Humic Acids Mitigate Salt and Drought Stress in Soybean (Glycine max (L.) Merr.) in vitro Cultures

Humic Acids Mitigate Salt and Drought Stress in Soybean (Glycine max (L.) Merr.) in vitro Cultures | 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 Research Article Humic Acids Mitigate Salt and Drought Stress in Soybean (Glycine max (L.) Merr.) in vitro Cultures Danuta Kulpa, Renata Matuszak-Slamani, Małgorzata Włodarczyk, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7352179/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract The aim of this study was to evaluate the protective effect of humic acids (HA) with different molecular weight fractions on the soybean Progres cultivar under drought and salinity stress in in vitro . HA were isolated from peat samples according to the International Humic Substances Society procedure. Three HA treatments were tested: HA 30kDa, and unfractionated HA (Mix). Sterilized soybean seeds were cultured on nutrient media supplemented with 100 mmol·dm⁻³ NaCl or 150 mmol·dm⁻³ Mannitol to simulate salinity and drought stress, respectively. HA fractions were added at 0.005 g C HA ·dm⁻³. No stress factors were used in the control samples. Biometric parameters (plant height, leaf number, root length, shoot and root biomass) and micromorphological traits (stomatal density and length) were measured. Micro- and macroelement contents in dry seedling matter were also analyzed. Soybean Progres exhibited greater sensitivity to salt than drought stress, shown by reduced biometric and micromorphological parameters and altered element contents. HA treatments demonstrated a protective role, which was dependent on the molecular fraction. The HA > 30kDa fraction and HA Mix provided the greatest protective and, at times, stimulatory effects, notably increasing stomatal density and biometric values under stress. HA, in the presence of salt and drought stress, did not specifically affect the uptake of the analysed micro- and macroelements by soybeans. The fractions HA 30kDa caused a decrease in the uptake of most analyzed elements. Unfractionated HA predominantly mitigated the effects of applied stresses. For HA Mix, the levels of micro- and macroelements in soybean seedlings were generally comparable to those in control plants. abiotic stress NaCl Mannitol humic acids soybean growth nutrients Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Traditional agriculture is at the forefront of climate change, being particularly affected by the increasing intensity and frequency of extreme events, such as floods, droughts, and rising temperatures (Canellas et al., 2024). Throughout their life cycle, plants are exposed to various stresses, including both biotic and abiotic factors. Salinity and drought are among the most significant abiotic stressors affecting plant growth and development. These stressors trigger morphological, biochemical, and molecular changes that severely limit crop production worldwide (Liu et al. 2017; Sahin et al. 2018; Bai et al. 2019; Singha et al. 2024; Akbari et al. 2024). Early responses to drought and salinity are similar, as both induce water stress that leads to reduced growth, decreased stomatal aperture, and nutrient deficiencies (such as K⁺ and Ca²⁺). However, long-term salt stress, in addition to causing dehydration, also leads to ionic stress, which contributes to leaf senescence and impaired photosynthesis (Feng et al. 2020; Ma et al. 2020; Cao et al. 2023). Soybean ( Glycine max (L.) Merr.) is one of the most essential leguminous crops, widely cultivated across diverse climates. It plays a pivotal role in global nutrition and food security, serving as a major source of both edible oil and plant-derived protein for humans and livestock. Globally, approximately 70% of plant-based proteins and 29% of edible oil are derived from soybeans (Hossain et al. 2024). In addition to its nutritional value, soybeans hold significant economic importance, providing raw materials for the animal feed and food industries. In recent years, soybeans have attracted increasing attention due to their high protein content, vitamin C, rich mineral composition, and reported anti-cancer properties (Cao et al. 2017; Feng et al. 2020). Moreover, soybean oil is considered a promising future biofuel source, with ongoing efforts to enhance soybean-derived biodiesel production (Santos et al. 2024). Under natural conditions, soybeans are exposed to various abiotic stress factors, including salinity and water deficiency. To achieve high yields, soybeans require an adequate water supply throughout their growth cycle (Buezo et al. 2019; Dong et al. 2019; Feng et al. 2020). Studies have demonstrated that both water deficiency and salinity stress negatively affect soybean growth and development (Matuszak‑Slamani et al. 2017; Bai et al. 2019; Khattab et al. 2019; Matuszak‑Slamani et al. 2022). Soybeans are moderately salt-tolerant and can be cultivated in light to moderately saline soils; however, their yields decline under salinity stress (Anwar-ul-Haq et al. 2023; Nasution et al. 2024). To mitigate the negative impacts of abiotic stresses, various agronomic and biological strategies are employed. One example is the use of rhizosphere microorganisms, such as plant growth-promoting rhizobacteria, which enhance nutrient uptake, reduce the toxic effects of salts, and support root development under challenging conditions (Khan et al. 2021; Kang et al. 2023; Handayani et al. 2024). Another approach involves appropriate soil management practices, such as mulching and balanced fertilization, which improve moisture conditions and alleviate stress caused by soil salinity (Jabeen et al. 2021). One of the most effective solutions is the application of biostimulants, which enhance plant physiological responses by strengthening their adaptive capabilities under stressful conditions. For instance, salicylic acid and algal extracts have been shown to significantly improve soybean drought tolerance by supporting antioxidant mechanisms (Ren et al. 2023). Humic substances (HS) are among the most active components of soil organic matter. As polymeric substances with complex structures, they are formed through the condensation of biomass from microorganisms, plants, and animals. HS influence on the physical, chemical, and biological properties of soil by improving aggregate stability, buffering capacity, sorption of hydrophobic organic compounds, nutrient transport and bioavailability, as well as environmental metal complexation. In addition, they enhance the soil’s ion exchange capacity and water retention (Ouni et al. 2014). According to their solubility in water at various pH, HS can be divided into three components: fulvic acids (FA, soluble at all pH values), humic acids (HA, soluble in alkaline media and insoluble at pH 1.0), and humin (HN, insoluble at all pH values) (Ukalska-Jaruga et al. 2021; Nardi et al. 2021; Ore et al. 2023; Canellas et al. 2024). The effect of HS on plant growth depends on several factors, including their origin, concentration, molecular weight, application method, plant species, and developmental stage (Nardi et al. 2002; Nardi et al. 2021). Humic acids (HA) are considered a potential strategy to mitigate the harmful effects of abiotic stress. They can be applied as plant growth stimulants and soil conditioners, and they can also reduce irrigation frequency, improve water use efficiency, and mitigate the effects of drought stress on plants (Olk et al. 2018; Franzoni et al. 2022; Matuszak‑Slamani et al. 2022; Rakkammal et al. 2023; Canellas et al. 2024; Maffia et al. 2025). Due to the complexity of HS structures and their certain fractions, it is important to identify which of them play a key role in plant growth and development under abiotic stress conditions, as the mechanisms of HS action are complex and require further research. Therefore, the aim of this study was to evaluate the protective effect of humic acids and their molecular fractions on the growth of soybean seedlings in in vitro cultures under laboratory-induced salinity and drought stress conditions. Material and methods Extraction and fractionation of humic substances The starting material for obtaining humic acids fractions was peat soils in the active accumulation stage of organic matter, located in Babia Góra National Park, Poland GPS: N 49°35′45′–E 19°30′21′′. Peat samples were collected from the subsurface layer up to a depth of 50 cm. The air-dried peat samples, with a moisture content not exceeding 12%, were homogenized after removing undecomposed plant residues. Humic acids were extracted using the method recommended by the International Humic Substances Society (Swift 1996). The main steps of HA extraction includes: decalcification of peat samples using HCl, extraction with 0.1 mol·dm⁻³ NaOH, precipitation of HA with 6 mol·dm⁻³ HCl, and purification of HA through deashing with an HCl/HF mixture, followed by washing with distilled water until no chlorides were detected. The purified HA preparations were frozen and lyophilized to achieve a constant mass. The fractionation of HA into molecular weight fractions was carried out using an Amicon model 8400 apparatus equipped with Millipore filters with a 30kDa molecular weight cut-off. The device comprises three main modules (Fig. 1 ). Module 1 supplies nitrogen under controlled pressure, which drives the separation of the polymer into distinct molecular fractions. Module 2 is a container equipped with a magnetic stirrer, where the solution to be fractionated is placed. Module 3 contains the Millipore membrane mount, and a nozzle connected to a hose that directs the filtered solution to an external collection container. Millipore membranes can only be used with solutions within a pH range of 2 to 10. Therefore, appropriate amounts of humic acids were first dissolved in 0.1 mol·dm⁻³ NaOH and then diluted fivefold. The solution was then passed through the Amberlite H⁺120 ion-exchange resin to replace Na⁺ ions with H⁺ ions. The resulting acidic solution was transferred to the container (Module 2) equipped with a magnetic stirrer. Module 3, containing a Millipore filter with a 30kDa cut-off, was mounted at the bottom. Module 1, supplying nitrogen, was positioned above Module 2 (the container holding the sample to be separated). The filtrate containing the fraction with a molecular weight below 30kDa (HA < 30kDa) was collected in a separate vessel and concentrated at a temperature not exceeding + 40°C. The concentrated solution was then frozen using a dry ice–alcohol mixture at − 70°C and lyophilized to constant weight. The supernatant containing the fraction with a molecular weight > 30kDa (HA > 30kDa) was similarly frozen using a dry ice–alcohol mixture at − 70°C and lyophilized to constant weight. The resulting HA preparation - HA > 30kDa and HA < 30kDa fractions, were dissolved in Michaelis buffer at pH 7.17. The unfractionated humic acids solution was referred to as HA Mix. Chemical and spectral analyses of the molecular HA fractions and the unfractionated preparation were presented in our previous studies: Gawlik et al. (2016), Matuszak-Slamani et al. (2017, 2022). In vitro cultures condition Soybean seedlings of the Progres cultivar were analyzed under laboratory-induced stress conditions to evaluate the effects of salinity and drought, as well as the protective role of humic acids against these abiotic stresses. Soybean seeds of the Progres cultivar used in the in vitro studies were sterilized prior to the experiment. They were immersed in 70% ethanol for 10 seconds and then treated with 0.2% mercuric chloride (HgCl 2 ) for 12 minutes. To prevent contamination, all subsequent procedures were carried out under sterile conditions in a laminar airflow chamber (Caetano-Anolléset al. 1990). The Murashige and Skoog (1962) medium was used in the experiment. The pH of the medium was adjusted to 5.7 using 0.1 mol·dm⁻³ solutions of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The media were supplemented with 8 g·dm⁻³ agar, 30 g·dm⁻³ sucrose, and 100 mg·dm⁻³ inositol. After the addition of the dry components, the media were heated to allow polymerization and solidification, poured into 900 cm 3 jars, and sterilized in an autoclave at 121°C for 20 minutes. In the first stage of the in vitro study, the effects of salinity and drought on soybean growth and development were assessed. For this purpose, sterilized soybean seeds were placed on a nutrient medium, to which 100 mmol·dm⁻³ NaCl and 150 mmol·dm⁻³ mannitol were added to simulate salinity and drought stress, respectively. Simultaneously, the protective effect of humic acids (HA) on soybean growth and development under these abiotic stress conditions was investigated. Molecular fractions of HA were added to the nutrient medium containing the appropriate stress factor (NaCl or mannitol): humic acids with molecular weights less than 30kDa (HA 30kDa), or unfractionated humic acids (HA Mix). For all tested molecular fractions, the concentration of HA in the nutrient medium was identical, amounting to 0.005 g C HA ·dm⁻³. The control group consisted of sterilized soybean seeds of the Progres cultivar placed on Murashige