An experiment using Petri dishes and hydroponics was performed at the Agronomy Laboratory at Khulna Agricultural University, Khulna, from December 2022 to January 2023. The popular “BARI Soybea-5” type of high-yielding and moderately susceptible soybean was collected for the experiment from the Bangladesh Agricultural Research Institute, Gazipur, Bangladesh. The seeds (12% moisture content) were pre-treated with 1% NaOCl for 5 min to eliminate microorganisms from the seed surface. Seeds of soybean were primed with water or with 1- or 2-mM salicylic acid (SA) at the root temperature for 60 min. Thereafter, distilled water was used to wash the seeds. The seeds were dried back to their initial moisture content at room temperature. Based on preliminary screening results, the SA levels were chosen (Fig. A1). Based on previous research, 150 mM NaCl stress was applied [12,23]. For each treatment, thirty soybean seeds were used for priming and placed on 150 mm × 25 mm diameter Petri dishes. Three-layered tissue paper was used in the Petri dishes. Each dish was moistened with 5 mL of 150 mM NaCl for the salinity treatment and the 1- and 2-mM SA treatments, and one dish was filled with water (8 mL) for non-saline conditions. All the treatments consisted of three replications. The study included the following treatments: control (C), 1 mM SA (SA1), 2 mM SA (SA2), 150 mM NaCl (salt), 1 mM SA+150 mM NaCl (SA1+salt), and 2 mM SA+150 mM NaCl (SA2+salt).

The germinated seeds were counted every 24 h from initial germination to the seventh day. The GR (germination rate), FGP (final germination %), MGT (mean germination time), GI (germination index), and GE (germination energy) were calculated from the obtained data as described previously [24–26]. Using plant height, the SVI (seed vigour index) was calculated [27]. The following formulas were used for the GR, FGP, MGT, GI, GE and SVI calculations: GR =GP11+−−−−−+GPxx where GP1 = germination percentage on the 1st day after sowing.

GPx = germination percentage on the x^th day after sowing. FGP =No. of total seeds germinatedNo. of total used seeds×100 MGT=ΣDaNN where N = No. of seeds on day Da.

Da = No. of days from the 1st germination. GI = No.  of seeds germinatedDay of 1st count+…+No.  of seeds germinatedDay of last count GE=Tj1Nj×100 where Tj1 = number of seeds germinated on the 1st day.

Nj = No. of total seeds.

SVI = final germination percentage × plant height (cm)

where plant height = length of shoot + length of root.

A modified hydroponic method was utilized for growing sprouted seeds in 3 L pots as previously described [23]. Each pot contained three seedlings. Thirty-day-old seedlings were subsequently exposed to a ten-day period of stress with 150 mM NaCl. During the period of ten days of stress, 1- or 2-mM SA was sprayed on the plants (sprayed every day at 10:00 and 22:00; 3 mL per spray per plant). Following ten days of SA spraying, one seedling per pot was used to measure the length of the roots (RL), fresh weight of the roots (RFW), length of the shoots (SL), fresh weight of the shoots (SFW), and relative water content (RWC). Fresh leaves were collected from another plant to analyse plant pigments, H₂O₂, and the MDA content. Upon completion of a 72-h drying period at 60°C, the root and shoot dry weight (RDW and SDW) were determined via an electric balance.

The remaining one seedling from per pot was used to measure the leaf water status. The relative water content (RWC) was calculated as previously described [28]. The fresh weight (FLW) of a single leaf per treatment was noted, and then the leaf was overwhelmed for 1 and 2 h in distilled water. The turgid weight (TLW) was quickly noted after all water was blotted with the same tissue. The dry weight (DLW) of the leaves was noted after 48 h of drying at 72°C. The following formula was used to calculate the RWC: RWC (%) = FLW−DLWTLW−DLW×100

Subsequent to the relative water content calculation, the relative water loss (RWL) was derived via the bellow formula [29]: RWL(%)=1−[ FLW−DLWTLW−DLW×100]

Approximately 500 mg of leaf material was put into a 15 mL tube containing 80% ethanol (10 mL). For pigment extraction, the tubes were left in darkness for 10 days. Photosynthetic pigments were quantified via spectrophotometers operated at 663, 645, and 480 nm (Shimadzu UV-2550, Kyoto, Japan) via the Ibiang et al. [30] and Ridley [31] methods:

Chlorophyll a = (Abs. at 663 × 0.999 − Abs. at 645 × 0.0989)

Chlorophyll b = {Abs. at 663 x (−0.328) + Abs. at 645 × 1.77}

Total Chlorophyll = Chlorophyll a + Chlorophyll b

Carotenoids = {(Abs. at 663 × 0.114 − Abs. at 645 × 0.638) + Abs. at 480}

Malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were assessed in soybean seedlings as described by Zhang et al. [32] and Velikova et al. [33], respectively. A UV–VIS spectrophotometer with wavelengths of 532 and 390 nm (T80, PG Instruments, China) was used to measure the MDA and H₂O₂ contents, respectively.

