Evaluation and selection of potato varieties (Solanum tuberosum L.) in vitro and study of their tolerance to osmotic stress

Evaluation and selection of potato varieties (Solanum tuberosum L.) in vitro and study of their tolerance to osmotic stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evaluation and selection of potato varieties (Solanum tuberosum L.) in vitro and study of their tolerance to osmotic stress Lama Laila, Salim Zaid, Fahed Al-Biski, Sumaya Jabal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8641040/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The experiments were conducted in the tissue culture laboratory of the National Commission for Biotechnology (NCBT) in Damascus, and at the Faculty of Science - University of Damascus with the aim of evaluating the response of nine introduced potato varieties resulting from in vitro tissue culture (Montereal, Salvador, Synergy, Yalas, Agria, Arizona, Everest, Evora, Hind) to artificial osmotic stress using sorbitol. This was based on some growth indicators and certain biochemical parameters (chlorophyll a and b, total carotenoids, and proline), to determine the cultivars most adapted to osmotic stress. The plants were treated by adding different concentrations of sorbitol (0, 100, 200, 300, 400 mM) to the growth medium. The experiment was designed according to a completely randomized block design, and significant differences were estimated at a 99% confidence level. The nine varieties varied in their response to osmotic stress, with an increase in the level of osmotic stress (sorbitol concentration) in the growth medium causing a significant decline in all growth parameters (plant length, root length, number of leaves and roots) compared to the control, as well as some biochemical traits (chlorophyll molecules types a and b, and carotenoids), and an increase in some biochemical parameters (proline), compared to the control. Cluster analysis based on the total relative values of growth parameters and biochemical parameters showed the distribution of the studied varieties into three groups depending on their tolerance to osmotic stress. The results indicated that the varieties (Yalas and Salvador) are among the tolerant varieties, while the varieties (Arizona, Hind, Synergy) are moderately tolerant, and the varieties (Agria, Everest, Monteral, Evora) are considered sensitive to osmotic stress. The results indicate the possibility of evaluation and selection in the laboratory as a rapid and effective method to explore the genetic variation for osmotic stress tolerance in potatoes. Biological sciences/Physiology Biological sciences/Plant sciences Potatoes osmotic stress growth parameters proline cluster analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The potato (Solanum tuberosum L .) is one of the most important vegetable crops in the world. It belongs to the genus Solanum and the family Solanaceae . Potatoes are grown in about 150 countries 1 , and they are the most important non-cereal food crop globally 2 , 3 . They are cultivated on all continents in vast areas and are a staple food in Europe, the Americas, and developing countries. FAO statistics show that potatoes are grown on an area of ​​more than 20.7 million hectares, with global production estimated at about 437 million tons, and they are consumed by billions of people as a staple food. China is one of the world's largest potato producers 4 . Potatoes are a rich source of carbohydrates, proteins, and dietary fiber, and have numerous industrial uses. Starch is extracted from potatoes and used in the manufacture of paper, textiles, and adhesives. Potatoes are also used in fermentation and the extraction of alcohols such as ethanol and butanol, as well as acids like citric and lactic acid 5 . Land occupies approximately one-quarter of the Earth's surface, and nearly half of its arable land suffers from water scarcity, sometimes reaching the point of drought. This has led to a 25% decrease in arable land 6 . Therefore, water stress is considered one of the most important factors threatening food security globally 7 . Stress tolerance is defined as the ability to grow, flower, and provide a good economic return under unfavorable environmental conditions. Stress-tolerant plants are defined as plants that adapt to survive under stress conditions by inducing morphological, physiological, chemical, and molecular responses 8 . Water stress is the main factor limiting the yield of all agricultural crops, including potatoes. Plants are exposed to periods of water deficit due to a decrease in the amount of water absorbed by the roots, which is insufficient to compensate for the water lost through transpiration, leading to the cessation of plant growth 9 . Potato breeding and genetic improvement programs have helped develop new varieties that meet market demands in terms of stress resistance, improved quality and yield. However, genetic progress in osmotic stress tolerance remains slow due to complex quantitative traits and the lack of complete understanding of the genetic basis of drought tolerance, especially under field conditions, due to the significant interaction between environmental and genetic factors. Therefore, many scientific studies have highlighted the importance of screening to reveal genetic variation among drought tolerance genotypes. Consequently, tissue culture technology has emerged as an alternative and effective method for developing stress-tolerant plants in recent years, as it can be achieved under controlled conditions in less time and space 10 . The effect of osmotic stress on plant growth using tissue culture technology is similar to its effect under field conditions; therefore, in vitro -grown plants can be used as an alternative to stressful and costly field trials 8 . Drought is a major abiotic stress that negatively impacts the performance and productivity of various plants by inhibiting photosynthesis. Drought causes stomata to close, reducing water loss through transpiration and maintaining the hydration of plant cells. However, this is usually accompanied by a decrease in the diffusion rate of carbon dioxide (CO 2 ), which is the precursor to sugar synthesis in the Calvin cycle 11 . Potatoes adapt to stress conditions by enhancing their enzymatic and non-enzymatic antioxidant activities. They synthesize protective substances, such as proline and polyols, to counteract the damaging effects of drought 12 . These substances contribute to changes in cellular osmotic pressure and ensure membrane integrity 13 . Chlorophyll pigments are also important for plants because they harvest light energy necessary for the synthesis of energy-rich compounds during photochemical reactions (NADPH, ATP). Both chlorophyll a and chlorophyll b molecules are significantly damaged when plants are exposed to drought, as the ratio of chlorophyll a to chlorophyll b changes 14 . Manoj et al. (2011) 15 , proved that tissue culture is a practical technique for producing stress-tolerant plants, both biotic and abiotic, by applying selection agents such as NaCl (for salinity tolerance) and PEG, mannitol, or sorbitol (for osmotic stress tolerance) to the culture medium. Only plants capable of surviving in such media are selected. In a study conducted by Sobh (2020) 16 to determine the effect of varying levels of sorbitol-induced osmotic stress on certain morphological traits in eleven potato varieties, using plant tissue culture techniques, increasing the osmotic stress level (sorbitol concentration) in the growth medium resulted in a significant decline in all morphological traits compared to the stress-free control, This included a decrease in the concentration of chlorophyll a and b molecules and total carotenoids, and an increase in proline and oxygenated water. Mehmandar et al. (2023) 17 , also demonstrated that in vitro assessment can be a precise, rapid, and reliable method for evaluating and identifying drought-tolerant genotypes. Results and discussion Growth parameters under the influence of osmotic stress in vitro : Plant height (cm): The results of the statistical analysis showed significant differences in the average plant height among the varieties and different levels of sorbitol. The average plant height decreased with increasing sorbitol concentration for each variety. The highest plant length was observed in the control treatment (13.01 cm), with a significant difference compared to the other treatments in all studied varieties. The lowest significant value was at the concentration of 400 mM (1.71 cm). The longest plant height was recorded for the Evora variety (8.67 cm), while the shortest was for the Everest variety (4.52 cm), (Figs. 1–2). Number of leaves: The results of the statistical analysis showed significant differences in the average number of leaves between the different varieties and levels of sorbitol. The average number of leaves per plant decreased directly and significantly with increasing sorbitol concentration in the growth medium. The highest number of leaves was observed in the control treatment (11.5 leaves/plant) with a significant difference compared to the other treatments, while the lowest number of leaves was observed in the sorbitol 400 mM treatment (1.82 leaves/plant). The highest number of leaves was observed in the Evora variety (8.87 