and Skoog nutrient medium without stress factors. Each experimental variant for each stress condition consisted of control ( 1 ) and four treatments: stress ( 2 ), stress + HA 30kDa ( 4 ), and stress + HA Mix ( 5 ). All tested stresses and their combinations were applied simultaneously under controlled in vitro culture conditions. Ten jars were prepared for each combination, with ten seeds placed in each jar, resulting in a total of 100 seeds per combination (Fig. 2 ). The experiment was conducted in a phytotron at 25 ± 1°C, under a light intensity of 40 µmol·m⁻²·s⁻¹ and a 16-hour photoperiod. After 21 days of soybean growth, biometric parameters, micromorphological features, and the content of micro- and macroelements in the dry mass of soybean seedlings were measured. Biometric measurements Biometric measurements included plant height, number of leaves, root length, fresh weight, and the weight of the overground parts of soybean seedlings were conducted using an electronic caliper (Varel, accuracy ± 0.01 mm), an analytical balance (Radwag AS 82/220.R2 PLUS), and a moisture analyzer (Radwag MA 50.R). Micromorphological analysis Leaves isolated from the middle part of soybean stems grown on media enriched with NaCl, mannitol, and humic acids were subjected to micromorphological analysis using a scanning electron microscope (SEM). The material was dried using a Critical Point Dryer (Quorum Technologies) and then coated with a gold layer using a Sputter Coater (Quorum Technologies). Observations were conducted with a Carl Zeiss EVO LS 10 microscope at an accelerating voltage of 15 kV. Determined stomatal length (µm) and stomatal density (the number of stomata per mm²) on the surface of soybean leaf blades. Measurements were made on the abaxial (lower) surface of the soybean leaves. Micro- and macroelement analysis The content of five microelements (Cu, Mn, Zn, Fe, and Co) and four macroelements (Mg, Ca, K, and Na) in the dry matter of soybean seedlings of the Progres cultivar grown in vitro was determined. These elements were present in the basic medium (according to Murashige and Skoog, 1962). The soybean seedlings were dried to constant weight at 105°C, ground, and mineralized in a mixture of concentrated HNO 3 and HClO 4 acids in a 3:1 ratio. All determinations were performed in triplicate. Quantitative analysis was performed using the Solar S4 Atomic Absorption Spectrophotometer with an acetylene-air flame atomizer. Statistical analysis The experiment with plants in vitro cultures was conducted with 10 replications, each containing 10 plants (a total of 100 plants per experimental variant). Data for micro- and macrominerals are presented as the mean ± SD from three replicates. During the microorphological analysis, 20 photos of leaf blades were taken for each combination, determining the stomatal density/mm2. The length of 10 stomata randomly selected from each photo was determined, for a total of 200 for each combination. One-way analysis of variance (ANOVA) was used to compare mean values. The analysis formed the basis for identifying homogeneous groups using Tukey’s test at a significance level of p ≤ 0.05. Means that differed significantly were marked with different letters. The results are presented in graphs and tables separately for each stress factor (mannitol or NaCl). Statistical analysis was performed using Statistica 13.1 PL (StatSoft, Kraków, Poland). Results and Discussion Research on the influence of humic acids on plant development has so far focused mainly on field, pot, or hydroponic experiments. In contrast, conducting research in vitro cultures allows for better standardization and more precise assessment of the effects of the studied factors on changes in plant morphological traits. In our study, we assessed the protective effects of humic acids and their molecular fractions on the growth of soybean seedlings in in vitro cultures under laboratory-induced salinity and drought stress conditions. Growth in in vitro cultures. Salt stress, primarily induced by sodium chloride (NaCl), is one of the major abiotic factors affecting plant growth and development under both natural conditions and in vitro cultures (Parida and Das 2005). According to our experiment, the best growth parameters - longer shoots and roots, the highest shoot and root biomass, and the greatest number of leaves - were observed in soybean plants grown on control medium without stress factors (NaCl and mannitol). These differences were statistically significant for both stresses. Under salt stress, plant height decreased by 80%, root length by 65%, number of leaves by 79%, root weight by 51%, and above-ground biomass by 85% (Fig. 3 ). The results confirm that soybean is a salt-sensitive legume, and salinity impairs seed germination, seedling growth, and overall plant development (Rasheed et al. 2022). Under salt stress conditions, plants exhibit morphological changes such as reduced leaf size, delayed root growth, decreased biomass, and shortened shoot length. In in vitro cultures, salt stress can also hinder tissue regeneration processes, including somatic embryogenesis and rooting ability (Begum et al. 2022; Açıkbaş et al. 2023; Gobade et al. 2024). Salinity inhibits overall plant growth by reducing the osmotic potential of the growing medium, causing specific ion toxicity, inducing oxidative stress, and decreasing nutrient uptake (Parida and Das 2005; Liu et al. 2017; Atero-Calvo et al. 2024; Hossain et al. 2024). The effect of mannitol on agricultural plant development has been widely studied, mainly due to its ability to regulate water management (Khatoon et al. 2018). Mannitol is commonly used as an osmotic stress agent, limiting water availability, disrupting metabolic processes, and reducing the rates of photosynthesis, growth, and protein synthesis in plants (Adrees et al. 2015; Saadaoui et al. 2023). Under drought stress, plant height decreased by 24%, root length by 55%, number of leaves by 36%, root weight by 50%, and above-ground biomass by 43% (Fig. 4 ). These results are consistent with our previous studies (Matuszak-Slamani et al. 2022) concerning soybean, as well as with studies by Ahmad et al. (2022) and Abu-Ria et al. (2024), which focused on the growth of sorghum and maize under drought stress conditions. The decrease in the analyzed biometric parameters is mainly attributed to drought-induced disruptions in water absorption, which leads to increased cell dehydration and inhibits cell division, expansion, and proliferation (Wahab et al. 2022). Recent studies emphasize the key role of humic substances (HS), particularly humic acids (HA), in mitigating the adverse effects of abiotic stress and enhancing plant resilience (Nardi et al. 2021; Canellas et al. 2024; Nabi et al. 2025; Maffia et al. 2025). Our research demonstrated that under salt stress, it is possible to enhance plant tolerance using humic acids. This is confirmed by the increased values of the analyzed parameters in soybean seedlings, particularly in the aboveground parts (Fig. 3 ). Among the three HA combinations tested, the > 30 kDa fraction and the unfractionated preparation (Mix) had the most beneficial effects. Compared to the NaCl treatment alone, a significant increase was observed in plant height (58–156%), number of leaves (119–165%), and fresh mass of the overground parts (44–122%) (Fig. 3 ). For the root-related parameters, the protective effect of HA was less pronounced. Root mass and length increased by 16–39% and 14–30%, respectively, compared to plants subjected to salt stress alone (Fig. 4 ). The obtained results indicate that the addition of humic acids activates many components of the ion homeostasis machinery in the presence of NaCl ions, thus highlighting their protective effect (Khaleda et al. 2017; Souza et al. 2021; Canellas et al. 2024; Nabi et al. 2025). In the case of the second stress factor – drought, the protective effect of humic acids was less pronounced. However, similarly to salinity stress, their protective effect was most significant for the HA > 30 kDa fraction and the HA Mix. Under drought conditions, the addition of humic acids led to an average increase of 20–40% in the analyzed biometric parameters. An exception was observed for the HA Mix, where the number of leaves increased by 87% (Fig. 4 ). Research shows that humic acids (HA) significantly alleviate drought-related stress in various plant species (sorghum, maize, rice, and wheat) by enhancing growth characteristics and biomass accumulation in leaves, stems, and roots (Ahmad et al. 2022; Mutlu et al. 2022; Abu-Ria et al. 2024). Humic acids exhibit pleiotropic effects, influencing plant growth, nutrient uptake, and stress tolerance (Nardi et al. 2021; Matuszak-Slamani et al. 2022; Abu-Ria et al. 2024; Maffia et al. 2025). Nardi et al. (2021) concluded that humic acids can stimulate chlorophyll synthesis and the activity of enzymes responsible for regulating osmotic pressure in plant cells. Humic substances also support the reconstruction of cellular structures, resulting in improved plant growth under osmotic stress conditions. Micromorphological analysis. Stomata regulate the exchange of CO₂ and water vapor between the leaves and the atmosphere, serving as key indicators of a plant’s resilience to environmental changes. Stomatal density directly influences photosynthesis and transpiration processes (Sultana et al. 2024). Example photos of the leaf surface of control soybean plants grown in vitro cultures are shown in Fig. 7 . The average stomatal length determined for control plants was 118.72 µm, and the average stomatal density was 296.85 mm⁻². It was found that the analyzed stresses (salinity and drought) had a negative effect on stomata. Compared to the control of soybean plants, salinity stress significantly reduced stomatal length by 16%, while stomatal density decreased by 40%. In the case of drought, changes in the analyzed stomatal parameters also showed significant decreases, amounting to an 11% reduction in stomatal length and a 61% reduction in stomatal density (Fig. 5 , Fig. 6 ). Stomatal development adapts to environmental conditions throughout the plant growth cycle (Sultana et al. 2024), often by altering the density and size of stomata on the plant epidermis. Abiotic stresses, such as drought, decrease stomatal length and density (Han and Torii 2016; Lawson and Vialet-Chabrand 2019; Mohi-Ud-Din et al. 2025). Hughes et al. (2017) suggest that this is associated with the potential for reduced transpiration and improved water management by plants. The use of humic acids in combination with salinity stress has a positive effect on stomata. The most beneficial effect was observed for the HA > 30kDa fraction, where stomatal length increased significantly by 15%. Stomatal density increased by 144% compared to stress treatment and by 47% compared to control. For the other fractions, the changes were smaller but comparable to the control values (Fig. 6 ). In the case of drought stress, a significant increase in stomatal length ranging from 6–12% was observed only for two fractions: HA > 30 kDa and HA Mix, reaching values comparable to those of control plants. A significant effect of humic acids was also found in the case of stomatal density. This parameter increased markedly for all analyzed HA fractions, ranging from up to 182% (HA < 30 kDa) to 234% (HA Mix). The obtained values were also higher than those of the control plants from 11% (HA < 30 kDa, not statistically significant) to 31% (HA Mix, statistically significant) (Fig. 7 ). Soybean is an amphistomatic species, with a higher stomatal density on the abaxial side than on the adaxial side. Stomata are distributed randomly across the leaf surface (Amaliah et al. 2019; Sultana et al. 2024). Based on our results, it can be concluded that humic acids fractions, particularly HA > 30 kDa and HA Mix exhibit not only protective but also stimulatory effects, increasing stomatal density on soybean leaves exposed to stress conditions. Micro- and macroelement analysis. The effect of humic acids (HA) on element uptake by plants depends on multiple factors, primarily the origin, type, dosage, and structural properties of HA, as well as the pH of the rooting medium, the concentration of exogenous elements, and the plant species (Nardi et al. 2017). The mechanism of element uptake by plant roots under natural conditions is complex, and the additional influence of humic substances and environmental stresses (such as drought and salinity) makes it even more intricate and dependent on multiple factors. The research conducted in this study on the effects of salt stress (NaCl), drought stress (mannitol), and humic acids fractions on the content of macro- and microelements in the leaves of the soybean cultivar Progres indicates differences in their accumulation. The impact of salt stress on the uptake of microelements from the medium by soybean seedlings of the Progres cultivar grown in in vitro cultures was observed (Table 1 ). According to Sahasakul et al. (2023), microelements are generally less susceptible to salt stress than macroelements. In our study, a decrease in the content of Mn, Zn, Fe, and Co was