our experiment spraying of melatonin in irriga- tion treatments of 90% FC improved carotenoid content compared with the control treatment by 40.32 percent and had the maximum carotenoid content (Table 4). In our study, although in irrigation treatment of 60% FC, there was no notable difference among the control treat- ment and elicitors, in the irrigation treatment of 40% FC, foliar spraying of chitosan, salicylic acid, and mela- tonin increased the carotenoid content by 77.41, 54.88, and 77.10 percent compared to the control. Phenol content Based on the results, with the intensification of water deficit stress, the phenol content was increased, so that the foliar spraying of chitosan and salicylic acid in irrigation treatment of 60% FC and the use of chitosan, salicylic acid, and melatonin in irrigation treatment of 40% FC had the highest phenol content and enhanced the amount of this trait remarkable compared with the control. The lowest phenol content was allocated to irri- gation treatment of 90% FC and the foliar spraying of salicylic acid and melatonin (Table 4). DPPH In our study water stress rose the amount of DPPH, so that the treatment of melatonin, chitosan, and control under irrigation treatment of 40% FC had the highest amount of DPPH activity. The minimum DPPH content was recorded in the control treatment under 90% FC irrigation conditions (Table 4). In our experiment, water shortage and foliar application of elicitors had a syner- gistic effect on DPPH activity. Rebaudioside A Results revealed that water deficit stress and foliar spraying of salicylic acid and melatonin had a synergis- tic effect on rebaudioside A content. Irrigation treatment with 40% FC with foliar spraying of salicylic acid and melatonin had the highest amount of glycoside rebau- dioside A. It should be noted that the application of these two treatments in the irrigation regime of 65% FC significantly increased the amount of rebaudioside A in comparison to the control and foliar spraying chitosan treatments (Table 4). The lowest amount of rebaudioside A was recorded under normal irrigation (90% FC) and salicylic acid application. Steviosid In our study irrigation regime of 40% FC along with melatonin foliar spraying had the highest steviosid gly- coside content, Furthermore foliar spraying of salicylic acid under 90% FC irrigation conditions, foliar spraying of salicylic acid and melatonin under 65% FC irrigation conditions, and application of melatonin under 40% FC irrigation conditions could significantly improved stevi- osid glycoside content compared with control and other treatments (Table 4). DISCUSSION Our research findings showed water deficit dimin- ished plant height, but spraying of salicylic acid, espe- cially in irrigation treatment of the 65% FC, was able to moderate the negative effect of water deficit on plant height. Under drought stress, cell elongation and cell differentiation are reduced due to decreased total water potential inside the plant (Xu et al., 2021). It seems that the application of salicylic acid can mitigate the adverse effect of drought stress on plant growth by preventing a reduction in cell divisions and cell size (Yan et al., 2018). In the study by Karimi et al. (Tardieu et al., 2000) on stevia, drought stress decreased plant height, while the use of external SVglys could not affect this trait. In this study, foliar application of melatonin under normal irrigation conditions produced the maximum leaf area index. leaf area index decreased with the inten- sification of water shortage. Results revealed the use of salicylic acid in the irrigation treatment of 40% FC, increased the leaf area index significantly compared to the control and other treatments. Probability salicyl- ic acid can improve nutrient uptake, especially under stress, which in turn can increase growth (25). It seems that salicylic acid can increase photosynthesis and, thus, increase growth by increasing the amount of chlorophyll in the leaves that are at the beginning of the aging pro- cess (26). Karimi et al. (21) showed that drought stress decreased leaf growth in stevia, but the external applica- tion of SVglys reduced the leaf losses. 21Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency In our research, the use of melatonin under normal irrigation had the highest biological yield, This trait was reduced by reducing the available water of the plant. The results also revealed that foliar application of mela- tonin under water shortage conditions increased bio- logical yield compared to the non-foliar spraying treat- ment. Increased biological yield in the present study can be due to the positive effect of foliar application on leaf area index and photosynthetic pigments under different irrigation, leaf area development and photosynthetic pig- ments increased the rate of photosynthesis and acceler- ate plant vegetative growth. One of the adverse effects of drought stress is accelerating the production of reactive oxygen species (ROS) (Ucar et al., 2016), which leads to cell damage, reduced growth and biomass production, and ultimately cell death. The result showed that under irrigation conditions of the 40% FC with the use of melatonin had the high- est proline content. Results also revealed that the use of melatonin in both 65% FC and 40% FC conditions was able to raise the proline content compared to the con- trol treatment. Proline acts as an osmolytic / sprotactant factor under water deficit stress (Zheng et al., 2018). The results of our study showed a significant increase in pro- line content under water deficit stress treatments. It has been reported that the use of melatonin under water deficit conditions induces drought resistance in plants by increasing proline biosynthesis (Zhang et al., 2015). Consistent with the results of the present study, Liang et al. (Zhao et al., 2017) in Actinidia cinnis, Campos et al. (2019) in Brassica napus, and Li et al. (2019) in Cophea Arabica have found that melatonin application signifi- cantly increases leaf proline content in these plants. In the study by Ghanbari et al., the use of chitosan and salicylic acid in milk thistle (silybum marianum L.) remarkably raised the proline content under drought stress. Water deficit causes the ROS accumulation, resulting in the degradation of the molecular structure of chloro- phyll, and finally declining plant photosynthesis. Under moisture shortage conditions, a decrease in Chl content was considered a typical sign of oxidative stress. The decrease in Chl content under drought stress is mainly due to degradation of Chl as the result of ROS activity (Zhang, et al ,2022). Reduction of chlorophyll content due to water deficiency, in chickpea, maize , and basil , has been documented in previous investigations. In the present study, the use of melatonin had the highest leaf chlorophyll a and b content. Similar to other photosyn- thetic pigments, water deficit decreases carotenoid con- tent, but the use of all three elicitors increased carote- noid compared with non-foliar spraying treatment (con- trol) under irrigation treatment of 40% FC. Under water deficit conditions stress, the use of elicitors can prevent the degradation of pigments molecules. Decreased pig- ments molecule degradation after melatonin treatment may be due to decreased down-regulation of genes of chlorophyll-degrading enzymes such as Chlase, PPH, and Chl-PRX. The results showed that induction of water deficit stress and foliar application of elicitors increased leaf phenol content so that the use of chitosan and salicylic acid under irrigation treatment of 60% FC and the use of chitosan, salicylic acid, and melatonin in irrigation treatment of 40% FC showed the highest amount of phenol content. As mentioned, drought stress increases the accumulation of ROS in cells that can damage cel- lular structures. The use of elicitors can increase the activity of antioxidant enzymes to strengthen the plant’s defense system and prevent accumulation of ROS in cells. Increased phenol content in response to melatonin treatment under drought stress had been reported in the study by Sharma et al. (2017) on Grafted (Carya cathay- ensis). In the study by Karimi et al. (2017), the highest values of antioxidant capacity in stevia were allocated to water deficit conditions and plants treated with SVglys, while the lowest values were reported under normal irrigation conditions and no use of SVglys. Application of salicylic acid by regulating the activity of antioxidant enzymes can improve plant tolerance to adverse condi- tions (Li, et al, 2021; Sun, et al. 2021; Xu, et al, 2021; Zhang, et al. 2022). The results showed the spraying of melatonin and chitosan under irrigation treatment of 40% FC showed the maximum amount of DPPH activity. Consistent with our