leaves/plant), and the lowest in the Everest variety (4.30 leaves/plant). (Fig. 1–2). Root Length: Statistical analysis revealed significant differences in average root length between different varieties and sorbitol levels. Average root length decreased proportionally and significantly with increasing sorbitol concentration in the growth medium. The control treatment exhibited a significantly higher average root length (5.85 cm) compared to the other treatments, while the shortest root length was observed in the 400 mM treatment (0.57 cm). The highest average root length was observed in the Salvador variety (4.52 cm), and the shortest in the Arizona variety (2.29 cm), (Fig. 1–2). Number of Roots: Statistical analysis revealed significant differences in the average number of roots between different varieties and sorbitol levels. The average number of roots per plant decreased proportionally and significantly with increasing sorbitol concentration in the growth medium. The highest average number of roots was observed in the control treatment (8.39 roots/plant), significantly higher than the other treatments, while the lowest was in the 400 mM treatment (0.6 roots/plant). The highest average number of roots was observed in the Hind and Evora varieties (6.03 and 5.9 roots/plant, respectively), while the lowest was observed in the Arizona variety (2.69 roots/plant), (Fig. 1–2). Figure (1) Effect of osmotic stress coefficients on growth parameters of the studied potato varieties Figure (2) The effect of osmotic stress on growth indicators in nine studied potato varieties Figure (3 ). The effect of different concentrations of sorbitol on growth indicators in the studied potato varieties: A- Salvador, B- Yalas, C- Arizona, D- Hind, E- Everest,F- Evora, G- Montereal, H- Agria, I- Synergy. Biochemical Indicators: Leaf Chlorophyll a content (mg. g⁻¹ wet weight): Statistical analysis revealed significant differences (P ≤ 0.01) in the mean leaf chlorophyll a content. The average leaf content of chlorophyll a was significantly higher in thecontrol (0.76 mg. g − 1 wet weight)، and it decreased proportionally with increasing sorbitol concentration in the growth medium. The leaf content of chlorophyll a was significantly lower at the highest osmotic concentration of 400 mM (0.19 mg. g − 1 wet weight) Figure (4). The highest average leaf content of chlorophyll a was found in the Salvador variety (0.67 mg. g − 1 wet weight)، while it was significantly lowest in the Everst variety (0.24 mg. g − 1 wet weight)، Figure (5). Leaf Chlorophyll b Content (mg. g wet weight): Statistical analysis revealed significant differences (P ≤ 0.01) in the average leaf content of chlorophyll b. The average leaf content of chlorophyll b was significantly higher in the control treatmen (0.62 mg. g − 1 wet weight)، and it decreased proportionally with the increase in sorbitol concentration concentration in the growth medium. The leaf content of chlorophyll b was significantly lower at the highest osmotic concentration of 400 mM (0.21 mg. g − 1 wet weight). The highest average leaf content of chlorophyll b was found in theSalvador variety (0.53 mg. g − 1 wet weight)، while the lowest was in the Synergy variety (0.27 mg. g − 1 wet weight) (Fig. 4–5). Leaf Carotenoid Content (mg. g⁻¹ wet weight): Statistical analysis revealed significant differences (P ≤ 0.01) in the average leaf content of carotenoids. The average leaf content of carotenoids was significantly higher in the control treatmen (0.24 mg. g − 1 wet weight)، and it decreased proportionally with increasing sorbitol concentration in the growth medium. The leaf content of carotenoids was significantly lower at the highest osmotic concentration of 400 mM (0.13 mg. g − 1 wet weight). The highest average leaf content of carotenoids was significantly higher in the Arizona variety (0.25 mg. g − 1 wet weight), while the lowest was in the Salvador variety (0.13 mg. g − 1 wet weight), (Fig. 4–5). Leaf proline content (µg. g⁻¹ green matter): Statistical analysis revealed significant differences (P ≤ 0.01) in the mean leaf proline content. The mean leaf proline content was significantly highest at the highest osmotic concentration of 400 mM (58.67 µg. g⁻¹ green matter), while the mean leaf proline content was significantly lowest in the control treatment (34.11 µg. g⁻¹ green matter). The highest mean leaf proline content was found in the Arizona cultivar (53.93 µg. g⁻¹ green matter), and the lowest in the Everest cultivar (34.82 µg. g⁻¹ green matter), (Fig. 4–5). Figure (4) Effect of osmotic stress coefficients on biochemical indicators in the studied potato varieties Figure (5) Effect of osmotic stress on biochemical indicators in nine studied potato varieties Osmotic Stress Levels and Morphological Changes: Osmotic stress levels showed significant changes in some morphological plant growth indicators, significantly reducing plant height, number of leaves, and root length and number. This reduction was directly proportional to increasing sorbitol concentration. Some studies have attributed the decrease in these indicators to osmotic stress, which inhibits root elongation and number due to high osmotic stress in the growing medium. This leads to a decrease in water potential (becoming more negative), thus reducing the amount of free water available to the plant. Consequently, the rate of water uptake by the root system is negatively affected, and the amount of water absorbed becomes insufficient to compensate for the water lost through transpiration by the aerial parts. As a result, plant elongation and cell division are inhibited, affecting plant diameter and reducing the number of nodes and leaves formed on the stem. This results from a decrease in the filling potential within leaf cells, which inhibits leaf cell elongation and growth, leading to water deficit in plant cells due to reduced water content 18 . These results are consistent with those of Wishart et al. (2014) 19 and Bundig et al. (2016) 20 , who found genetic variation in response to drought conditions, Potato varieties depend on some morphological characteristics, such as root length and aerial parts. Similarly, the study by Sajid (2022) 21 showed a decrease in most growth indicators when treated with sorbitol and mannitol as osmotic stress factors for two potato varieties. Biochemical Indicators: The variation in the leaf content of chlorophyll a and chlorophyll b molecules among the studied varieties is attributed to the genetic factors responsible for the stability of chlorophyll molecules. All the genetic varieties in which the leaf content of chlorophyll a molecule was significantly higher also had a significantly higher leaf content of chlorophyll b the leaf content of chlorophyll b. Similarly, varieties that had significantly lower chlorophyll a also had lower chlorophyll b content. Osmotic stress affected the biochemical processes of potato varieties, leading to a decrease in the chlorophyll and carotenoids content of the leaves. Osmotic stress contributes to a decline in the level of gene expression of genes responsible for proteins associated with chlorophyll molecules (Chlorophyll a-b), and increases the activity of the enzyme Chlorophyllase, which breaks down chlorophyll molecules. This leads to a decrease in the chlorophyll content of the leaves and also affects the basic enzymes in the photosynthesis process and the enzymes responsible for sucrose synthesis. Dehydration increases the level of gene expression of the enzymes responsible for breaking down sucrose 22 . Our results are consistent with those of Mehmandar et al. (2023) 17 , where the addition of sorbitol reduced the chlorophyll content while increasing the proline and malondialdehyde content. The addition of sorbitol to MS medium mimics dehydration stress better than polyethylene glycol. The decrease in carotenoids under stress conditions is attributed to the breakdown of beta-carotene and the formation of zeaxanthins, which play a role in protecting against photoinhibition. These findings align with those of Farooq et al. (2009) 14 , who indicated that carotenoids (orange pigments) regulate the uptake of light energy by chlorophyll molecules to prevent photoinhibition when light utilization efficiency is reduced in stressful environments. Additionally, crops produce proline under drought stress conditions, which protects drought-sensitive cell enclosures from damage 23 . These substances play a key role in stress tolerance 24 . These findings are consistent with those of Wellpott et al. (2024) 25 , where potato genotypes showed genetic variation in the rate of synthesis and accumulation of compatible organic solutes, which explains the genetic variation in drought tolerance. Al-Hadid et al. (2023) 26 , showed that proline concentration increased with increasing salt stress compared to the control for both C3 and C4 plants. Proline accumulation under abiotic stress conditions in many plant species is associated with enhanced stress tolerance. Its concentrations are typically higher in stress-tolerant plants than in sensitive ones. However, stress-sensitive genotypes can sometimes synthesize larger quantities of proline, in which case proline accumulation is related to the extent of damage rather than the level of tolerance 27 . Thus, we observe that sorbitol led to