observed compared to the control, ranging from 15.41% (Mn) to 4.02% (Fe). A significant increase in Cu uptake by soybean seedlings was noted, exceeding 140%. On the other hand, Celik et al. (2011) and Katkat et al. (2009) reported that the foliar application of humic acids had a statistically significant positive effect on the uptake of Cu, Zn, Mn, Mg, and Fe by wheat. Table 1 The content of microelements in soybean seedlings of Progres cultivar under salt stress conditions Combination Microelements ±SD [mg·kg − 1 DM] Cu Mn Zn Fe Co Control 16.57 ± 2.66 *a 69.65 ± 1.58 a 61.68 ± 2.01 a 128.50 ± 2.22 a 0.434 ± 0.025 a Salinity (NaCl) 40.02 ± 6.67 b 58.92 ± 1.06 b 54.57 ± 3.12 b 123.34 ± 0.82 b 0.408 ± 0.018 a NaCl + HA 30kDa 14.93 ± 1.69 a 56.43 ± 0.85 c,b 59.43 ± 0.62 c 232.62 ± 3.73 c 0.510 ± 0.043 b NaCl + HA Mix 15.71 ± 1.03 a 53.26 ± 1.41 c 52.75 ± 1.92 b 133.15 ± 2.99 a 0.416 ± 0.011 a SD – standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p ≤ 0.05) The humic acids fractions applied in combination with NaCl had a highly variable effect on the content of the analyzed microelements in soybean seedlings of the Progres cultivar. For Mn and Zn, a significant decrease in their content was observed for all analyzed molecular fractions of humic acids compared to the control, and these changes were statistically significant for both elements. However, compared to soybean seedlings subjected to salt stress alone, the tested molecular fractions of humic acids did not significantly affect the uptake of Mn and Zn. The measured Mn content was lower and statistically significant in the NaCl + HA 30kDa. It was found that the HA > 30kDa fraction reduced the negative effect of NaCl on Zn uptake by soybean, as the Zn content in the NaCl + HA > 30kDa treatment was 8.9% higher than in the NaCl-only treatment and similar to the control level (61.68 ± 2.01 mg/g dry matter) (Table 1 ). Humic acids fractions did not significantly affect Cu uptake by soybean seedlings of the Progres cultivar. The observed differences between the control and the HA treatments were statistically insignificant. However, it was found that the addition of humic acids significantly reduced the plant’s affinity for Cu under salt stress. Compared to the NaCl treatment, a significantly lower Cu content was measured in all HA variants, ranging from a 42% reduction for the HA 30kDa fraction. In the case of Fe, the effect of HA was most pronounced for the > 30kDa fraction. When applied in combination with NaCl, this fraction led to a significant increase in Fe uptake—by approximately 80% compared to the control. Humic substances can enhance iron availability, possibly through chelation or by improving root capacity to absorb microelements from the soil (Zanin et al. 2019; Alvarez et al. 2023). For the < 30kDa fraction, the Fe content was comparable to that of soybean seedlings under salt stress, while in the NaCl + HA Mix treatment, it was similar to the level observed in control seedlings. The HA 30kDa fractions applied in combination with NaCl also influenced Co uptake by the test plant. For both fractions, an increase in Co content of approximately 20% was observed compared to the other treatments (control, NaCl, and NaCl + HA Mix), in which the differences in measured Co content were statistically insignificant. Salt stress also affected the uptake of the analyzed macroelements. Among the four elements examined Na, K, Mg, and Ca, only Na showed a significant increase in content compared to the control, with an approximately 350% rise in soybean seedlings. In contrast, the remaining macroelements K, Mg, and Ca experienced significant reductions in content by 19.3%, 27.5%, and 28.9%, respectively, confirming the harmful effects of salt stress on the uptake of essential nutrients by plants. Salt stress results in ionic imbalance associated with high cytosolic accumulation of Na⁺ and Cl⁻, which disrupts cellular homeostasis and interferes with essential metabolic processes in plants (Matuszak et al. 2009; Malekzadeh et al. 2024; Pour-Aboughadareh et al. 2024). Excessive sodium ions can displace other essential elements such as K, P, and Mg, which are crucial for enzyme activation and osmotic regulation, leading to impaired nutrient uptake and reduced plant growth (Atero-Calvo et al. 2024; Joshi et al. 2025). Furthermore, elevated chloride levels contribute to toxicity symptoms by affecting photosynthesis and cellular membrane integrity, ultimately compromising plant vitality and productivity (Liu et al. 2017). The effect of HA fractions applied in combination with NaCl on the macroelement content of soybean seedlings of the Progres cultivar is variable (Table 2 ). For Ca and Mg, the amounts measured in soybean seedlings were similar to those found in plants under salt stress (lower than the control), and the differences between the analyzed HA molecular fractions were insignificant. However, it was found that HA fractions mitigate the negative effects of stress on the uptake of Na and K. The ability of HA to adsorb exchangeable cations such as Mg, Ca, and K, preventing their leaching by percolating water, helps ensure the availability of these elements for plant uptake (Yang et al. 2021). Table 2 The content of macroelements in soybean seedlings of Progres cultivar under salt stress conditions Combination Macroelements ±SD [mg·kg − 1 d.m.] Na K Mg Ca Control 3.35 ± 0.52 a 17.79 ± 0.97 a 2.769 ± 0.191 a 1.772 ± 0.203 a NaCl 15.18 ± 0.79 b 14.36 ± 0.07 b 2.007 ± 0.093 b 1.260 ± 0.148 b NaCl + HA 30kDa 13.39 ± 0.21 c 17.79 ± 0.06 a 2.148 ± 0.067 b 1.292 ± 0.183 b NaCl + HA Mix 11.03 ± 0.22 d 18.93 ± 0.48 a 2.124 ± 0.028 b 1.131 ± 0.167 b SD – standard deviation, *values marked with the same letters do not differ significantly according to Tukey's test (p ≤ 0.05) The measured K content in soybean seedlings treated with NaCl combined with humic acids molecular fractions 30kDa, and the HA Mix was comparable to the control level. In the case of Na, high content was observed in all tested plants subjected to salt stress, but this was significantly reduced by all analyzed humic acids molecular fractions. The greatest reduction in Na uptake by soybean seedlings was observed with the HA Mix, which decreased Na content by more than 30% (Table 2 ). A similar decrease in Na content in the shoots and roots of pepper with increasing doses of humic acids was reported by Çimrin et al. (2010). Based on plant growth parameters and element content, they concluded that humic acids application could mitigate the harmful effects of salt stress on pepper plants. The analysis of micro- and macroelements in soybean seedlings of the Progres cultivar grown in in vitro cultures indicates a low sensitivity of this cultivar to drought stress. Drought induced under controlled conditions did not significantly affect the uptake of Cu, Zn, Fe, and Co from the medium by soybean seedlings (Table 3 ). The amounts of these microelements in control plants and those grown under drought stress conditions did not differ significantly. However, it was found that soybean seedlings subjected to drought stress had a significantly lower Mn content, reduced by 25.1%. Hu and Schmidhalter (2005) stated that drought stress can significantly affect the uptake and translocation of mineral elements within plants. Under drought conditions, reduced water availability limits the mass flow of nutrients from the medium to the roots, leading to decreased nutrient absorption (Nieves-Cordones et al. 2019). The humic acids fractions applied under drought stress did not significantly affect the uptake of the analyzed microelements. In the case of Cu, Zn, and Fe, it was observed that the molecular fractions of humic acids slightly reduced their uptake. However, the differences between the HA treatments and the control or drought combinations were statistically insignificant. The exception was the M + HA > 30kDa treatment for Zn, where the measured content of this microelement 45.90 ± 0.70 mg/kg dry matter was significantly lower by 25.6% compared to the control and by 20.6% compared to the drought-only treatment. Table 3 The content of microelements in soybean seedlings of Progres cultivar under drought stress conditions Combination Microelements ±SD [mg·kg − 1 d.m.] Cu Mn Zn Fe Co Control 16.57 ± 2.66 *a 69.65 ± 1.58 a 61.68 ± 2.01 a 128.50 ± 2.22 a 0.434 ± 0.025 a Mannitol (M) 14.34 ± 0.66 a 52.17 ± 1.15 b 57.87 ± 1.67 a 132.95 ± 5.54 a 0.383 ± 0.038 a,b M + HA 30kDa 13.37 ± 0.03 a 56.23 ± 0.05 b 45.90 ± 0.70 b 120.88 ± 0.86 a 0.456 ± 0.070 a,c M + HA Mix 12.95 ± 2.35 a 62.94 ± 0.08 c 55.93 ± 2.71 a 121.39 ± 6.40 a 0.487 ± 0.008 c SD – standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p ≤ 0.05) The effect of humic acids on the uptake of Co by soybean seedlings varies depending on the analyzed HA molecular fraction. The HA 30kDa fraction and the HA Mix mitigated the effects of drought, reflected by a higher cobalt content in soybean seedlings, which was significant for the M + HA Mix treatment. For Mn, lower contents were measured in soybean seedlings for all analyzed combinations compared to the control. However, the addition of humic acids appeared to mitigate the effect of drought on Mn uptake. Higher Mn levels were observed in soybean seedlings for all combinations with HA compared to plants grown under drought conditions. The differences for HA 30kDa fractions were statistically insignificant, amounting to approximately 7.5%, whereas for the unfractionated humic acids (M + HA Mix), the increase was significant, reaching 20.6%. Drought stress induced by the addition of mannitol also affected the uptake of macroelements by soybean seedlings of the Progres cultivar grown in in vitro cultures. Drought caused a significant decrease in the uptake of Na, Mg, and Ca by the test plants, with reductions of 33.8%, 20.3%, and 48.8%, respectively. No significant effect of mannitol on K content in soybean seedlings was observed. The measured K amounts were 17.79 ± 0.97 mg/kg in the control and 16.69 ± 0.45 mg/kg under drought conditions, a difference that was statistically insignificant. Chen et al. (2011) reported contrasting findings compared to our studies, observing that drought stress led to an increase in the content of several key macroelements, including K, Na, Ca, Mg, and Fe, in rice plants. This suggests that rice may activate specific physiological or biochemical mechanisms under drought conditions to enhance the uptake or retention of these essential nutrients, possibly as a strategy to maintain cellular homeostasis and metabolic functions. Humic acids fractions affect the uptake of the analyzed macroelements by soybean seedlings from in vitro cultures under drought stress in different ways. The HA fractions did not cause any changes in the K content of soybean seedlings. The HA 30kDa fractions maintained Na and Mg contents at levels similar to those in plants subjected to drought stress. In contrast, for Ca, these fractions alleviated the effects of stress, resulting in a significant increase in Ca content by 25% and 30%, respectively, compared to drought alone. Unfractionated humic acids (HA Mix) significantly alleviated drought stress. Na and Mg contents were maintained at levels comparable to those of control plants. In contrast, Ca content increased significantly by 65.6% compared to soybean seedlings subjected to drought stress; however, it was still slightly lower by 15% than that of the control plants. Tokarz et al. (2020) and Solek-Podwika et al. (2023) reported that accumulation of inorganic ions (e.g., K⁺, Ca²⁺, Na⁺, Mg²⁺, and Cl⁻) and dissolved organic substances (such as sucrose, polyols, glycine betaine, and proline) is a key osmotic adjustment mechanism, serving as one of the plant’s defenses against drought stress. Table 4 The content of macroelements in soybean seedlings of Progres cultivar under drought stress conditions Combination Macroelements ±SD [mg·kg − 1 d.m.] Na K Mg Ca Control 3.35 ± 0.52 *a 17.79 ± 0,97 a 2.77 ± 0.19 a 1.772 ± 0,20 a Mannitol (M) 2.22 ± 0.40 b 16.69 ± 0.45 a 2.21 ± 0.02 b 0.907 ± 0.08 d M + HA 30kDa 2.35 ± 0.10 b 17.53 ± 0.96 a 2.27 ± 0.01 b 1.171 ± 0.10 c M + HA Mix 3.29 ± 0.20 a 17.59 ± 0.97 a 2.58 ± 0.07 a 1.502 ± 0.05 b SD – standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p ≤ 0.05) Humic acids can influence plant nutrition directly by promoting nutrient uptake through interaction with specific nutrient master regulators and nonspecific targets, particularly at the plant cell membrane. Indirectly, HA affect the substrate and, under field conditions, the soil, altering its chemical, physical, and biological properties (Nardi 2017, Olivares et al. 2017). Humic acids-based biostimulants improve nutrient availability to plant roots, mainly by chelating metal ions and forming stable complexes with micronutrients like iron and phosphorus (Zanin et al. 2019; Alvarez et al. 2023). These interactions enhance iron uptake and increase nutrient use efficiency (Nardi et al. 2017, 2018). Additionally, humic substances facilitate iron and phosphate absorption by creating stable humic-metal complexes, boosting nutrient availability in the soil (Gerke 2021). The chelating