Six different soybean varieties were used to optimize nutrient seed priming in soybean underlying seed drought tolerance ( Table 1 ). The evaluated varieties have different growth periods, seed size, and other morphological traits. All the soybean varieties were obtained from Chinese National Center for Soybean Improvement (CNCSI), Nanjing Agricultural University, Jiangsu, China. Seed priming used two biopolymers SCMC (CAS: 9004–32-4, viscosity: 300–800 mPa·s) was purchased from Sino Pharm Chemical Reagent Co., Ltd. (Shanghai, China). GA, molecular weight 250 kDa was obtained from the Shanghai Ryon Biological Technology Co., Ltd. (Shanghai, China). All other reagents used were of analytical grade.

In a preliminary experiment, various concentrations were tested to determine the optimal concentration for enhancing seed performance. Through systematic evaluation, 10% CG was identified as the most effective concentration. Soybean seeds were surface sterilized using 20% Clorox bleach, and 70% ethyl alcohol (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). These seeds were then washed ten times with 0.2 µm Millipore membrane (Millipore Corporation, MA, USA) filter-sterilized distilled water.

Soybean seeds were soaked in 10% CG for 6 h. After priming, the seeds were left to dry before germination, four interventions were included in the experiment, each replicated three times in a completely randomized design (CRD).

Seed germination was applied using the paper roll method employing a solution of polyethylene glycol (PEG, Sigma-Aldrich) to induce drought stress during a seven-day cultivation period. Fifteen seeds of each variety were wrapped in Whatman filter paper (Whatman, Maidstone, England) and immersed in a 20% (w/v) solution of PEG 6000 to simulate drought conditions. For the control treatment, both primed and non-primed seeds were grown under normal conditions without any imposed stress. Each treatment was replicated three times to ensure reliability.

The paper rolls were randomly arranged in a growth chamber set to maintain an average day/night temperature of 22/19°C and 75% relative humidity for seven days (Vassilevska-Ivanova et al., 2014; Salleh et al., 2020). All soybean varieties were phenotypically evaluated for various traits including germination percentage (GP), GR, shoot length (ShL), root length (RL), seedling fresh weight (SFW), and seedling dry weight (SDW). The GP and GR of the samples at three and seven days were calculated as follows:

High-quality seeds were surface sterilized as described above. The experiment was organized into four treatment groups in CRD. Twenty seeds from each variety were placed in 350 mL plastic cups containing 50 mL of distilled water, covered with sterilized Petri dishes, and incubated for three days in a germination cabinet maintained at 25°C, following the protocol of Ali et al. (2018).

During this incubation period, electrical conductivity (EC) was measured daily to assess membrane integrity by monitoring ion leakage, such as potassium and sodium, which serves as an indicator of membrane damage and cell permeability. After three days, seeds demonstrating intact membranes, indicative of high quality and viability, were selected and transferred onto germination paper. The paper was then rolled to enclose the seeds, and the rolls were maintained for an additional seven days. Subsequently, various parameters were measured, including GR, ShL, RL, SFW, and SDW under both control and seed flooding conditions.

The activities of POD, SOD, and CAT, along with the malondialdehyde (MDA) content, were measured using a POD assay kit (A084-3), SOD assay kit (T-SOD, A001-1), CAT assay kit (A007-1), and MDA assay kit (A003), respectively.

All assays were conducted according to the manufacturer instructions provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China), with each biological replicate performed in triplicate. In brief, 1.0 g of fresh root tissue was ground into a fine paste using a mortar and pestle. The paste was then mixed with 9 mL of ice-cold 20× phosphate-buffered saline (PBS) solution (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with a pH of 7.2–7.4. The resulting homogenates were centrifuged at 3500 rpm for 10 min at 4°C. The supernatants were collected and used as crude extracts for the assays, with measurements conducted using a UV-1800 (Shimadzu Corporation, Analytical Instruments Division, Kyoto, Japan).

All experiments were replicated thrice and expressed as mean ± SD. Experimental data were statistically analyzed using SPSS 16.0 software (SPSS Inc., IBM SPSS Statistics, Chicago, IL, USA). Analysis of a two-factor completely randomized was used to evaluate the significance. Data were analyzed using Duncan’s multiple range test at 1% level of significance.

Figure 1A depicts the microstructure of the biopolymer composed of SCMC and GA (CG). The arrows indicate areas that exhibit essential surface attributes, including texture uniformity and particle dispersion, which are vital for evaluating material homogeneity and smoothness. The FT-IR spectra of SCMC and GA biocomposite biopolymer and SCMC biocomposite containing 10% GA are depicted in Figure 1B .