a decrease in leaf chlorophyll and carotenoid content and an increase in proline content with increasing osmotic stress. Results of Agglomerative Hierarchical Clustering (AHC): The cluster analysis, based on the sum of the relative values of the studied parameters (growth indicators and biochemical parameters), led to the division of the studied potato varieties, according to their tolerance to the sum of osmotic stress levels, into three groups, (Fig. 6): -Group 1 (G1): Plants tolerant to osmotic stress, including the cultivars Yalas and Salvador. -Group 2 (G2): Plants with moderate tolerance to osmotic stress, including the cultivars Arizona, Hind, and Synergy. -Group 3 (G3): Plants intolerant to osmotic stress, including the cultivars Agria, Everest, Monteral, and Evora. Since tolerance to abiotic stresses is a complex quantitative trait 28 , and there is a significant interaction between the environment and specific genetic factors associated with economic yield, many studies have focused on studying genetic diversity in potatoes and determining the extent of tolerance to abiotic stresses. These studies often use cluster analysis, which relies on various indicators such as morphological and physiological traits 29 , 30 Figure (6) Cluster analysis of nine potato varieties according to their tolerance to osmotic stress, based on the sum of the relative values of all the studied indicators (growth indicators, chemical parameters). Conclusion Many scientific studies indicate the importance of tissue culture technology as an alternative and effective method for developing stress-tolerant plants. This technology can be implemented under controlled conditions in less time and space. Furthermore, the effect of osmotic stress on plant growth using tissue culture technology is similar to its effect under field conditions. Therefore, this research aimed to investigate the effect of osmotic stress on certain morphological characteristics and chemical parameters, and to select varieties tolerant to osmotic stress. This research is significant in its screening and evaluation of the tolerance of potato ( Solanum tuberosum L. ) varieties to osmotic stress in vitro . The results showed a decrease in most growth indicators and some biochemical properties (chlorophyll molecules of both types (a,b), and carotenoids), and an increase in some biochemical properties (proline) compared to the control when treated with sorbitol as an osmotic stress-causing agent. The nine varieties also varied in their response to osmotic stress, as cluster analysis showed the distribution of the studied varieties into three groups based on their tolerance to osmotic stress. The results showed that the two varieties Yalas, Salvador are considered tolerant varieties, while the varieties Arizona, Hind, Synergy are considered moderately tolerant varieties, and the varieties Agria, Everest Monteral, Evora are considered sensitive varieties to osmotic stress. Materials and Methods Place and Time of Research This research was carried out in the laboratories of the Faculty of Science, Damascus University and the laboratories of the National Commission for Biotechnology in Damascus, during the period (2024–2025). Plant material : Nine varieties of commercial starchy potatoes, preferred by farmers were used in this research: (Montereal, Salvador, Synergy, Yalas, Agria, Arizona, Everest, Evora, Hind), These varieties were recently approved in Syria and were obtained from the Seed Multiplication Foundation in Damascus. Primary Culture and Propagation Rosettes taken from tubers were cultured in plastic pots containing sterile peat moss. After 45 days, the resulting shoots were collected and cut into 2–3 cm single cuttings, each containing one lateral bud. These cuttings were soaked in a 3% sodium hypochlorite solution for 10 minutes as recommended by previous studies 31 , and then washed three times consecutively with sterile distilled water for 5 minutes each time. After disinfection, the cuttings were cultured in test tubes containing 18 ml of MS medium 32 , as shown in table 1, supplemented with 30 g. L⁻¹ sucrose and 7 g. L⁻¹ agar at pH 5.8. The cultured tubes were incubated in a growth chamber at 22 ± 2C°, with a light cycle of 16 hours light /8 hours of darkness, and a light intensity of 3000 lux 33 . Following this, the plant propagation stage began. This stage aims to obtain the largest possible number of well-developed vegetative growths. The propagation process was repeated several times consecutively to obtain the sufficient quantity of plant material for the research. Table (1) Composition of the nutrient medium for Murashige and Skoog (MS). Concentration (mg.l − 1 ) Chemical Composition Compound 1650 NH 4 NO 3 Ammonium Nitrate 1900 KNO 3 Potassium Nitrate 440 CaCl 2 .2H 2 O Calcium Chloride Hydrate 370 MgSO 4 .7H 2 O Magnesium Sulfate Hydrate 170 KH 2 PO 4 Potassium Phosphate 27.85 FeSO 4 .7H 2 O Iron Sulfate Hydrate 37.25 Na 2 EDTA Sodium Chelate 22.3 MnSO 4 .7H 2 O Manganese Sulfate Hydrate 8.6 ZnSO 4 .7H 2 O Zinc Sulfate 6.2 H 3 BO 3 Boric Acid 6.6 KI Potassium Iodide 0.83 Na 2 MoO 4 .2H 2 O Ammonium Molybdate Hydrate 0.025 CuSO 4 .2H 2 O Cupressus Sulfate Hydrate 0.025 CoCl 2 .6H 2 O Cobalt Chloride Hydrate 1 B1 Thiamin 100 Myo-inositol 30000 Sucrose 7000 Agar 5.8 pH Stress treatments Plant samples from the propagation stage were divided into small cuttings containing a lateral bud, and drought stress was applied using different concentrations of sorbitol (0, 100, 200, 300, and 400 mM) added to the growth medium. Each treatment consisted of three replicates, with an average of four plants per replicate, compared to the control (sorbitol-free medium). The studied indicators The results were recorded after 45 days of applying different stress treatments to laboratory plants. The following indicators were estimated Growth Parameters • Stem length (cm): The stem length was measured using a measuring tape, from the base of the stem to the apical bud. • Number of leaves (leaves/plant). • Root length (cm): Measured using a measuring tape, from the base of the root to the tip of the longest root. • Number of roots (roots/plant) 34 . Biochemical indicators: Estimation of leaf content of chlorophyll and total carotenoids: Pigments were extracted by grinding 1 g of frozen plant leaves treated with sorbitol, in the presence of 4 ml of 80% acetone. The extract was then separated using a centrifuge at 10,000 rpm for 10 minutes. The supernatant phase was transferred to new test tubes, and the absorbance was measured using a spectrophotometer at wavelengths of 645 and 662 nm to estimate the amounts of chlorophyll a and b, respectively, and at a wavelength of 470 nm to estimate the total carotenoids 35 . Estimation of the leaf proline content : Proline was extracted by crushing 0.5 g of plant leaves after 45 days of exposure to osmotic stress in the presence of 2 ml of a 3% aqueous solution of sulfosalicylic acid. The extract was then separated by centrifugation at 10,000 rpm for 10 minutes, and its volume was adjusted to 5 ml using 3% sulfosalicylic acid. To estimate the proline content of the extract, 2 ml were taken and 2 ml of ninhydrin solution were added to activate the reaction, along with 2 ml of glacial acetic acid. The tubes were placed in a boiling water bath for 1 hour, then removed and rapidly cooled, 4 ml of toluene was added to each tube, and the tubes were shaken and allowed to separate into two phases. The upper phase was taken, and its optical absorption was measured at a wavelength of 520 nm, according to the concentration of proline in the plant sample compared to a standard curve of known concentrations of commercial proline 36 . Experimental design and statistical analysis: The experiment was designed using a completely randomized block design, with measurements taken after 30–45 days of applying the stress treatments. Each treatment including the control, had three replicates, with each replicate consisting of 4 plants in test tubes. The results were analyzed using the statistical program XLSTAT to compare the means and calculate the least significant difference (LSD) at a significance level of 1%, along with the coefficient of variation (CV%). The Agglomerative Hierarchical Clustering (AHC) method, which classifies variable data into several subgroups such that they are homogeneous within one group (cluster) and different with respect to other clusters, was used to determine the tolerance of varieties to osmotic stress. The cluster analysis followed the equation of Vreugdenhil et al. (2007) 37 , and Al-Biski (2018) 29 based on the sum of the relative values of the studied parameters, the control and stress coefficients were determined as follows: 𝑅𝑉 plant status= \(\:\sum\:\frac{(\text{S}\text{p}1\to\:\text{p}8\text{*}100)}{\text{C}\text{p}1\to\:\text{p}8}\) Where 𝑅𝑉 plant status : is the sum of the relative values specific to the variety, 𝑆𝑝1→𝑝8 is the value of the studied indicator in the stressed plant, 𝐶𝑝1→𝑝8 is the value of the indicator in the control plant. Data availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. ( Lama Laila, PhD in Plant Biology. Email; [email protected] [email protected] ) Abbreviations NCBT - National Commission for Biotechnology; MS- Murashige and Skoog medium;AHC- Agglomerative Hierarchical Clustering ; NaCl- Sodium hypo Chloride;PEG - Poly Ethylene glycol. Declarations Competing interests L. laila, S. Zaid, F. Al-Biski & S. Jabal declare that they have no competing interests. Funding Not applicable. Author Contribution L. laila, PhD in Plant Biology. Department of plant Biology, Faculty of Sciences , Damascus University, Syria , was carried out this research in the field and at the laboratory of plant biotechnology and the wrote of this scientific research was done by the help of supervisors Dr. S. Zaid, Dr. F. Al-Biski & Dr. S. Jabal . In addition, designed the experiment, analyzed the data, drafted and improved the manuscript were carried out by all of us. Acknowledgements The authors express their gratitude to thank staff in Plant Biology Department, Faculty of Sciences, Damascus University, and in Biotechnology Department, the National Commission for Biotechnology. Syria. Data Availability The datasets used and analysed during the current study available from the corresponding author on reasonable request. References Basera, M., Chandra, A., Kumar, V. A., & Kumar, A. (Effect of brassinosteroids on in vitro proliferation and vegetative growth of potato. Pharma Innovation J , 7 (4), 4-9, (2018). Bamberg, J. B., Martin, M. W., Abad, J., Jenderek, M. M., Tanner, J., Donnelly, D. J., & Novy, R. G. In vitro technology at the US Potato Genebank. In Vitro Cellular & Developmental Biology-Plant , 52, 213-225, ‏ (2016). Nikitin, M. M., Statsyuk, N. V., Frantsuzov, P. A., Dzhavakhiya, V. G., & Golikov, A. G. Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR. 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Hwang, E.W., Shin, S.J., Yu, B.K., Byun, M.O., and Kwon, H.B. miR171 family members are involved in drought response in Solanum tuberosum. J. Plant Biol . 54 , 43–48. (2011)b. Chen, Z., Cuin, T. A., Zhou, M., Twomey, A., Naidu, B. P., and Shabala, S. Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. J. Exp. Bot. 58 , 4245–4255. (2007). Munns, R. and Tester, M. Mechanisms of salt tolerance. Annual Review Plant Biology, 59 , 372 651-681. (2008). Wellpott,k., Herde,M.,Winkelmann,T., Bündig,C. Liquid in vitro culture system allows gradual intensification of osmotic stress in Solanum tuberosum through sorbitol. Plant Cell , Tissue and Organ Culture Journul, vol 157 :12. (2024). Al-Hadid, Hanin; Zaid, Salim; Ibrahim, Amina. A study of the effect of salinity on some physiological parameters of a trivalent and a quaternary plant. Damascus University Journal of Basic Sciences , 39 , 10-1:2. (2023). KaviKishor, P.B., Sangam, S., Amrutha, R.N., Sri Laxmi, P., Naidu, K.R., Rai, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P. and Sreenivasulu, N. Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Current Science 88 , 424–438. (2005). Scarano, A., Olivieri, F., Gerardi, C., Liso, M., Chiesa, M., Chieppa, M., Frusciante, L., Barone, A., Santino, A., Rigano, M.M. .Selection of tomato landraces with high fruit yield and nutritional quality under elevated temperatures. J. Sci. Food Agric ., 100 : 2791–2799. (2020). Albiski, F. Screening of some Potato( Solanum tuberosum ) varieties for osmotic stress tolerance using growth parameters in vitro . Damascus University Journal of Agricultural Sciences ., 34 :99-124. ‏ (2018). Ahmad, Najwa. Effect of grafting two hybrids onto two tomato rootstocks grown in greenhouses under saline stress conditions. Master's thesis. Department of Horticultural Sciences. Faculty of Agriculture. Damascus University. (2022). Murshed, R; Najla, S; Albiski, F; Kassem, I; Jbour, M., and Al-Said, H. Using growth parameters for in-vitro screening of potato varieties tolerant to salt stress .J. Agr. Sci.Tech., 17 : 483-494. (2015). Murashige, T. and Skoog, F. A .revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum ., 15 : 473-497 . (1962). Aazami, M. A., Torabi, M. and Jalili, E. In vitro response of promising tomato genotypes for tolerance to osmotic stress. African Journal of Biotechnology , 9 (26): 4014-4017. (2010). Albiski, F., Najla, S., Sanoubar, R., Alkabani, N., & Murshed, R. In vitro screening of potato lines for drought tolerance. Physiology and Molecular biology of plants , 18 , 315-321. ‏ (2012). Lichtenthaler, H. K. and Buschmann, C. Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. Current Protocols in Food Analytical Chemistry, Unit F4. 3 : 1-8. (2001). Bates, L. S.; Waldren, R. P. and Teare, I. D. Rapid determination of free proline for water-stress studies. Plant and Soil ., 39 : 205–207. (1973). Vreugdenhil, D., Bradshaw, J., Gebhardt, C., Govers, F., Taylor, M., acKerron, D. & Ross, H. Water Availability and Potato Crop Performance, Potato Biology and Biotechnology. Advances and Perspectives, Elsevier, Amsterdam. Pp: 333-351. (2007). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8641040","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":588092756,"identity":"dab912b7-dba1-4c26-9ef0-6876736da52a","order_by":0,"name":"Lama Laila","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIie2RsWrDMBBALwSURY1WQ4r9BQWFQOkQ3L/oLGOwlwgyGjpUU76h4EJ/IcHgWUFDh/oDCl2SvQGNHXvSGuy2W6F6w+kk7nE6CSAQ+INMfVwDdYm21TJ2e30YUIiPHCgmo/1jVyy8In6guGRsLjYmU+5gUJm8ZhZ4ekkmGgztivL5zhyxSxpfqR6FyiYCnlNCBeyfqqXcvRcclXxxrfsuJreojHEWAfqjK+SuFk7RWdunsFPzCfyBEnYAjbOU87q0w0okW+xiKInwYqiIZLb6pkt0am8Ef0HlqNwjz7ez1VoL3j8LY7J5s9X9LWO5sfiVSVKXDSZp3Kd4hI8j5RfuK/lA+RmJ+k11IBAI/Ae+ABTyXn/IYP2HAAAAAElFTkSuQmCC","orcid":"","institution":"Damascus University","correspondingAuthor":true,"prefix":"","firstName":"Lama","middleName":"","lastName":"Laila","suffix":""},{"id":588092757,"identity":"d2a014b3-9412-4df6-8899-9670733126d4","order_by":1,"name":"Salim Zaid","email":"","orcid":"","institution":"Damascus University","correspondingAuthor":false,"prefix":"","firstName":"Salim","middleName":"","lastName":"Zaid","suffix":""},{"id":588092758,"identity":"721a9a56-8428-4671-bb7b-0a47644ee6f5","order_by":2,"name":"Fahed Al-Biski","email":"","orcid":"","institution":"National Commission for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Fahed","middleName":"","lastName":"Al-Biski","suffix":""},{"id":588092759,"identity":"917d8e53-11f9-4e11-a546-5745d5ec5fb0","order_by":3,"name":"Sumaya Jabal","email":"","orcid":"","institution":"Researcher at the General Commission for Scientific Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Sumaya","middleName":"","lastName":"Jabal","suffix":""}],"badges":[],"createdAt":"2026-01-19 15:41:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8641040/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8641040/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102615113,"identity":"9e71250f-475b-416e-8ca2-e116bddb517d","added_by":"auto","created_at":"2026-02-13 15:28:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71543,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of osmotic stress coefficients on growth parameters of the studied potato varieties\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/0de108f3fbc05db8b0453ff9.png"},{"id":102748534,"identity":"f4292855-c8f6-453e-b6ba-3e7aa8b6f6b8","added_by":"auto","created_at":"2026-02-16 09:11:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110188,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of osmotic stress on growth indicators in nine studied potato varieties\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/d43ddc2b3adbc0f58359b42f.png"},{"id":102615117,"identity":"9f42a214-c72a-49d2-a49a-b30338b6eab4","added_by":"auto","created_at":"2026-02-13 15:28:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":731085,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of different concentrations of sorbitol on growth indicators in the studied potato varieties: A- Salvador\u003cem\u003e, \u003c/em\u003eB- Yalas, C- Arizona, D- Hind, E- Everest,F- Evora, G- Montereal, H- Agria, I- Synergy.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/edd9159cf841e5583596b195.png"},{"id":102615116,"identity":"d56c9ebc-7da2-4b1e-8a9a-1ec7474793cd","added_by":"auto","created_at":"2026-02-13 15:28:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71013,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of osmotic stress coefficients on biochemical indicators in the studied potato varieties\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/4e5f8b44e97751235da0b192.png"},{"id":102747650,"identity":"e75942c7-08cc-4e4d-a5ef-e8217869dc79","added_by":"auto","created_at":"2026-02-16 09:05:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117578,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of osmotic stress on biochemical indicators in nine studied potato varieties\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/d08faac72614e8fa7cf2ac4a.png"},{"id":102615114,"identity":"2f777761-d4b9-4b0f-8093-08dab3ec9b0f","added_by":"auto","created_at":"2026-02-13 15:28:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35473,"visible":true,"origin":"","legend":"\u003cp\u003eCluster analysis of nine potato varieties according to their tolerance to osmotic stress, based on the sum of the relative values ​​of all the studied indicators (growth indicators, chemical parameters).