properties of humic acids are key factors in improving element assimilation by crops (Zhang et al. 2013). Conclusions Plants are exposed to various environmental stresses during their growth and development under both natural and agricultural conditions. Stress is generally defined as an external factor that negatively affects plant growth and development, including crop quality and yield. Soybean Progres derived from in vitro cultures exhibits greater sensitivity to salt stress than to drought stress. This is evidenced by significantly lower values of the analyzed biometric parameters (i.e., fresh mass of shoots and roots, plant height, number of leaves, and root length), as well as micromorphological traits related to stomatal density and aperture. Additionally, significant changes were observed in the content of analyzed micro- and macroelements in the dry matter of soybean seedlings. Humic acids (HA) play an important role in plant growth, yield, and resilience to abiotic stress. Our research confirms their protective role in counteracting salinity and drought stress; however, this effect depends on their molecular fractions. Analysis of biometric and micromorphological parameters of soybean seedlings of the Progres variety derived from in vitro cultures indicates that the greatest protective and, in some cases even stimulatory effect (e.g., on stomatal density) was observed for the HA > 30kDa fraction and unfractionated (Mix) humic acids. This is supported by their significantly higher values compared to plants grown under stress conditions. Humic acids, in the presence of salt and drought stress, did not specifically affect the uptake of analyzed micro- and macroelements by soybean seedlings of the Progres cultivar grown in vitro. The molecular fractions of humic acids (HA 30kDa) caused a decrease in the uptake of most analyzed elements by these seedlings. Unfractionated humic acids predominantly mitigated the effects of the applied salt and drought stresses. The levels of micro- and macroelements measured in the dry matter of Progres soybean seedlings were generally comparable to those of control plants. Declarations Conflicts of Interest: The authors declare that they have no conflicts of interest. Funding: Grant of Polish Scientific Research Committee (N N310 162338). Author Contributions: Conceptualization: D.K., M.W., and R.B.; Methodology: D.K., M.W., and R.B.; Investigation: D.K., M.W., R.M.-S., R.B., A.G., S.Z.; Data curation: D.K., M.W., R.M.-S., R.B., A.G., S.Z., and D.G.; Writing—review and editing: D.K., M.W. R. M.-S., and R.B. All authors have read and agreed to the published version of the manuscript. Data availability: Data will be made available on request References Abu-Ria ME, Elghareeb EM, Shukry WM, Abo-Hamed SA, Farag I (2024) Mitigation of drought stress in maize and sorghum by humic acid: differential growth and physiological responses. 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In: Sparks DL, Page AL, Helmke PA, Loeppert RH, Soltanpour PN, Tabatabai MA, Johnston CT, Sumner ME (eds) Methods of Soil Analysis: Part 3, Chemical Methods. 5.3; Eds. American Society of Agronomy, Madison, WI, USA, pp 1018–1020 Tokarz B, Wójtowicz T, Makowski W, Jedrzejczyk RJ, Tokarz KM (2020) What is the difference between the response of grass pea ( Lathyrus sativus L.) to salinity and drought stress? - A physiological study. Agron 10(6):833. 10.3390/agronomy10060833 Ukalska-Jaruga A, Bejger R, Debaene G, Smreczak B (2021) Characterization of Soil Organic Matter Individual Fractions (Fulvic Acids, Humic Acids, and Humins) by Spectroscopic and Electrochemical Techniques in Agricultural Soils. Agron 11:1067. https://doi.org/10.3390/agronomy11061067 Wahab A, Abdi G, Saleem MH, Ali B, Ullah S, Shah W, Shah W, Mumtaz S, Yasin G, Muresan CC, Marc RA (2022) Plants physiobiochemical and phyto-hormonal responses to alleviate the adverse effects of drought stress: a comprehensive review. Plants 11:1620. https://doi.org/10.3390/plants11131620 Yang F, Tang C, Antonietti M (2021) Natural and artificial humic substances to manage minerals, ions, water, and soil microorganisms. Chem Soc Rev 50(10):6221–6239 Zanin L, Tomasi N, Cesco S, Varanini Z, Pinton R (2019) Humic substances contribute to plant iron nutrition acting as chelators and biostimulants. Front Plant Sci 10:675. https://doi:10.3389/fpls.2019.00675 Zhang L, Gao M, Zhang L, Li B, Han M, Alva AK, Ashraf M (2013) Role of exogenous glycinebetaine and humic acid in mitigating drought stress-induced adverse effects in Malus robusta seedlings. Turk J Bot 37:920–929. htpps://doi.org/10.3906/bot-1212-21 Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 25 Sep, 2025 Reviewers invited by journal 20 Aug, 2025 Editor assigned by journal 14 Aug, 2025 First submitted to journal 12 Aug, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7352179","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503233020,"identity":"42acdad1-e421-4463-9acb-d9bd2fdc36fc","order_by":0,"name":"Danuta Kulpa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDCCAxDKgIEdSH6AcRgYLPBrOQBSxczAwDiDgUECqkWCOC3MPMRo4Tt+9pj0hxoGY4PDvAc/2/w5XMfA3rxNgnEHbi2SZ/LSJA4cYzAzOMyXLJ3Dc1iCgedYmQTjGdxaDA7kmEkcYGOwMTjMYyCdIwHUIgEUYWzDo+X8G6CWf2Atxr8tDIBa5N8Q0HIDaObBNpDDeMykGRJAtvDg1yJ5442xxdk+CWPJw3xplj0H0iXbeNKKLRLx+IXvfI7hjYpvNoZ9x3sP3/jxx5qfn/3wxhsfd9jg1AIFIDN5IEw2EJHYQEgHGPAgsRmJ0zIKRsEoGAUjAwAAdiNMaCJyddQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2352-7588","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":true,"prefix":"","firstName":"Danuta","middleName":"","lastName":"Kulpa","suffix":""},{"id":503233021,"identity":"67e7f468-ce7a-40cb-a886-73d77b5dd732","order_by":1,"name":"Renata Matuszak-Slamani","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Renata","middleName":"","lastName":"Matuszak-Slamani","suffix":""},{"id":503233022,"identity":"dec011c0-323e-439c-8651-4e8b012f1a61","order_by":2,"name":"Małgorzata Włodarczyk","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Małgorzata","middleName":"","lastName":"Włodarczyk","suffix":""},{"id":503233023,"identity":"14607b09-eab8-434e-ad39-e7f0e8bece10","order_by":3,"name":"Romualda Bejger","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Romualda","middleName":"","lastName":"Bejger","suffix":""},{"id":503233024,"identity":"43191961-94a3-42ab-b0b6-8aed158bad94","order_by":4,"name":"Andrzej Gawlik","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Andrzej","middleName":"","lastName":"Gawlik","suffix":""},{"id":503233025,"identity":"43aae014-22ce-4a08-adf4-55ab99fdb043","order_by":5,"name":"Sylwia Zarówna","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Sylwia","middleName":"","lastName":"Zarówna","suffix":""},{"id":503233026,"identity":"72001d01-7b60-459a-83f0-09405b34527c","order_by":6,"name":"Dorota Gołębiowska","email":"","orcid":"","institution":"West Pomeranian University of Technology: Zachodniopomorski Uniwersytet Technologiczny w Szczecinie","correspondingAuthor":false,"prefix":"","firstName":"Dorota","middleName":"","lastName":"Gołębiowska","suffix":""}],"badges":[],"createdAt":"2025-08-12 06:46:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7352179/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7352179/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03319-5","type":"published","date":"2025-12-17T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90177015,"identity":"d1f85be0-4b2b-4455-9442-83186ca02581","added_by":"auto","created_at":"2025-08-29 12:40:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33978,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the Amicon 8400 device\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/6ba0fef8b3b9cd79f22d16e9.png"},{"id":90177442,"identity":"c95f1c96-5d7f-4852-8c4a-94e32cd5981c","added_by":"auto","created_at":"2025-08-29 12:48:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":239982,"visible":true,"origin":"","legend":"\u003cp\u003eSoybeans growing on medium with the addition of (from the left): NaCl; NaCl and HA\u0026lt;30 kDa; NaCl and HA\u0026gt;30 kDa, NaCl and unfractionated humic acids (HA Mix) and control medium\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/78508b98ade6189ef14a0981.jpg"},{"id":90177437,"identity":"ca7bf2ac-b8bb-4b2f-a4b8-71ed49654cdc","added_by":"auto","created_at":"2025-08-29 12:48:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96439,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of humic acids on biometric parameters of overground parts of Progres soybean seedlings under salt stress conditions (Combinations: NaCl - salt stress; NaCl+HA\u0026lt;30kDa – salt stress + humic acids fraction \u0026lt;30kDa; NaCl+HA\u0026gt;30kDa – salt stress + humic acids fraction \u0026lt;30kDa; NaCl+HA Mix – salt stress + unfractionated humic acids; C – control – medium). Values marked with the same letters do not differ significantly according to Tukey's test (p≤0.05)\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/ad9aa4336cb5c38e0120631a.jpg"},{"id":90178319,"identity":"b81716bd-8c79-4142-afb7-93c693cf9dbe","added_by":"auto","created_at":"2025-08-29 12:56:02","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":108958,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of humic acids on biometric parameters of overground parts of Progres soybean seedlings under drought stress conditions (Combinations: M (Mannitol) - drought stress, M+HA\u0026lt;30kDa – drought stress + humic acids fraction \u0026lt;30kDa; M+HA\u0026gt;30kDa – drought stress + humic acids fraction \u0026lt;30kDa; M+HA Mix – drought stress + unfractionated humic acids; C – control – medium). Values marked with the same letters do not differ significantly according to Tukey's test (p≤0.05)\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/29be14416057ebaa8b792bb4.jpeg"},{"id":90177021,"identity":"857a3823-57b8-4e7c-854c-d24847a7884f","added_by":"auto","created_at":"2025-08-29 12:40:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":756196,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf surface of soybean cultured on control medium with visible stomata (a- magnification x 300; b- magnification x 1000)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/0f35152088573f223e7cdf2a.png"},{"id":90177440,"identity":"16fafd92-ded1-4e99-bd3b-d8c6a3d31f92","added_by":"auto","created_at":"2025-08-29 12:48:02","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52315,"visible":true,"origin":"","legend":"\u003cp\u003eThe length and stomatal density on the surface of soybean leaf blades under salt stress conditions. Explanations as in Fig. 3\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/fed484cd33a6b0118123801f.jpeg"},{"id":90178320,"identity":"e7dcb1d5-04da-4634-8ae8-870dcdab0cb2","added_by":"auto","created_at":"2025-08-29 12:56:02","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":59691,"visible":true,"origin":"","legend":"\u003cp\u003eThe length and density of stomata on the surface of soybean leaf blades under drought stress conditions. Explanations as in Fig. 4\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/0b3f0ee4705464f28a5ef4c3.jpeg"},{"id":98814038,"identity":"c70020d8-3da1-4fee-a74e-a2cb4c44afb6","added_by":"auto","created_at":"2025-12-22 16:09:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2332432,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7352179/v1/027fbd37-fd85-4b2a-8044-d61a9a2c01a6.pdf"}],"financialInterests":"","formattedTitle":"Humic Acids Mitigate Salt and Drought Stress in Soybean (Glycine max (L.) Merr.) in vitro Cultures","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraditional agriculture is at the forefront of climate change, being particularly affected by the increasing intensity and frequency of extreme events, such as floods, droughts, and rising temperatures (Canellas et al., 2024). Throughout their life cycle, plants are exposed to various stresses, including both biotic and abiotic factors. Salinity and drought are among the most significant abiotic stressors affecting plant growth and development. These stressors trigger morphological, biochemical, and molecular changes that severely limit crop production worldwide (Liu et al. 2017; Sahin et al. 2018; Bai et al. 2019; Singha et al. 2024; Akbari et al. 2024). Early responses to drought and salinity are similar, as both induce water stress that leads to reduced growth, decreased stomatal aperture, and nutrient deficiencies (such as K⁺ and Ca\u0026sup2;⁺). However, long-term salt stress, in addition to causing dehydration, also leads to ionic stress, which contributes to leaf senescence and impaired photosynthesis (Feng et al. 2020; Ma et al. 2020; Cao et al. 2023).\u003c/p\u003e\u003cp\u003eSoybean (\u003cem\u003eGlycine max (L.) Merr.)