Broadband emission near 1590 cm⁻¹ was attributed to the N–H bending resonance in the case of SCMC, which overlapped the amide II vibration. The bands detected at 1419 and 1053 cm⁻¹ were caused by the stretching vibration of the C–O–C bond in the glycosidic linkage of the polymer and the scissoring vibration of the NH₂ group, respectively ( Figure 1B ). The spectra of the GA and the CG biocomposite biopolymer were nearly identical due to the structural similarity of the polysaccharides. In contrast, the introduction of GA into SCMC resulted in a modification of the wavelength values when compared to GA (3287 cm⁻¹), thereby establishing a robust absorption band at 3276 cm⁻¹ that facilitated O–H stretching ( Figure 1B ).

The expansive bond observed at 3276 cm⁻¹ was determined to result from elongation and unconstrained hydroxyl group vibrations within and between molecules. Furthermore, the presence of an additional peak (2878 cm⁻¹) in the spectrum, which was absent in both the SCMC and GA spectra, suggested the possibility of a cross-linked reaction between the two substances. The peak shifted marginally from 3277 to 3282 cm⁻¹ following the addition of GA, indicating that the surface OH of GA and the OH of CG molecules were in interaction. Collectively, these findings indicated that the incorporation of GA compounds into the SCMC was useful for enhancing the mechanical and barrier properties of the biocomposite.

In addition, XRD analysis shows the successful incorporation of GA into the SCMC biopolymer ( Figure 1C ). The region in question exhibits no discernible peaks besides the peak at a diffraction angle of 20 degrees resulting in a decrease in crystallinity. In addition, the results of the DSC analysis showed an improvement in the thermal stability of the biopolymer after adding GA, which could decrease the quantity of hydroxyl groups present in polysaccharides ( Figure 1D ). This causes the biopolymer’s structure to be enhanced.

The analysis of variance (ANOVA) results presented in Table 2 revealed significant effects of assessed varieties and treatments on the studied traits of soybean under drought and flooding stress conditions. Under drought stress, highly significant differences (P< 0.01) were observed among varieties and treatments for all parameters, including GR, GP, ShL, RL, SFW, SDW, MDA content, CAT activity, SOD activity, and POD activity ( Table 2 ). The interaction between varieties and treatments was also significant for most traits, such as ShL, RL, SFW, SDW, MDA content, CAT activity, SOD activity, and POD activity, indicating that the response to drought stress varied across the different soybean varieties ( Table 2 ).

Under flooding stress, highly significant differences differences (P< 0.01) were also observed for GR, GP, ShL, RL, SFW, and SDW among varieties and treatments ( Table 2 ). The interaction between soybean varieties and treatments was significant for ShL, RL, SFW, and SDW, suggesting variability in the response of different soybean varieties to flooding stress ( Table 2 ). Seed priming with 10% CG under drought stress treatment demonstrated a significant improvement in the germination parameters of assessed soybean varieties compared to the untreated control. Under normal conditions without any induced drought stress, seeds pretreated with 10% CG exhibited higher GR and GP than the untreated seeds, highlighting the effectiveness of the seed priming strategy ( Table 2 ). This indicates that 10% CG priming enhanced the seeds water uptake and metabolic activation, leading to more efficient germination.

Under drought stress, seeds pretreated with 10% CG maintained relatively higher GR and GP compared to the control ( Figures 2A, B ). This suggests that seed priming provides a protective mechanism, possibly through osmotic adjustment or the activation of stress-related pathways, that enables seeds to better withstand drought conditions.

In contrast, the physiological response to flooding stress revealed a different pattern. Under normal conditions, 10% CG seed priming still enhanced GR as observed in the control ( Figure 2C ). However, when exposed to flooding stress, the GP of seeds pretreated with 10% CG was slightly lower than the control group. This indicates that while seed priming generally boosts germination under optimal conditions, its efficacy diminishes under flooding stress.

Flooding can reduce seed respiration and oxygen availability, which 10% CG priming cannot fully address. The essential contention is that although seed priming has certain metabolic advantages, its effects are less significant under flooding stress than under drought conditions. This suggests that flooding presents a more complex challenge requiring additional or alternative strategies, such as the development of varieties with enhanced anaerobic germination tolerance or improved field management practices to prevent waterlogging.