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/200caa98d55628031e8a0b77.png"},{"id":108569780,"identity":"0858d2db-8794-43b4-951c-94e2209c51b2","added_by":"auto","created_at":"2026-05-06 05:56:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1630763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8641040/v1/4496abd6-1623-4cb2-a0fa-0a4c019d073a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation and selection of potato varieties (Solanum tuberosum L.) in vitro and study of their tolerance to osmotic stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe potato \u003cem\u003e(Solanum tuberosum L\u003c/em\u003e.) is one of the most important vegetable crops in the world. It belongs to the genus \u003cem\u003eSolanum\u003c/em\u003e and the family \u003cem\u003eSolanaceae\u003c/em\u003e. Potatoes are grown in about 150 countries \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and they are the most important non-cereal food crop globally \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. They are cultivated on all continents in vast areas and are a staple food in Europe, the Americas, and developing countries. FAO statistics show that potatoes are grown on an area of ​​more than 20.7\u0026nbsp;million hectares, with global production estimated at about 437\u0026nbsp;million tons, and they are consumed by billions of people as a staple food. China is one of the world's largest potato producers \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePotatoes are a rich source of carbohydrates, proteins, and dietary fiber, and have numerous industrial uses. Starch is extracted from potatoes and used in the manufacture of paper, textiles, and adhesives. Potatoes are also used in fermentation and the extraction of alcohols such as ethanol and butanol, as well as acids like citric and lactic acid \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Land occupies approximately one-quarter of the Earth's surface, and nearly half of its arable land suffers from water scarcity, sometimes reaching the point of drought. This has led to a 25% decrease in arable land \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, water stress is considered one of the most important factors threatening food security globally \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Stress tolerance is defined as the ability to grow, flower, and provide a good economic return under unfavorable environmental conditions. Stress-tolerant plants are defined as plants that adapt to survive under stress conditions by inducing morphological, physiological, chemical, and molecular responses \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Water stress is the main factor limiting the yield of all agricultural crops, including potatoes. Plants are exposed to periods of water deficit due to a decrease in the amount of water absorbed by the roots, which is insufficient to compensate for the water lost through transpiration, leading to the cessation of plant growth \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003ePotato breeding and genetic improvement programs have helped develop new varieties that meet market demands in terms of stress resistance, improved quality and yield. However, genetic progress in osmotic stress tolerance remains slow due to complex quantitative traits and the lack of complete understanding of the genetic basis of drought tolerance, especially under field conditions, due to the significant interaction between environmental and genetic factors. Therefore, many scientific studies have highlighted the importance of screening to reveal genetic variation among drought tolerance genotypes. Consequently, tissue culture technology has emerged as an alternative and effective method for developing stress-tolerant plants in recent years, as it can be achieved under controlled conditions in less time and space\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The effect of osmotic stress on plant growth using tissue culture technology is similar to its effect under field conditions; therefore, \u003cem\u003ein vitro\u003c/em\u003e-grown plants can be used as an alternative to stressful and costly field trials \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDrought is a major abiotic stress that negatively impacts the performance and productivity of various plants by inhibiting photosynthesis. Drought causes stomata to close, reducing water loss through transpiration and maintaining the hydration of plant cells. However, this is usually accompanied by a decrease in the diffusion rate of carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e), which is the precursor to sugar synthesis in the Calvin cycle \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Potatoes adapt to stress conditions by enhancing their enzymatic and non-enzymatic antioxidant activities. They synthesize protective substances, such as proline and polyols, to counteract the damaging effects of drought \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These substances contribute to changes in cellular osmotic pressure and ensure membrane integrity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Chlorophyll pigments are also important for plants because they harvest light energy necessary for the synthesis of energy-rich compounds during photochemical reactions (NADPH, ATP). Both chlorophyll a and chlorophyll b molecules are significantly damaged when plants are exposed to drought, as the ratio of chlorophyll a to chlorophyll b changes \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Manoj et al. (2011) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, proved that tissue culture is a practical technique for producing stress-tolerant plants, both biotic and abiotic, by applying selection agents such as NaCl (for salinity tolerance) and PEG, mannitol, or sorbitol (for osmotic stress tolerance) to the culture medium. Only plants capable of surviving in such media are selected.\u003c/p\u003e \u003cp\u003eIn a study conducted by Sobh (2020)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e to determine the effect of varying levels of sorbitol-induced osmotic stress on certain morphological traits in eleven potato varieties, using plant tissue culture techniques, increasing the osmotic stress level (sorbitol concentration) in the growth medium resulted in a significant decline in all morphological traits compared to the stress-free control, This included a decrease in the concentration of chlorophyll a and b molecules and total carotenoids, and an increase in proline and oxygenated water. Mehmandar et al. (2023)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, also demonstrated that \u003cem\u003ein vitro\u003c/em\u003e assessment can be a precise, rapid, and reliable method for evaluating and identifying drought-tolerant genotypes.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eGrowth parameters under the influence of osmotic stress\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e:\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant height (cm):\u003c/h2\u003e\n \u003cp\u003eThe results of the statistical analysis showed significant differences in the average plant height among the varieties and different levels of sorbitol. The average plant height decreased with increasing sorbitol concentration for each variety. The highest plant length was observed in the control treatment (13.01 cm), with a significant difference compared to the other treatments in all studied varieties. The lowest significant value was at the concentration of 400 mM (1.71 cm). The longest plant height was recorded for the Evora variety (8.67 cm), while the shortest was for the Everest variety (4.52 cm), (Figs.\u0026nbsp;1\u0026ndash;2).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eNumber of leaves:\u003c/h3\u003e\n\u003cp\u003eThe results of the statistical analysis showed significant differences in the average number of leaves between the different varieties and levels of sorbitol. The average number of leaves per plant decreased directly and significantly with increasing sorbitol concentration in the growth medium. The highest number of leaves was observed in the control treatment (11.5 leaves/plant) with a significant difference compared to the other treatments, while the lowest number of leaves was observed in the sorbitol 400 mM treatment (1.82 leaves/plant). The highest number of leaves was observed in the Evora variety (8.87 leaves/plant), and the lowest in the Everest variety (4.30 leaves/plant). (Fig.\u0026nbsp;1\u0026ndash;2).\u003c/p\u003e\n\u003ch3\u003eRoot Length:\u003c/h3\u003e\n\u003cp\u003eStatistical analysis revealed significant differences in average root length between different varieties and sorbitol levels. Average root length decreased proportionally and significantly with increasing sorbitol concentration in the growth medium. The control treatment exhibited a significantly higher average root length (5.85 cm) compared to the other treatments, while the shortest root length was observed in the 400 mM treatment (0.57 cm). The highest average root length was observed in the Salvador variety (4.52 cm), and the shortest in the Arizona variety (2.29 cm), (Fig.\u0026nbsp;1\u0026ndash;2).