\u003c/em\u003e is one of the most essential leguminous crops, widely cultivated across diverse climates. It plays a pivotal role in global nutrition and food security, serving as a major source of both edible oil and plant-derived protein for humans and livestock. Globally, approximately 70% of plant-based proteins and 29% of edible oil are derived from soybeans (Hossain et al. 2024). In addition to its nutritional value, soybeans hold significant economic importance, providing raw materials for the animal feed and food industries. In recent years, soybeans have attracted increasing attention due to their high protein content, vitamin C, rich mineral composition, and reported anti-cancer properties (Cao et al. 2017; Feng et al. 2020). Moreover, soybean oil is considered a promising future biofuel source, with ongoing efforts to enhance soybean-derived biodiesel production (Santos et al. 2024). Under natural conditions, soybeans are exposed to various abiotic stress factors, including salinity and water deficiency. To achieve high yields, soybeans require an adequate water supply throughout their growth cycle (Buezo et al. 2019; Dong et al. 2019; Feng et al. 2020). Studies have demonstrated that both water deficiency and salinity stress negatively affect soybean growth and development (Matuszak‑Slamani et al. 2017; Bai et al. 2019; Khattab et al. 2019; Matuszak‑Slamani et al. 2022). Soybeans are moderately salt-tolerant and can be cultivated in light to moderately saline soils; however, their yields decline under salinity stress (Anwar-ul-Haq et al. 2023; Nasution et al. 2024).\u003c/p\u003e\u003cp\u003eTo mitigate the negative impacts of abiotic stresses, various agronomic and biological strategies are employed. One example is the use of rhizosphere microorganisms, such as plant growth-promoting rhizobacteria, which enhance nutrient uptake, reduce the toxic effects of salts, and support root development under challenging conditions (Khan et al. 2021; Kang et al. 2023; Handayani et al. 2024). Another approach involves appropriate soil management practices, such as mulching and balanced fertilization, which improve moisture conditions and alleviate stress caused by soil salinity (Jabeen et al. 2021). One of the most effective solutions is the application of biostimulants, which enhance plant physiological responses by strengthening their adaptive capabilities under stressful conditions. For instance, salicylic acid and algal extracts have been shown to significantly improve soybean drought tolerance by supporting antioxidant mechanisms (Ren et al. 2023).\u003c/p\u003e\u003cp\u003eHumic substances (HS) are among the most active components of soil organic matter. As polymeric substances with complex structures, they are formed through the condensation of biomass from microorganisms, plants, and animals. HS influence on the physical, chemical, and biological properties of soil by improving aggregate stability, buffering capacity, sorption of hydrophobic organic compounds, nutrient transport and bioavailability, as well as environmental metal complexation. In addition, they enhance the soil\u0026rsquo;s ion exchange capacity and water retention (Ouni et al. 2014). According to their solubility in water at various pH, HS can be divided into three components: fulvic acids (FA, soluble at all pH values), humic acids (HA, soluble in alkaline media and insoluble at pH 1.0), and humin (HN, insoluble at all pH values) (Ukalska-Jaruga et al. 2021; Nardi et al. 2021; Ore et al. 2023; Canellas et al. 2024). The effect of HS on plant growth depends on several factors, including their origin, concentration, molecular weight, application method, plant species, and developmental stage (Nardi et al. 2002; Nardi et al. 2021). Humic acids (HA) are considered a potential strategy to mitigate the harmful effects of abiotic stress. They can be applied as plant growth stimulants and soil conditioners, and they can also reduce irrigation frequency, improve water use efficiency, and mitigate the effects of drought stress on plants (Olk et al. 2018; Franzoni et al. 2022; Matuszak‑Slamani et al. 2022; Rakkammal et al. 2023; Canellas et al. 2024; Maffia et al. 2025).\u003c/p\u003e\u003cp\u003eDue to the complexity of HS structures and their certain fractions, it is important to identify which of them play a key role in plant growth and development under abiotic stress conditions, as the mechanisms of HS action are complex and require further research. Therefore, the aim of this study was to evaluate the protective effect of humic acids and their molecular fractions on the growth of soybean seedlings in \u003cem\u003ein vitro\u003c/em\u003e cultures under laboratory-induced salinity and drought stress conditions.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExtraction and fractionation of humic substances\u003c/h2\u003e\u003cp\u003eThe starting material for obtaining humic acids fractions was peat soils in the active accumulation stage of organic matter, located in Babia G\u0026oacute;ra National Park, Poland GPS: N 49\u0026deg;35\u0026prime;45\u0026prime;\u0026ndash;E 19\u0026deg;30\u0026prime;21\u0026prime;\u0026prime;. Peat samples were collected from the subsurface layer up to a depth of 50 cm. The air-dried peat samples, with a moisture content not exceeding 12%, were homogenized after removing undecomposed plant residues. Humic acids were extracted using the method recommended by the International Humic Substances Society (Swift 1996). The main steps of HA extraction includes: decalcification of peat samples using HCl, extraction with 0.1 mol\u0026middot;dm⁻\u0026sup3; NaOH, precipitation of HA with 6 mol\u0026middot;dm⁻\u0026sup3; HCl, and purification of HA through deashing with an HCl/HF mixture, followed by washing with distilled water until no chlorides were detected. The purified HA preparations were frozen and lyophilized to achieve a constant mass. The fractionation of HA into molecular weight fractions was carried out using an Amicon model 8400 apparatus equipped with Millipore filters with a 30kDa molecular weight cut-off. The device comprises three main modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Module 1 supplies nitrogen under controlled pressure, which drives the separation of the polymer into distinct molecular fractions. Module 2 is a container equipped with a magnetic stirrer, where the solution to be fractionated is placed. Module 3 contains the Millipore membrane mount, and a nozzle connected to a hose that directs the filtered solution to an external collection container.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMillipore membranes can only be used with solutions within a pH range of 2 to 10. Therefore, appropriate amounts of humic acids were first dissolved in 0.1 mol\u0026middot;dm⁻\u0026sup3; NaOH and then diluted fivefold. The solution was then passed through the Amberlite H⁺120 ion-exchange resin to replace Na⁺ ions with H⁺ ions. The resulting acidic solution was transferred to the container (Module 2) equipped with a magnetic stirrer. Module 3, containing a Millipore filter with a 30kDa cut-off, was mounted at the bottom. Module 1, supplying nitrogen, was positioned above Module 2 (the container holding the sample to be separated). The filtrate containing the fraction with a molecular weight below 30kDa (HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa) was collected in a separate vessel and concentrated at a temperature not exceeding\u0026thinsp;+\u0026thinsp;40\u0026deg;C. The concentrated solution was then frozen using a dry ice\u0026ndash;alcohol mixture at \u0026minus;\u0026thinsp;70\u0026deg;C and lyophilized to constant weight. The supernatant containing the fraction with a molecular weight\u0026thinsp;\u0026gt;\u0026thinsp;30kDa (HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa) was similarly frozen using a dry ice\u0026ndash;alcohol mixture at \u0026minus;\u0026thinsp;70\u0026deg;C and lyophilized to constant weight. The resulting HA preparation - HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa and HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa fractions, were dissolved in Michaelis buffer at pH 7.17. The unfractionated humic acids solution was referred to as HA Mix. Chemical and spectral analyses of the molecular HA fractions and the unfractionated preparation were presented in our previous studies: Gawlik et al. (2016), Matuszak-Slamani et al. (2017, 2022).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecultures condition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSoybean seedlings of the Progres cultivar were analyzed under laboratory-induced stress conditions to evaluate the effects of salinity and drought, as well as the protective role of humic acids against these abiotic stresses. Soybean seeds of the Progres cultivar used in the \u003cem\u003ein vitro\u003c/em\u003e studies were sterilized prior to the experiment. They were immersed in 70% ethanol for 10 seconds and then treated with 0.2% mercuric chloride (HgCl\u003csub\u003e2\u003c/sub\u003e) for 12 minutes. To prevent contamination, all subsequent procedures were carried out under sterile conditions in a laminar airflow chamber (Caetano-Anoll\u0026eacute;set al. 1990).\u003c/p\u003e\u003cp\u003eThe Murashige and Skoog (1962) medium was used in the experiment. The pH of the medium was adjusted to 5.7 using 0.1 mol\u0026middot;dm⁻\u0026sup3; solutions of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The media were supplemented with 8 g\u0026middot;dm⁻\u0026sup3; agar, 30 g\u0026middot;dm⁻\u0026sup3; sucrose, and 100 mg\u0026middot;dm⁻\u0026sup3; inositol. After the addition of the dry components, the media were heated to allow polymerization and solidification, poured into 900 cm\u003csup\u003e3\u003c/sup\u003e jars, and sterilized in an autoclave at 121\u0026deg;C for 20 minutes.\u003c/p\u003e\u003cp\u003eIn the first stage of the \u003cem\u003ein vitro\u003c/em\u003e study, the effects of salinity and drought on soybean growth and development were assessed.\u003c/p\u003e\u003cp\u003eFor this purpose, sterilized soybean seeds were placed on a nutrient medium, to which 100 mmol\u0026middot;dm⁻\u0026sup3; NaCl and 150 mmol\u0026middot;dm⁻\u0026sup3; mannitol were added to simulate salinity and drought stress, respectively. Simultaneously, the protective effect of humic acids (HA) on soybean growth and development under these abiotic stress conditions was investigated. Molecular fractions of HA were added to the nutrient medium containing the appropriate stress factor (NaCl or mannitol): humic acids with molecular weights less than 30kDa (HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa), greater than 30kDa (HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa), or unfractionated humic acids (HA Mix). For all tested molecular fractions, the concentration of HA in the nutrient medium was identical, amounting to 0.005 g C\u003csub\u003eHA\u003c/sub\u003e\u0026middot;dm⁻\u0026sup3;. The control group consisted of sterilized soybean seeds of the Progres cultivar placed on Murashige and Skoog nutrient medium without stress factors.\u003c/p\u003e\u003cp\u003eEach experimental variant for each stress condition consisted of control (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and four treatments: stress (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), stress\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), stress\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), and stress\u0026thinsp;+\u0026thinsp;HA Mix (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). All tested stresses and their combinations were applied simultaneously under controlled \u003cem\u003ein vitro\u003c/em\u003e culture conditions. Ten jars were prepared for each combination, with ten seeds placed in each jar, resulting in a total of 100 seeds per combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The experiment was conducted in a phytotron at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, under a light intensity of 40 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1; and a 16-hour photoperiod. After 21 days of soybean growth, biometric parameters, micromorphological features, and the content of micro- and macroelements in the dry mass of soybean seedlings were measured.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBiometric measurements\u003c/h3\u003e\n\u003cp\u003eBiometric measurements included plant height, number of leaves, root length, fresh weight, and the weight of the overground parts of soybean seedlings were conducted using an electronic caliper (Varel, accuracy\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mm), an analytical balance (Radwag AS 82/220.R2 PLUS), and a moisture analyzer (Radwag MA 50.R).\u003c/p\u003e\n\u003ch3\u003eMicromorphological analysis\u003c/h3\u003e\n\u003cp\u003eLeaves isolated from the middle part of soybean stems grown on media enriched with NaCl, mannitol, and humic acids were subjected to micromorphological analysis using a scanning electron microscope (SEM). The material was dried using a Critical Point Dryer (Quorum Technologies) and then coated with a gold layer using a Sputter Coater (Quorum Technologies). Observations were conducted with a Carl Zeiss EVO LS 10 microscope at an accelerating voltage of 15 kV. Determined stomatal length (\u0026micro;m) and stomatal density (the number of stomata per mm\u0026sup2;) on the surface of soybean leaf blades. Measurements were made on the abaxial (lower) surface of the soybean leaves.