results, the use of melatonin had a protective role against water shortage in corn and oats . In the study by Ahmed et al. (42), water deficit increased rebau- dioside A and stevioside content in steva. compatible sol- utes such as soluble sugars under melatonin treatment increase, These substances are responsible for main- taining the turgor and osmotic pressure of plant cells under water deficit conditions. In the study by Jalal et al. (2018), salicylic acid treatment increased sugar content on plant in both drought stress and control treatments. Salicylic acid increases plant resistance to drought stress by stimulating sugar production in the cell. Karimi et al. (2019) showed that drought stress increased rebaudioside A content in stevia (Bi, et al., 2021; Duan, et al., 2022; Guo, et al, 2021; Guo, et al, 2022). The results showed that application of salicylic acid under 90% and 65% FC irrigation conditions and appli- cation of melatonin under 40% FC irrigation had a positive effect on increasing steviosid glycoside content, 22 Basoz Sadiq Muhealdin et al. therefore, the content of this substance increased in all irrigation conditions in response to different elicitors. In a study on milk thistle (silybum marianum L.), the highest soluble sugars was reported in the application of chitosan and salicylic acid under water stress condi- tions. The positive effect of melatonin on increasing the amount of soluble sugars has also been reported in the studies by Liang et al. (2011), Campos et al. (2012), and Li et al. (2018). CONCLUSION To summarize, the findings of this research revealed that water deficit stress markedly decreased plant growth and increased the proline, phenol, DPPH, rebaudioside, and steviosid contents. 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Opti- mal model evaluation of the proton-exchange mem- brane fuel cells based on deep learning and modified African Vulture Optimization Algorithm. Energy Sources, Part A: Recovery, Utilization, and Environ- mental Effects, 44(1):287-305. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 4 - 2022 Firenze University Press Avicennia genus molecular phylogeny and barcoding: A multiple approach Laleh Malekmohammadi1, Masoud Sheidai1,*, Farrokh Ghahremaninejad2, Afshin Danehkar3, Fahimeh Koohdar1 Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency Basoz Sadiq Muhealdin1, Sahar Hussein Hamarashid1,*, Fairuz Ibrahim Ali1, Nakhshin Omer Abdulla1, Syamand Ahmad Qadir2 Morphological and cytogenetic characterization in experimental hybrid Aloe jucunda Reyn. x Aloe vera (L.) Burm. f. (Asphodelaceae) Wendy Ozols-Narbona*, José Imery-Buiza Assessment of the absorption ability of nitrate and lead by japanese raisin under salt stress conditions Seyedeh Mahsa Hosseini1, Sepideh Kalatejari1, Mohsen Kafi2,*, Babak Motesharezadeh3 Assessment of protein and DNA polymorphisms in corn (Zea mays) under the effect of non-ionizing electromagnetic radiation Ekram M. Abdelhaliem1,*, Hanan M.Abdalla1, Ahmed A. Bolbol1, Rania S. Shehata1,2 Chromosome counts of some species of wetland plants from Northwest Iran Saeedeh Sadat Mirzadeh Vaghefi*, Adel Jalili Delimiting species using DNA and morphological variation in some Alcea (Malvaceae) species based on SRAP markers Chnar Hama Noori Meerza1, Basoz Sadiq Muhealdin2, Sahar Hussein Hamarashid2,*, Syamand Ahmad Qadir3, Yusef Juan4 Mapping CAP-A satellite DNAs by FISH in Sapajus cay paraguay and S. macrocephalus (Platyrrhini, Primates) Simona Ceraulo, Francesca Dumas* Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry Peng Zhou1, Jiao Li2, Jing Huang1, Fei Li1, Qiang Zhang2,*, Min Zhang1,* First report of chromosome and karyological analysis of Gekko nutaphandi (Gekkonidae, Squamata) from Thailand: Neo-diploid chromosome number in genus Gekko Weera Thongnetr1, Suphat Prasopsin2, Surachest Aiumsumang3,*, Sukhonthip Ditcharoen4, Alongklod Tanomtong5, Prayoon Wongchantra6, Wutthisak Bunnaen7, Sumalee Phimphan3 Intraspecific karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida (Ferula assa-foetida L., Apiaceae) Narges Firoozi, Ghasem Karimzadeh*, Mohammad Sadegh Sabet, Vahid Sayadi Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) Seyed Mahmood Ghaffari¹,*, Seyed Mohsen Hesamzadeh Hejazi² Karyomorphology, genome size, and variation of antioxidant in twelve berry species from Iran Saeed Mohammadpour1, Ghasem Karimzadeh1,*, Seyed Mahmood Ghaffari2