\u003c/p\u003e\n\u003ch3\u003eNumber of Roots:\u003c/h3\u003e\n\u003cp\u003eStatistical analysis revealed significant differences in the average number of roots between different varieties and sorbitol levels. The average number of roots per plant decreased proportionally and significantly with increasing sorbitol concentration in the growth medium. The highest average number of roots was observed in the control treatment (8.39 roots/plant), significantly higher than the other treatments, while the lowest was in the 400 mM treatment (0.6 roots/plant). The highest average number of roots was observed in the Hind and Evora varieties (6.03 and 5.9 roots/plant, respectively), while the lowest was observed in the Arizona variety (2.69 roots/plant), (Fig. 1\u0026ndash;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure (1)\u003c/strong\u003e Effect of osmotic stress coefficients on growth parameters of the studied potato varieties\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure (2)\u003c/strong\u003e The effect of osmotic stress on growth indicators in nine studied potato varieties\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure (3\u003c/strong\u003e). The effect of different concentrations of sorbitol on growth indicators in the studied potato varieties: A- Salvador, B- Yalas, C- Arizona, D- Hind, E- Everest,F- Evora, G- Montereal, H- Agria, I- Synergy.\u003c/p\u003e\n\u003ch3\u003eBiochemical Indicators:\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eLeaf Chlorophyll a content (mg. g⁻\u0026sup1; wet weight):\u003c/h2\u003e\n \u003cp\u003eStatistical analysis revealed significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) in the mean leaf chlorophyll a content. The average leaf content of chlorophyll a was significantly higher in thecontrol (0.76 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، and it decreased proportionally with increasing sorbitol concentration in the growth medium. The leaf content of chlorophyll a was significantly lower at the highest osmotic concentration of 400 mM (0.19 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight) Figure (4). The highest average leaf content of chlorophyll a was found in the Salvador variety (0.67 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، while it was significantly lowest in the Everst variety (0.24 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، Figure (5).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eLeaf Chlorophyll b Content (mg. g wet weight):\u003c/h3\u003e\n\u003cp\u003eStatistical analysis revealed significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) in the average leaf content of chlorophyll b. The average leaf content of chlorophyll b was significantly higher in the control treatmen (0.62 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، and it decreased proportionally with the increase in sorbitol concentration concentration in the growth medium. The leaf content of chlorophyll b was significantly lower at the highest osmotic concentration of 400 mM (0.21 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight). The highest average leaf content of chlorophyll b was found in theSalvador variety (0.53 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، while the lowest was in the Synergy variety (0.27 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight) (Fig.\u0026nbsp;4\u0026ndash;5).\u003c/p\u003e\n\u003ch3\u003eLeaf Carotenoid Content (mg. g⁻\u0026sup1; wet weight):\u003c/h3\u003e\n\u003cp\u003eStatistical analysis revealed significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) in the average leaf content of carotenoids. The average leaf content of carotenoids was significantly higher in the control treatmen (0.24 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight)، and it decreased proportionally with increasing sorbitol concentration in the growth medium. The leaf content of carotenoids was significantly lower at the highest osmotic concentration of 400 mM (0.13 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight). The highest average leaf content of carotenoids was significantly higher in the Arizona variety (0.25 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight), while the lowest was in the Salvador variety (0.13 mg. g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wet weight), (Fig.\u0026nbsp;4\u0026ndash;5).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eLeaf proline content (\u0026micro;g. g⁻\u0026sup1; green matter):\u003c/h2\u003e\n \u003cp\u003eStatistical analysis revealed significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) in the mean leaf proline content. The mean leaf proline content was significantly highest at the highest osmotic concentration of 400 mM (58.67 \u0026micro;g. g⁻\u0026sup1; green matter), while the mean leaf proline content was significantly lowest in the control treatment (34.11 \u0026micro;g. g⁻\u0026sup1; green matter). The highest mean leaf proline content was found in the Arizona cultivar (53.93 \u0026micro;g. g⁻\u0026sup1; green matter), and the lowest in the Everest cultivar (34.82 \u0026micro;g. g⁻\u0026sup1; green matter), (Fig. 4\u0026ndash;5).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure (4)\u003c/strong\u003e Effect of osmotic stress coefficients on biochemical indicators in the studied potato varieties\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure (5)\u003c/strong\u003e Effect of osmotic stress on biochemical indicators in nine studied potato varieties\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eOsmotic Stress Levels and Morphological Changes:\u003c/h2\u003e\n \u003cp\u003eOsmotic stress levels showed significant changes in some morphological plant growth indicators, significantly reducing plant height, number of leaves, and root length and number. This reduction was directly proportional to increasing sorbitol concentration. Some studies have attributed the decrease in these indicators to osmotic stress, which inhibits root elongation and number due to high osmotic stress in the growing medium. This leads to a decrease in water potential (becoming more negative), thus reducing the amount of free water available to the plant. Consequently, the rate of water uptake by the root system is negatively affected, and the amount of water absorbed becomes insufficient to compensate for the water lost through transpiration by the aerial parts. As a result, plant elongation and cell division are inhibited, affecting plant diameter and reducing the number of nodes and leaves formed on the stem. This results from a decrease in the filling potential within leaf cells, which inhibits leaf cell elongation and growth, leading to water deficit in plant cells due to reduced water content \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThese results are consistent with those of Wishart et al. (2014)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and Bundig et al. (2016)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, who found genetic variation in response to drought conditions, Potato varieties depend on some morphological characteristics, such as root length and aerial parts. Similarly, the study by Sajid (2022)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e showed a decrease in most growth indicators when treated with sorbitol and mannitol as osmotic stress factors for two potato varieties.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eBiochemical Indicators:\u003c/h2\u003e\n \u003cp\u003eThe variation in the leaf content of chlorophyll a and chlorophyll b molecules among the studied varieties is attributed to the genetic factors responsible for the stability of chlorophyll molecules. All the genetic varieties in which the leaf content of chlorophyll a molecule was significantly higher also had a significantly higher leaf content of chlorophyll b the leaf content of chlorophyll b. Similarly, varieties that had significantly lower chlorophyll a also had lower chlorophyll b content. Osmotic stress affected the biochemical processes of potato varieties, leading to a decrease in the chlorophyll and carotenoids content of the leaves. Osmotic stress contributes to a decline in the level of gene expression of genes responsible for proteins associated with chlorophyll molecules (Chlorophyll a-b), and increases the activity of the enzyme Chlorophyllase, which breaks down chlorophyll molecules. This leads to a decrease in the chlorophyll content of the leaves and also affects the basic enzymes in the photosynthesis process and the enzymes responsible for sucrose synthesis. Dehydration increases the level of gene expression of the enzymes responsible for breaking down sucrose \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eOur results are consistent with those of Mehmandar et al. (2023)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, where the addition of sorbitol reduced the chlorophyll content while increasing the proline and malondialdehyde content. The addition of sorbitol to MS medium mimics dehydration stress better than polyethylene glycol.\u003c/p\u003e\n \u003cp\u003eThe decrease in carotenoids under stress conditions is attributed to the breakdown of beta-carotene and the formation of zeaxanthins, which play a role in protecting against photoinhibition. These findings align with those of Farooq et al. (2009)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, who indicated that carotenoids (orange pigments) regulate the uptake of light energy by chlorophyll molecules to prevent photoinhibition when light utilization efficiency is reduced in stressful environments.