\u003c/p\u003e\n\u003ch3\u003eMicro- and macroelement analysis\u003c/h3\u003e\n\u003cp\u003eThe content of five microelements (Cu, Mn, Zn, Fe, and Co) and four macroelements (Mg, Ca, K, and Na) in the dry matter of soybean seedlings of the Progres cultivar grown \u003cem\u003ein vitro\u003c/em\u003e was determined. These elements were present in the basic medium (according to Murashige and Skoog, 1962). The soybean seedlings were dried to constant weight at 105\u0026deg;C, ground, and mineralized in a mixture of concentrated HNO\u003csub\u003e3\u003c/sub\u003e and HClO\u003csub\u003e4\u003c/sub\u003e acids in a 3:1 ratio. All determinations were performed in triplicate. Quantitative analysis was performed using the Solar S4 Atomic Absorption Spectrophotometer with an acetylene-air flame atomizer.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe experiment with plants \u003cem\u003ein vitro\u003c/em\u003e cultures was conducted with 10 replications, each containing 10 plants (a total of 100 plants per experimental variant). Data for micro- and macrominerals are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three replicates. During the microorphological analysis, 20 photos of leaf blades were taken for each combination, determining the stomatal density/mm2. The length of 10 stomata randomly selected from each photo was determined, for a total of 200 for each combination. One-way analysis of variance (ANOVA) was used to compare mean values. The analysis formed the basis for identifying homogeneous groups using Tukey\u0026rsquo;s test at a significance level of p\u0026thinsp;\u0026le;\u0026thinsp;0.05. Means that differed significantly were marked with different letters. The results are presented in graphs and tables separately for each stress factor (mannitol or NaCl). Statistical analysis was performed using Statistica 13.1 PL (StatSoft, Krak\u0026oacute;w, Poland).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eResearch on the influence of humic acids on plant development has so far focused mainly on field, pot, or hydroponic experiments. In contrast, conducting research \u003cem\u003ein vitro\u003c/em\u003e cultures allows for better standardization and more precise assessment of the effects of the studied factors on changes in plant morphological traits. In our study, we assessed the protective effects of humic acids and their molecular fractions on the growth of soybean seedlings in \u003cem\u003ein vitro\u003c/em\u003e cultures under laboratory-induced salinity and drought stress conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth in\u003c/b\u003e\u003cb\u003ein vitro\u003c/b\u003e\u003cb\u003ecultures.\u003c/b\u003e Salt stress, primarily induced by sodium chloride (NaCl), is one of the major abiotic factors affecting plant growth and development under both natural conditions and \u003cem\u003ein vitro\u003c/em\u003e cultures (Parida and Das 2005). According to our experiment, the best growth parameters - longer shoots and roots, the highest shoot and root biomass, and the greatest number of leaves - were observed in soybean plants grown on control medium without stress factors (NaCl and mannitol). These differences were statistically significant for both stresses. Under salt stress, plant height decreased by 80%, root length by 65%, number of leaves by 79%, root weight by 51%, and above-ground biomass by 85% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results confirm that soybean is a salt-sensitive legume, and salinity impairs seed germination, seedling growth, and overall plant development (Rasheed et al. 2022). Under salt stress conditions, plants exhibit morphological changes such as reduced leaf size, delayed root growth, decreased biomass, and shortened shoot length. In \u003cem\u003ein vitro\u003c/em\u003e cultures, salt stress can also hinder tissue regeneration processes, including somatic embryogenesis and rooting ability (Begum et al. 2022; A\u0026ccedil;ıkbaş et al. 2023; Gobade et al. 2024). Salinity inhibits overall plant growth by reducing the osmotic potential of the growing medium, causing specific ion toxicity, inducing oxidative stress, and decreasing nutrient uptake (Parida and Das 2005; Liu et al. 2017; Atero-Calvo et al. 2024; Hossain et al. 2024).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effect of mannitol on agricultural plant development has been widely studied, mainly due to its ability to regulate water management (Khatoon et al. 2018). Mannitol is commonly used as an osmotic stress agent, limiting water availability, disrupting metabolic processes, and reducing the rates of photosynthesis, growth, and protein synthesis in plants (Adrees et al. 2015; Saadaoui et al. 2023). Under drought stress, plant height decreased by 24%, root length by 55%, number of leaves by 36%, root weight by 50%, and above-ground biomass by 43% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results are consistent with our previous studies (Matuszak-Slamani et al. 2022) concerning soybean, as well as with studies by Ahmad et al. (2022) and Abu-Ria et al. (2024), which focused on the growth of sorghum and maize under drought stress conditions. The decrease in the analyzed biometric parameters is mainly attributed to drought-induced disruptions in water absorption, which leads to increased cell dehydration and inhibits cell division, expansion, and proliferation (Wahab et al. 2022).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRecent studies emphasize the key role of humic substances (HS), particularly humic acids (HA), in mitigating the adverse effects of abiotic stress and enhancing plant resilience (Nardi et al. 2021; Canellas et al. 2024; Nabi et al. 2025; Maffia et al. 2025). Our research demonstrated that under salt stress, it is possible to enhance plant tolerance using humic acids. This is confirmed by the increased values of the analyzed parameters in soybean seedlings, particularly in the aboveground parts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the three HA combinations tested, the \u0026gt;\u0026thinsp;30 kDa fraction and the unfractionated preparation (Mix) had the most beneficial effects. Compared to the NaCl treatment alone, a significant increase was observed in plant height (58\u0026ndash;156%), number of leaves (119\u0026ndash;165%), and fresh mass of the overground parts (44\u0026ndash;122%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the root-related parameters, the protective effect of HA was less pronounced. Root mass and length increased by 16\u0026ndash;39% and 14\u0026ndash;30%, respectively, compared to plants subjected to salt stress alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The obtained results indicate that the addition of humic acids activates many components of the ion homeostasis machinery in the presence of NaCl ions, thus highlighting their protective effect (Khaleda et al. 2017; Souza et al. 2021; Canellas et al. 2024; Nabi et al. 2025).\u003c/p\u003e\u003cp\u003eIn the case of the second stress factor \u0026ndash; drought, the protective effect of humic acids was less pronounced. However, similarly to salinity stress, their protective effect was most significant for the HA\u0026thinsp;\u0026gt;\u0026thinsp;30 kDa fraction and the HA Mix. Under drought conditions, the addition of humic acids led to an average increase of 20\u0026ndash;40% in the analyzed biometric parameters. An exception was observed for the HA Mix, where the number of leaves increased by 87% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResearch shows that humic acids (HA) significantly alleviate drought-related stress in various plant species (sorghum, maize, rice, and wheat) by enhancing growth characteristics and biomass accumulation in leaves, stems, and roots (Ahmad et al. 2022; Mutlu et al. 2022; Abu-Ria et al. 2024). Humic acids exhibit pleiotropic effects, influencing plant growth, nutrient uptake, and stress tolerance (Nardi et al. 2021; Matuszak-Slamani et al. 2022; Abu-Ria et al. 2024; Maffia et al. 2025). Nardi et al. (2021) concluded that humic acids can stimulate chlorophyll synthesis and the activity of enzymes responsible for regulating osmotic pressure in plant cells. Humic substances also support the reconstruction of cellular structures, resulting in improved plant growth under osmotic stress conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicromorphological analysis.\u003c/b\u003e Stomata regulate the exchange of CO₂ and water vapor between the leaves and the atmosphere, serving as key indicators of a plant\u0026rsquo;s resilience to environmental changes. Stomatal density directly influences photosynthesis and transpiration processes (Sultana et al. 2024). Example photos of the leaf surface of control soybean plants grown \u003cem\u003ein vitro\u003c/em\u003e cultures are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The average stomatal length determined for control plants was 118.72 \u0026micro;m, and the average stomatal density was 296.85 mm⁻\u0026sup2;. It was found that the analyzed stresses (salinity and drought) had a negative effect on stomata.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCompared to the control of soybean plants, salinity stress significantly reduced stomatal length by 16%, while stomatal density decreased by 40%. In the case of drought, changes in the analyzed stomatal parameters also showed significant decreases, amounting to an 11% reduction in stomatal length and a 61% reduction in stomatal density (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Stomatal development adapts to environmental conditions throughout the plant growth cycle (Sultana et al. 2024), often by altering the density and size of stomata on the plant epidermis. Abiotic stresses, such as drought, decrease stomatal length and density (Han and Torii 2016; Lawson and Vialet-Chabrand 2019; Mohi-Ud-Din et al. 2025). Hughes et al. (2017) suggest that this is associated with the potential for reduced transpiration and improved water management by plants.\u003c/p\u003e\u003cp\u003eThe use of humic acids in combination with salinity stress has a positive effect on stomata. The most beneficial effect was observed for the HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction, where stomatal length increased significantly by 15%. Stomatal density increased by 144% compared to stress treatment and by 47% compared to control. For the other fractions, the changes were smaller but comparable to the control values (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the case of drought stress, a significant increase in stomatal length ranging from 6\u0026ndash;12% was observed only for two fractions: HA\u0026thinsp;\u0026gt;\u0026thinsp;30 kDa and HA Mix, reaching values comparable to those of control plants. A significant effect of humic acids was also found in the case of stomatal density. This parameter increased markedly for all analyzed HA fractions, ranging from up to 182% (HA\u0026thinsp;\u0026lt;\u0026thinsp;30 kDa) to 234% (HA Mix). The obtained values were also higher than those of the control plants from 11% (HA\u0026thinsp;\u0026lt;\u0026thinsp;30 kDa, not statistically significant) to 31% (HA Mix, statistically significant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSoybean is an amphistomatic species, with a higher stomatal density on the abaxial side than on the adaxial side. Stomata are distributed randomly across the leaf surface (Amaliah et al. 2019; Sultana et al. 2024). Based on our results, it can be concluded that humic acids fractions, particularly HA\u0026thinsp;\u0026gt;\u0026thinsp;30 kDa and HA Mix exhibit not only protective but also stimulatory effects, increasing stomatal density on soybean leaves exposed to stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicro- and macroelement analysis.\u003c/b\u003e The effect of humic acids (HA) on element uptake by plants depends on multiple factors, primarily the origin, type, dosage, and structural properties of HA, as well as the pH of the rooting medium, the concentration of exogenous elements, and the plant species (Nardi et al. 2017). The mechanism of element uptake by plant roots under natural conditions is complex, and the additional influence of humic substances and environmental stresses (such as drought and salinity) makes it even more intricate and dependent on multiple factors.\u003c/p\u003e\u003cp\u003eThe research conducted in this study on the effects of salt stress (NaCl), drought stress (mannitol), and humic acids fractions on the content of macro- and microelements in the leaves of the soybean cultivar Progres indicates differences in their accumulation. The impact of salt stress on the uptake of microelements from the medium by soybean seedlings of the Progres cultivar grown in \u003cem\u003ein vitro\u003c/em\u003e cultures was observed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to Sahasakul et al. (2023), microelements are generally less susceptible to salt stress than macroelements. In our study, a decrease in the content of Mn, Zn, Fe, and Co was observed compared to the control, ranging from 15.41% (Mn) to 4.02% (Fe). A significant increase in Cu uptake by soybean seedlings was noted, exceeding 140%. On the other hand, Celik et al. (2011) and Katkat et al. (2009) reported that the foliar application of humic acids had a statistically significant positive effect on the uptake of Cu, Zn, Mn, Mg, and Fe by wheat.