Primed soybean seedlings demonstrated minimal phenotypic changes, maintaining fully expanded seedling structures across all priming treatments compared to the control (without seed stress) in six distinct soybean varieties ( Figures 3A, B ). These results suggest that seed priming effectively mitigates the adverse effects of water stress on the morphological traits of soybean seedlings. The ability to sustain fully expanded seedlings indicates that the priming treatments supported overall growth and development even under challenging drought and flooding conditions ( Figures 3A, B ).

Measurements of ShL and RL for different soybean varieties without seed priming showed a substantial decline after seven days of drought and flooding stress ( Figure 4 ). Statistical analysis revealed significant differences among the control group (CK) under normal conditions, CK+CG under normal conditions, and treatments under drought and flooding stress, as well as CG and drought stress under primed stress conditions. The results demonstrated that ShL and RL were significantly enhanced by all seed priming treatments under drought and flooding stress ( Figure 4 ).

Notably, ShL was considerably higher in all varieties with CK+CG, CG and drought stress, and CG and flooding stress seed priming treatments compared to CK, drought stress and flooding stress treatments without seed priming ( Figures 4A, C ). For RL, the T21R1001-02 variety displayed a significant increase when treated with CK+CG, CG and drought stress and CG and flooding stress, while untreated seeds (CK and S) exhibited lower RL values ( Figures 4B, D ). The findings confirmed that priming treatments significantly improved ShL and RL compared to the control (CK) and untreated drought and flooding stress treatments ( Figure 4 ). Specifically, CK+CG, CG and drought stress and CG and flooding stress seed priming treatments produced notably higher ShL values ( Figure 4 ).

Under drought stress, seeds primed with CG exhibited an increase of 16%, 54%, and 26% in both fresh and dry weight compared to unprimed seeds ( Figure 5 ).

Furthermore, SFW and SDW of all soybean varieties were significantly increased by all seed priming treatments under drought and flooding stress ( Figure 5 ). The highest ShL was observed in varieties treated with seed priming using SCMC and GA at a 10% concentration under water stress conditions ( Figure 4 ). Overall, the data indicated that seed priming interventions, particularly those using CG, substantially enhanced ShL, root development, and increased both SFW and SDW in soybean cultivars under drought stress. Seed priming with CG at a 10% concentration was particularly effective, yielding significantly higher SFW and SDW across all varieties in drought and flooding stress conditions ( Figure 5 ).

After seven days of germination under water-stress conditions, the seedlings exhibited a notable MDA accumulation ( Figure 6 ). However, it was observed that the CK+CG treatment (seed priming with 10% CG under normal conditions without exposure to stress) followed by CG and drought stress treatment (seed priming with 10% CG under drought stress) resulted in lower MDA content compared to the drought stress treatment (seeds exposed to drought stress without CG priming) across all varieties ( Figure 6 ). This finding suggests that seed priming with CG provides a protective effect against oxidative damage induced by drought stress, likely through enhanced stabilization of cellular membranes and mitigation of lipid peroxidation.

Furthermore, the activity of SOD, an antioxidant enzyme, significantly changed in response to water stress. Specifically, the drought stress treatment showed a significant increase in SOD activity compared to normal conditions. Additionally, the CG and drought stress treatment exhibited a further increase in SOD activity ( Figure 7A ). This indicates that seed priming with CG can enhance the antioxidant defense system, as reflected by the increased SOD activity under drought stress ( Figure 7 ).

Regarding other antioxidant enzymes, water stress was found to inhibit POD activity but improve CAT activity in both the drought stress, and CG and drought stress treatments ( Figures 7B, C ). However, seedlings germinated from seeds primed with CG showed significantly higher POD and CAT activities compared to seedlings without CG priming ( Figures 7B, C ). This suggests that CG priming can enhance the activities of these antioxidant enzymes, potentially contributing to better protection against oxidative damage caused by drought stress.

To evaluate the effects of the different seed priming treatments, the EC and GR of six different soybean varieties were examined under control and seed flooding stress. The highest EC was observed in soybean T21R1006-08 and T21R1009-11 under seed flooding stress without seed priming treatments ( Figure 8 ).

In seed priming treatments, the EC was significantly reduced in the case of all soybean varieties compared to unprimed seeds under seed flooding stress ( Figure 8 ). In the case of 10% CG treatment, the T21R1012-14 variety showed significantly lower EC. The other three varieties have higher EC but have no significant variation of EC among them ( Figure 8 ).