\u003c/p\u003e\n \u003cp\u003eAdditionally, crops produce proline under drought stress conditions, which protects drought-sensitive cell enclosures from damage \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These substances play a key role in stress tolerance \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These findings are consistent with those of Wellpott et al. (2024)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, where potato genotypes showed genetic variation in the rate of synthesis and accumulation of compatible organic solutes, which explains the genetic variation in drought tolerance.\u003c/p\u003e\n \u003cp\u003eAl-Hadid et al. (2023) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, showed that proline concentration increased with increasing salt stress compared to the control for both C3 and C4 plants.\u003c/p\u003e\n \u003cp\u003eProline accumulation under abiotic stress conditions in many plant species is associated with enhanced stress tolerance. Its concentrations are typically higher in stress-tolerant plants than in sensitive ones. However, stress-sensitive genotypes can sometimes synthesize larger quantities of proline, in which case proline accumulation is related to the extent of damage rather than the level of tolerance \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Thus, we observe that sorbitol led to a decrease in leaf chlorophyll and carotenoid content and an increase in proline content with increasing osmotic stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of Agglomerative Hierarchical Clustering (AHC):\u003c/h2\u003e\n \u003cp\u003eThe cluster analysis, based on the sum of the relative values of the studied parameters (growth indicators and biochemical parameters), led to the division of the studied potato varieties, according to their tolerance to the sum of osmotic stress levels, into three groups, (Fig. 6):\u003c/p\u003e\n \u003cp\u003e-Group 1 (G1): Plants tolerant to osmotic stress, including the cultivars Yalas and Salvador.\u003c/p\u003e\n \u003cp\u003e-Group 2 (G2): Plants with moderate tolerance to osmotic stress, including the cultivars Arizona, Hind, and Synergy.\u003c/p\u003e\n \u003cp\u003e-Group 3 (G3): Plants intolerant to osmotic stress, including the cultivars Agria, Everest, Monteral, and Evora.\u003c/p\u003e\n \u003cp\u003eSince tolerance to abiotic stresses is a complex quantitative trait \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and there is a significant interaction between the environment and specific genetic factors associated with economic yield, many studies have focused on studying genetic diversity in potatoes and determining the extent of tolerance to abiotic stresses. These studies often use cluster analysis, which relies on various indicators such as morphological and physiological traits \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure (6)\u0026nbsp;\u003c/strong\u003eCluster analysis of nine potato varieties according to their tolerance to osmotic stress, based on the sum of the relative values of all the studied indicators (growth indicators, chemical parameters).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMany scientific studies indicate the importance of tissue culture technology as an alternative and effective method for developing stress-tolerant plants. This technology can be implemented under controlled conditions in less time and space. Furthermore, the effect of osmotic stress on plant growth using tissue culture technology is similar to its effect under field conditions. Therefore, this research aimed to investigate the effect of osmotic stress on certain morphological characteristics and chemical parameters, and to select varieties tolerant to osmotic stress.\u003c/p\u003e \u003cp\u003eThis research is significant in its screening and evaluation of the tolerance of potato (\u003cem\u003eSolanum tuberosum L.\u003c/em\u003e) varieties to osmotic stress \u003cem\u003ein vitro\u003c/em\u003e. The results showed a decrease in most growth indicators and some biochemical properties (chlorophyll molecules of both types (a,b), and carotenoids), and an increase in some biochemical properties (proline) compared to the control when treated with sorbitol as an osmotic stress-causing agent. The nine varieties also varied in their response to osmotic stress, as cluster analysis showed the distribution of the studied varieties into three groups based on their tolerance to osmotic stress. The results showed that the two varieties Yalas, Salvador are considered tolerant varieties, while the varieties Arizona, Hind, Synergy are considered moderately tolerant varieties, and the varieties Agria, Everest Monteral, Evora are considered sensitive varieties to osmotic stress.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlace and Time of Research\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried out in the laboratories of the Faculty of Science, Damascus University and the laboratories of the National Commission for Biotechnology in Damascus, during the period (2024\u0026ndash;2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant material\u003c/strong\u003e: Nine varieties of commercial starchy potatoes, preferred by farmers were used in this research: (Montereal, Salvador, Synergy, Yalas, Agria, Arizona, Everest, Evora, Hind), These varieties were recently approved in Syria and were obtained from the Seed Multiplication Foundation in Damascus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Culture and Propagation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRosettes taken from tubers were cultured in plastic pots containing sterile peat moss. After 45 days, the resulting shoots were collected and cut into 2\u0026ndash;3 cm single cuttings, each containing one lateral bud. These cuttings were soaked in a 3% sodium hypochlorite solution for 10 minutes as recommended by previous studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and then washed three times consecutively with sterile distilled water for 5 minutes each time. After disinfection, the cuttings were cultured in test tubes containing 18 ml of MS medium \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, as shown in table 1, supplemented with 30 g. L⁻\u0026sup1; sucrose and 7 g. L⁻\u0026sup1; agar at pH 5.8. The cultured tubes were incubated in a growth chamber at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2C\u0026deg;, with a light cycle of 16 hours light /8 hours of darkness, and a light intensity of 3000 lux \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Following this, the plant propagation stage began. This stage aims to obtain the largest possible number of well-developed vegetative growths. The propagation process was repeated several times consecutively to obtain the sufficient quantity of plant material for the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;(1)\u003c/strong\u003e Composition of the nutrient medium for Murashige and Skoog (MS).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003cp\u003e(mg.l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChemical Composition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmmonium Nitrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotassium Nitrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCalcium Chloride Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMagnesium Sulfate Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotassium Phosphate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIron Sulfate Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eEDTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSodium Chelate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eManganese Sulfate Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZinc Sulfate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBoric Acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotassium Iodide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmmonium Molybdate Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCupressus Sulfate Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCobalt Chloride Hydrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eThiamin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMyo-inositol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSucrose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eStress treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant samples from the propagation stage were divided into small cuttings containing a lateral bud, and drought stress was applied using different concentrations of sorbitol (0, 100, 200, 300, and 400 mM) added to the growth medium. Each treatment consisted of three replicates, with an average of four plants per replicate, compared to the control (sorbitol-free medium).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe studied indicators\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results were recorded after 45 days of applying different stress treatments to laboratory plants. The following indicators were estimated\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eGrowth Parameters\u003c/h2\u003e\n \u003cp\u003e\u0026bull; Stem length (cm): The stem length was measured using a measuring tape, from the base of the stem to the apical bud.\u003c/p\u003e\n \u003cp\u003e\u0026bull; Number of leaves (leaves/plant).