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe content of microelements in soybean seedlings of Progres cultivar under salt stress conditions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCombination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eMicroelements \u0026plusmn;SD [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCu\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eMn\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eZn\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eFe\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eCo\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003c/b\u003e\u003csup\u003e\u003cb\u003e*a\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e69.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e61.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e128.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.434\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSalinity (NaCl)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40.02\u0026thinsp;\u0026plusmn;\u0026thinsp;6.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e123.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.408\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e120.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.516\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003ec,b\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e59.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e232.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.510\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA Mix\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e133.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.416\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eSD \u0026ndash; standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe humic acids fractions applied in combination with NaCl had a highly variable effect on the content of the analyzed microelements in soybean seedlings of the Progres cultivar. For Mn and Zn, a significant decrease in their content was observed for all analyzed molecular fractions of humic acids compared to the control, and these changes were statistically significant for both elements. However, compared to soybean seedlings subjected to salt stress alone, the tested molecular fractions of humic acids did not significantly affect the uptake of Mn and Zn. The measured Mn content was lower and statistically significant in the NaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and NaCl\u0026thinsp;+\u0026thinsp;HA Mix treatments. In contrast, significant changes in Zn content were observed only for the humic acids fraction\u0026thinsp;\u0026gt;\u0026thinsp;30kDa. It was found that the HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction reduced the negative effect of NaCl on Zn uptake by soybean, as the Zn content in the NaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa treatment was 8.9% higher than in the NaCl-only treatment and similar to the control level (61.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 mg/g dry matter) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Humic acids fractions did not significantly affect Cu uptake by soybean seedlings of the Progres cultivar. The observed differences between the control and the HA treatments were statistically insignificant. However, it was found that the addition of humic acids significantly reduced the plant\u0026rsquo;s affinity for Cu under salt stress. Compared to the NaCl treatment, a significantly lower Cu content was measured in all HA variants, ranging from a 42% reduction for the HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa fraction to a 62% reduction for the HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction.\u003c/p\u003e\u003cp\u003eIn the case of Fe, the effect of HA was most pronounced for the \u0026gt;\u0026thinsp;30kDa fraction. When applied in combination with NaCl, this fraction led to a significant increase in Fe uptake\u0026mdash;by approximately 80% compared to the control. Humic substances can enhance iron availability, possibly through chelation or by improving root capacity to absorb microelements from the soil (Zanin et al. 2019; Alvarez et al. 2023). For the \u0026lt;\u0026thinsp;30kDa fraction, the Fe content was comparable to that of soybean seedlings under salt stress, while in the NaCl\u0026thinsp;+\u0026thinsp;HA Mix treatment, it was similar to the level observed in control seedlings.\u003c/p\u003e\u003cp\u003eThe HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and \u0026gt;\u0026thinsp;30kDa fractions applied in combination with NaCl also influenced Co uptake by the test plant. For both fractions, an increase in Co content of approximately 20% was observed compared to the other treatments (control, NaCl, and NaCl\u0026thinsp;+\u0026thinsp;HA Mix), in which the differences in measured Co content were statistically insignificant.\u003c/p\u003e\u003cp\u003eSalt stress also affected the uptake of the analyzed macroelements. Among the four elements examined Na, K, Mg, and Ca, only Na showed a significant increase in content compared to the control, with an approximately 350% rise in soybean seedlings. In contrast, the remaining macroelements K, Mg, and Ca experienced significant reductions in content by 19.3%, 27.5%, and 28.9%, respectively, confirming the harmful effects of salt stress on the uptake of essential nutrients by plants.\u003c/p\u003e\u003cp\u003eSalt stress results in ionic imbalance associated with high cytosolic accumulation of Na⁺ and Cl⁻, which disrupts cellular homeostasis and interferes with essential metabolic processes in plants (Matuszak et al. 2009; Malekzadeh et al. 2024; Pour-Aboughadareh et al. 2024). Excessive sodium ions can displace other essential elements such as K, P, and Mg, which are crucial for enzyme activation and osmotic regulation, leading to impaired nutrient uptake and reduced plant growth (Atero-Calvo et al. 2024; Joshi et al. 2025). Furthermore, elevated chloride levels contribute to toxicity symptoms by affecting photosynthesis and cellular membrane integrity, ultimately compromising plant vitality and productivity (Liu et al. 2017).\u003c/p\u003e\u003cp\u003eThe effect of HA fractions applied in combination with NaCl on the macroelement content of soybean seedlings of the Progres cultivar is variable (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For Ca and Mg, the amounts measured in soybean seedlings were similar to those found in plants under salt stress (lower than the control), and the differences between the analyzed HA molecular fractions were insignificant. However, it was found that HA fractions mitigate the negative effects of stress on the uptake of Na and K. The ability of HA to adsorb exchangeable cations such as Mg, Ca, and K, preventing their leaching by percolating water, helps ensure the availability of these elements for plant uptake (Yang et al. 2021).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe content of macroelements in soybean seedlings of Progres cultivar under salt stress conditions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCombination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eMacroelements \u0026plusmn;SD [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.m.]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNa\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eK\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eMg\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eCa\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e17.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e2.769\u0026thinsp;\u0026plusmn;\u0026thinsp;0.191\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1.772\u0026thinsp;\u0026plusmn;\u0026thinsp;0.203\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.093\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.260\u0026thinsp;\u0026plusmn;\u0026thinsp;0.148\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.017\u0026thinsp;\u0026plusmn;\u0026thinsp;0.093\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.148\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.292\u0026thinsp;\u0026plusmn;\u0026thinsp;0.183\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u0026thinsp;+\u0026thinsp;HA Mix\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.124\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.167\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eSD \u0026ndash; standard deviation, *values marked with the same letters do not differ significantly according to Tukey's test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe measured K content in soybean seedlings treated with NaCl combined with humic acids molecular fractions\u0026thinsp;\u0026lt;\u0026thinsp;30kDa, \u0026gt;\u0026thinsp;30kDa, and the HA Mix was comparable to the control level. In the case of Na, high content was observed in all tested plants subjected to salt stress, but this was significantly reduced by all analyzed humic acids molecular fractions. The greatest reduction in Na uptake by soybean seedlings was observed with the HA Mix, which decreased Na content by more than 30% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A similar decrease in Na content in the shoots and roots of pepper with increasing doses of humic acids was reported by \u0026Ccedil;imrin et al. (2010). Based on plant growth parameters and element content, they concluded that humic acids application could mitigate the harmful effects of salt stress on pepper plants.\u003c/p\u003e\u003cp\u003eThe analysis of micro- and macroelements in soybean seedlings of the Progres cultivar grown in \u003cem\u003ein vitro\u003c/em\u003e cultures indicates a low sensitivity of this cultivar to drought stress. Drought induced under controlled conditions did not significantly affect the uptake of Cu, Zn, Fe, and Co from the medium by soybean seedlings (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The amounts of these microelements in control plants and those grown under drought stress conditions did not differ significantly. However, it was found that soybean seedlings subjected to drought stress had a significantly lower Mn content, reduced by 25.1%. Hu and Schmidhalter (2005) stated that drought stress can significantly affect the uptake and translocation of mineral elements within plants. Under drought conditions, reduced water availability limits the mass flow of nutrients from the medium to the roots, leading to decreased nutrient absorption (Nieves-Cordones et al. 2019).\u003c/p\u003e\u003cp\u003eThe humic acids fractions applied under drought stress did not significantly affect the uptake of the analyzed microelements. In the case of Cu, Zn, and Fe, it was observed that the molecular fractions of humic acids slightly reduced their uptake. However, the differences between the HA treatments and the control or drought combinations were statistically insignificant. The exception was the M\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa treatment for Zn, where the measured content of this microelement 45.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 mg/kg dry matter was significantly lower by 25.6% compared to the control and by 20.6% compared to the drought-only treatment.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe content of microelements in soybean seedlings of Progres cultivar under drought stress conditions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCombination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eMicroelements \u0026plusmn;SD [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.m.]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCu\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eMn\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eZn\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eFe\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eCo\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e16.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66\u003c/b\u003e\u003csup\u003e\u003cb\u003e*a\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e69.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e61.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e128.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.434\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMannitol (M)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e52.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e132.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.383\u0026thinsp;\u0026plusmn;\u0026thinsp;0.038\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e116.10\u0026thinsp;\u0026plusmn;\u0026thinsp;13.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.368\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e120.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.456\u0026thinsp;\u0026plusmn;\u0026thinsp;0.070\u003csup\u003ea,c\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA Mix\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e121.39\u0026thinsp;\u0026plusmn;\u0026thinsp;6.