\u003c/p\u003e\n \u003cp\u003e\u0026bull; Root length (cm): Measured using a measuring tape, from the base of the root to the tip of the longest root.\u003c/p\u003e\n \u003cp\u003e\u0026bull; Number of roots (roots/plant) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eBiochemical indicators:\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003eEstimation of leaf content of chlorophyll and total carotenoids:\u003c/h2\u003e\n \u003cp\u003ePigments were extracted by grinding 1 g of frozen plant leaves treated with sorbitol, in the presence of 4 ml of 80% acetone. The extract was then separated using a centrifuge at 10,000 rpm for 10 minutes. The supernatant phase was transferred to new test tubes, and the absorbance was measured using a spectrophotometer at wavelengths of 645 and 662 nm to estimate the amounts of chlorophyll a and b, respectively, and at a wavelength of 470 nm to estimate the total carotenoids \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003eEstimation of the leaf proline content\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eProline was extracted by crushing 0.5 g of plant leaves after 45 days of exposure to osmotic stress in the presence of 2 ml of a 3% aqueous solution of sulfosalicylic acid. The extract was then separated by centrifugation at 10,000 rpm for 10 minutes, and its volume was adjusted to 5 ml using 3% sulfosalicylic acid. To estimate the proline content of the extract, 2 ml were taken and 2 ml of ninhydrin solution were added to activate the reaction, along with 2 ml of glacial acetic acid. The tubes were placed in a boiling water bath for 1 hour, then removed and rapidly cooled, 4 ml of toluene was added to each tube, and the tubes were shaken and allowed to separate into two phases. The upper phase was taken, and its optical absorption was measured at a wavelength of 520 nm, according to the concentration of proline in the plant sample compared to a standard curve of known concentrations of commercial proline\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental design and statistical analysis:\u003c/h2\u003e\n \u003cp\u003eThe experiment was designed using a completely randomized block design, with measurements taken after 30\u0026ndash;45 days of applying the stress treatments. Each treatment including the control, had three replicates, with each replicate consisting of 4 plants in test tubes. The results were analyzed using the statistical program XLSTAT to compare the means and calculate the least significant difference (LSD) at a significance level of 1%, along with the coefficient of variation (CV%).\u003c/p\u003e\n \u003cp\u003eThe Agglomerative Hierarchical Clustering (AHC) method, which classifies variable data into several subgroups such that they are homogeneous within one group (cluster) and different with respect to other clusters, was used to determine the tolerance of varieties to osmotic stress.\u003c/p\u003e\n \u003cp\u003eThe cluster analysis followed the equation of Vreugdenhil et al. (2007)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and Al-Biski (2018) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e based on the sum of the relative values of the studied parameters, the control and stress coefficients were determined as follows:\u003c/p\u003e\n \u003cp\u003e𝑅𝑉 plant status=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sum\\:\\frac{(\\text{S}\\text{p}1\\to\\:\\text{p}8\\text{*}100)}{\\text{C}\\text{p}1\\to\\:\\text{p}8}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eWhere 𝑅𝑉 \u003csub\u003eplant status\u003c/sub\u003e: is the sum of the relative values specific to the variety, 𝑆𝑝1\u0026rarr;𝑝8 is the value of the studied indicator in the stressed plant, 𝐶𝑝1\u0026rarr;𝑝8 is the value of the indicator in the control plant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003cp\u003eData availability\u003c/p\u003e\n \u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003eLama Laila, PhD in Plant Biology. Email;\u003c/p\u003e\n \u003cp\[email protected] [email protected] )\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNCBT\u003cstrong\u003e-\u0026nbsp;\u003c/strong\u003eNational Commission for Biotechnology; MS- Murashige and Skoog medium;AHC- Agglomerative Hierarchical Clustering\u003cstrong\u003e;\u003c/strong\u003e NaCl- Sodium \u0026nbsp;hypo Chloride;PEG\u003cstrong\u003e-\u0026nbsp;\u003c/strong\u003ePoly Ethylene glycol.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eL. laila, S. Zaid, F. Al-Biski \u0026amp; S. Jabal declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL. laila, PhD in Plant Biology. Department of plant Biology, Faculty of Sciences , Damascus University, Syria , was carried out this research in the field and at the laboratory of plant biotechnology and the wrote of this scientific research was done by the help of supervisors Dr. S. Zaid, Dr. F. Al-Biski \u0026amp;amp; Dr. S. Jabal . In addition, designed the experiment, analyzed the data, drafted and improved the manuscript were carried out by all of us.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors express their gratitude to thank staff in Plant Biology Department, Faculty of Sciences, Damascus University, and in Biotechnology Department, the National Commission for Biotechnology. Syria.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBasera, M., Chandra, A., Kumar, V. A., \u0026amp; Kumar, A. (Effect of brassinosteroids on \u003cem\u003ein vitro\u003c/em\u003e proliferation and vegetative growth of potato. \u003cem\u003ePharma Innovation J\u003c/em\u003e, \u003cstrong\u003e7\u003c/strong\u003e(4), 4-9, (2018).\u003c/li\u003e\n \u003cli\u003eBamberg, J. B., Martin, M. W., Abad, J., Jenderek, M. M., Tanner, J., Donnelly, D. J., \u0026amp; Novy, R. G. In vitro technology at the US Potato Genebank. 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(1973).\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Vreugdenhil, D., Bradshaw, J., Gebhardt, C., Govers, F., Taylor, M., acKerron, D. \u0026amp; Ross, H. Water Availability and Potato Crop Performance, \u003cem\u003ePotato Biology and Biotechnology.\u003c/em\u003e Advances and Perspectives, Elsevier, Amsterdam. Pp: 333-351. (2007). \u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Potatoes, osmotic stress, growth parameters, proline, cluster analysis","lastPublishedDoi":"10.21203/rs.3.rs-8641040/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8641040/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe experiments were conducted in the tissue culture laboratory of the National Commission for Biotechnology (NCBT) in Damascus, and at the Faculty of Science - University of Damascus with the aim of evaluating the response of nine introduced potato varieties resulting from \u003cem\u003ein vitro\u003c/em\u003e tissue culture (Montereal, Salvador, Synergy, Yalas, Agria, Arizona, Everest, Evora, Hind) to artificial osmotic stress using sorbitol. This was based on some growth indicators and certain biochemical parameters (chlorophyll a and b, total carotenoids, and proline), to determine the cultivars most adapted to osmotic stress. The plants were treated by adding different concentrations of sorbitol (0, 100, 200, 300, 400 mM) to the growth medium.\u003c/p\u003e \u003cp\u003eThe experiment was designed according to a completely randomized block design, and significant differences were estimated at a 99% confidence level. The nine varieties varied in their response to osmotic stress, with an increase in the level of osmotic stress (sorbitol concentration) in the growth medium causing a significant decline in all growth parameters (plant length, root length, number of leaves and roots) compared to the control, as well as some biochemical traits (chlorophyll molecules types a and b, and carotenoids), and an increase in some biochemical parameters (proline), compared to the control. Cluster analysis based on the total relative values of growth parameters and biochemical parameters showed the distribution of the studied varieties into three groups depending on their tolerance to osmotic stress. The results indicated that the varieties (Yalas and Salvador) are among the tolerant varieties, while the varieties (Arizona, Hind, Synergy) are moderately tolerant, and the varieties (Agria, Everest, Monteral, Evora) are considered sensitive to osmotic stress. The results indicate the possibility of evaluation and selection in the laboratory as a rapid and effective method to explore the genetic variation for osmotic stress tolerance in potatoes.\u003c/p\u003e","manuscriptTitle":"Evaluation and selection of potato varieties (Solanum tuberosum L.) in vitro and study of their tolerance to osmotic stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 15:28:11","doi":"10.21203/rs.3.rs-8641040/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9524310e-84ef-44b9-9b90-57d5405b7cf6","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-06T05:37:57+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62559438,"name":"Biological sciences/Physiology"},{"id":62559439,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-06T05:54:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 15:28:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8641040","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8641040","identity":"rs-8641040","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}