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.487\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eSD \u0026ndash; standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe effect of humic acids on the uptake of Co by soybean seedlings varies depending on the analyzed HA molecular fraction. The HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa fraction increased the sensitivity of soybean seedlings to drought stress, resulting in a 15.2% decrease in Co content compared to the control. In contrast, the HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction and the HA Mix mitigated the effects of drought, reflected by a higher cobalt content in soybean seedlings, which was significant for the M\u0026thinsp;+\u0026thinsp;HA Mix treatment. For Mn, lower contents were measured in soybean seedlings for all analyzed combinations compared to the control. However, the addition of humic acids appeared to mitigate the effect of drought on Mn uptake. Higher Mn levels were observed in soybean seedlings for all combinations with HA compared to plants grown under drought conditions. The differences for HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fractions were statistically insignificant, amounting to approximately 7.5%, whereas for the unfractionated humic acids (M\u0026thinsp;+\u0026thinsp;HA Mix), the increase was significant, reaching 20.6%.\u003c/p\u003e\u003cp\u003eDrought stress induced by the addition of mannitol also affected the uptake of macroelements by soybean seedlings of the Progres cultivar grown in \u003cem\u003ein vitro\u003c/em\u003e cultures. Drought caused a significant decrease in the uptake of Na, Mg, and Ca by the test plants, with reductions of 33.8%, 20.3%, and 48.8%, respectively. No significant effect of mannitol on K content in soybean seedlings was observed. The measured K amounts were 17.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 mg/kg in the control and 16.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 mg/kg under drought conditions, a difference that was statistically insignificant. Chen et al. (2011) reported contrasting findings compared to our studies, observing that drought stress led to an increase in the content of several key macroelements, including K, Na, Ca, Mg, and Fe, in rice plants. This suggests that rice may activate specific physiological or biochemical mechanisms under drought conditions to enhance the uptake or retention of these essential nutrients, possibly as a strategy to maintain cellular homeostasis and metabolic functions.\u003c/p\u003e\u003cp\u003eHumic acids fractions affect the uptake of the analyzed macroelements by soybean seedlings from \u003cem\u003ein vitro\u003c/em\u003e cultures under drought stress in different ways. The HA fractions did not cause any changes in the K content of soybean seedlings. The HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fractions maintained Na and Mg contents at levels similar to those in plants subjected to drought stress. In contrast, for Ca, these fractions alleviated the effects of stress, resulting in a significant increase in Ca content by 25% and 30%, respectively, compared to drought alone. Unfractionated humic acids (HA Mix) significantly alleviated drought stress. Na and Mg contents were maintained at levels comparable to those of control plants. In contrast, Ca content increased significantly by 65.6% compared to soybean seedlings subjected to drought stress; however, it was still slightly lower by 15% than that of the control plants. Tokarz et al. (2020) and Solek-Podwika et al. (2023) reported that accumulation of inorganic ions (e.g., K⁺, Ca\u0026sup2;⁺, Na⁺, Mg\u0026sup2;⁺, and Cl⁻) and dissolved organic substances (such as sucrose, polyols, glycine betaine, and proline) is a key osmotic adjustment mechanism, serving as one of the plant\u0026rsquo;s defenses against drought stress.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe content of macroelements in soybean seedlings of Progres cultivar under drought stress conditions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCombination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eMacroelements \u0026plusmn;SD [mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.m.]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNa\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eK\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eMg\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eCa\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/b\u003e\u003csup\u003e\u003cb\u003e*a\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e17.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0,97\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1.772\u0026thinsp;\u0026plusmn;\u0026thinsp;0,20\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMannitol (M)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.907\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.129\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.171\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eM\u0026thinsp;+\u0026thinsp;HA Mix\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.502\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eSD \u0026ndash; standard deviation, *Values marked with the same letters do not differ significantly according to Tukey's test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHumic acids can influence plant nutrition directly by promoting nutrient uptake through interaction with specific nutrient master regulators and nonspecific targets, particularly at the plant cell membrane. Indirectly, HA affect the substrate and, under field conditions, the soil, altering its chemical, physical, and biological properties (Nardi 2017, Olivares et al. 2017). Humic acids-based biostimulants improve nutrient availability to plant roots, mainly by chelating metal ions and forming stable complexes with micronutrients like iron and phosphorus (Zanin et al. 2019; Alvarez et al. 2023). These interactions enhance iron uptake and increase nutrient use efficiency (Nardi et al. 2017, 2018). Additionally, humic substances facilitate iron and phosphate absorption by creating stable humic-metal complexes, boosting nutrient availability in the soil (Gerke 2021). The chelating properties of humic acids are key factors in improving element assimilation by crops (Zhang et al. 2013).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePlants are exposed to various environmental stresses during their growth and development under both natural and agricultural conditions. Stress is generally defined as an external factor that negatively affects plant growth and development, including crop quality and yield. Soybean Progres derived from \u003cem\u003ein vitro\u003c/em\u003e cultures exhibits greater sensitivity to salt stress than to drought stress. This is evidenced by significantly lower values of the analyzed biometric parameters (i.e., fresh mass of shoots and roots, plant height, number of leaves, and root length), as well as micromorphological traits related to stomatal density and aperture. Additionally, significant changes were observed in the content of analyzed micro- and macroelements in the dry matter of soybean seedlings.\u003c/p\u003e\u003cp\u003eHumic acids (HA) play an important role in plant growth, yield, and resilience to abiotic stress. Our research confirms their protective role in counteracting salinity and drought stress; however, this effect depends on their molecular fractions. Analysis of biometric and micromorphological parameters of soybean seedlings of the Progres variety derived from \u003cem\u003ein vitro\u003c/em\u003e cultures indicates that the greatest protective and, in some cases even stimulatory effect (e.g., on stomatal density) was observed for the HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction and unfractionated (Mix) humic acids. This is supported by their significantly higher values compared to plants grown under stress conditions. Humic acids, in the presence of salt and drought stress, did not specifically affect the uptake of analyzed micro- and macroelements by soybean seedlings of the Progres cultivar grown \u003cem\u003ein vitro.\u003c/em\u003e The molecular fractions of humic acids (HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa) caused a decrease in the uptake of most analyzed elements by these seedlings. Unfractionated humic acids predominantly mitigated the effects of the applied salt and drought stresses. The levels of micro- and macroelements measured in the dry matter of Progres soybean seedlings were generally comparable to those of control plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eGrant of Polish Scientific Research Committee (N N310 162338).\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\u003cp\u003eConceptualization: D.K., M.W., and R.B.; Methodology: D.K., M.W., and R.B.; Investigation: D.K., M.W., R.M.-S., R.B., A.G., S.Z.; Data curation: D.K., M.W., R.M.-S., R.B., A.G., S.Z., and D.G.; Writing\u0026mdash;review and editing: D.K., M.W. R. M.-S., and R.B. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eData will be made available on request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu-Ria ME, Elghareeb EM, Shukry WM, Abo-Hamed SA, Farag I (2024) Mitigation of drought stress in maize and sorghum by humic acid: differential growth and physiological responses. 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Turk J Bot 37:920\u0026ndash;929. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehtpps://doi.org/10.3906/bot-1212-21\u003c/span\u003e\u003cspan address=\"htpps://10.3906/bot-1212-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"abiotic stress, NaCl, Mannitol, humic acids, soybean, growth, nutrients","lastPublishedDoi":"10.21203/rs.3.rs-7352179/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7352179/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to evaluate the protective effect of humic acids (HA) with different molecular weight fractions on the soybean Progres cultivar under drought and salinity stress in \u003cem\u003ein vitro\u003c/em\u003e. HA were isolated from peat samples according to the International Humic Substances Society procedure. Three HA treatments were tested: HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa, HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa, and unfractionated HA (Mix). Sterilized soybean seeds were cultured on nutrient media supplemented with 100 mmol\u0026middot;dm⁻\u0026sup3; NaCl or 150 mmol\u0026middot;dm⁻\u0026sup3; Mannitol to simulate salinity and drought stress, respectively. HA fractions were added at 0.005 g C\u003csub\u003eHA\u003c/sub\u003e\u0026middot;dm⁻\u0026sup3;. No stress factors were used in the control samples. Biometric parameters (plant height, leaf number, root length, shoot and root biomass) and micromorphological traits (stomatal density and length) were measured. Micro- and macroelement contents in dry seedling matter were also analyzed. Soybean Progres exhibited greater sensitivity to salt than drought stress, shown by reduced biometric and micromorphological parameters and altered element contents. HA treatments demonstrated a protective role, which was dependent on the molecular fraction. The HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa fraction and HA Mix provided the greatest protective and, at times, stimulatory effects, notably increasing stomatal density and biometric values under stress. HA, in the presence of salt and drought stress, did not specifically affect the uptake of the analysed micro- and macroelements by soybeans. The fractions HA\u0026thinsp;\u0026lt;\u0026thinsp;30kDa and HA\u0026thinsp;\u0026gt;\u0026thinsp;30kDa caused a decrease in the uptake of most analyzed elements. Unfractionated HA predominantly mitigated the effects of applied stresses. For HA Mix, the levels of micro- and macroelements in soybean seedlings were generally comparable to those in control plants.\u003c/p\u003e","manuscriptTitle":"Humic Acids Mitigate Salt and Drought Stress in Soybean (Glycine max (L.) Merr.) in vitro Cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 12:39:57","doi":"10.21203/rs.3.rs-7352179/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-25T16:46:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-20T16:01:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T11:12:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-08-12T13:41:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5fca240c-194d-45a3-8fe6-3c6abb0f0789","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:03:37+00:00","versionOfRecord":{"articleIdentity":"rs-7352179","link":"https://doi.org/10.1007/s11240-025-03319-5","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-12-17 15:57:29","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-08-29 12:39:57","video":"","vorDoi":"10.1007/s11240-025-03319-5","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03319-5","workflowStages":[]},"version":"v1","identity":"rs-7352179","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7352179","identity":"rs-7352179","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}