The data were analysed via one-way ANOVA, and Tukey’s HSD test (p < 0.05) was employed to distinguish means. A heatmap was generated in R 4.2.3 via the ‘pheatmap’ package. Principal component analysis (PCA) was conducted via the ‘GGally’and ‘factoextra’ packages.

The effects of priming seeds with SA on the germination metrics of soybean seeds under NaCl stress are presented in Fig. 1. The results demonstrated that the germination rate (GR) substantially decreased under salt condition compared with the control condition. However, the results revealed that SA1 and SA2 priming under salt stress substantially increased GR (Fig. 1A). In terms of the final germination percentage (FGP), salt stress substantially abated the FGP in comparison with that in the control condition (Fig. 1B). Compared with salinity stress, the seeds primed with SA substantially increased the FGP (Fig. 1B). Moreover, the mean germination time (MGT) substantially increased under condition of salinity stress compared with that under non-stress condition. The results revealed that MGT substantially decreased under the SA1+salt and SA2+salt conditions compared with the salt condition (Fig. 1C). Salinity stress substantially decreased the germination index (GI); however, SA1 and SA2 priming substantially increased the GI during salt stress (Fig. 1D). Similarly, priming with SA1 and SA2 substantially increased the germination energy (GE) under salt stress and control condition compared with that under condition of salt stress alone (Fig. 1E). In contrast to the salt stress scenario, priming with SA increased the seed vigor index (SVI), whereas salt stress drastically abated the SVI. Compared with that under salt stress, the SVI substantially increased for SA1+salt and SA2+salt (Fig. 1F). These findings demonstrated that, under conditions of salinity stress, SA2 resulted in greater germination metrics than did SA1.

We measured the root length (RL), root fresh weight (RFW), root dry weight (RDW), shoot length (SL), shoot fresh weight (SFW), shoot dry weight (SDW), and plant height (PH) of the soybean seedlings to determine the consequences of NaCl stress and SA actions for stress mitigation (Table 1). Compared with non-stressed plants, salt-treated plants presented substantially reduced RL. Administering SA substantially increased RL in SA1+salt- and SA2+salt-treated plants. Moreover, RFW under condition of salt stress was substantially lower than that under the control condition, but supplementation with SA2 substantially increased RFW under salt condition. Moreover, the RDW was substantially greater in the SA1+salt- and SA2+salt-treated plants than in the salt-induced plants.

A substantial reduction in SL was noticed in salt-induced plants compared with non-stressed plants, and supplementation of plants with SA1 and SA2 substantially increased SL under salt condition. Additionally, SFW was substantially affected and reduced in salt-induced plants compared with non-stressed plants. Exogenous supplementation with SA1 and SA2 helped plants recover from injury and resulted in a substantial increase in SFW under salt condition. Moreover, the SDW was substantially increased by the application of SA1 and SA2 to the salt-stressed plants. Salt stress substantially affects PH. However, compared with salt conditions, supplementation with SA1 and SA2 substantially increased plant height under salt condition. The results also indicated that SA2 outperformed SA1 in terms of seedling growth traits under salt condition.

This study assessed RWC and RWL both in the presence and absence of NaCl using SA to determine the soybean leaf water status (Table 1). The results indicated that RWC was greatly decreased by salt addition relative to the control condition at both 1 and 2 h. The lowest RWC (46.677%) at 1 h and 44.636% at 2 h was measured for the salt-stressed plants compared with the non-stressed plants. However, during salt stress condition, the application of SA resulted in a substantial increase in RWC at both 1 and 2 h in the SA1+salt- and SA2+salt-treated plants compared with the salt-stressed plants. Alternatively, a substantial increase in RWL was also recorded in the salt-stressed plants at both 1 and 2 h. The highest RWL values (53.323%) at 1 h and 55.364% at 2 h were observed for the salt-treated plants, in contrast with those of the non-treated plants. Compared with those of salt-stressed plants, substantially lower RWLs were measured for SA1+salt- and SA2+salt-treated plants. These outcomes indicated that SA2 upheld better water status in soybean than did SA1 under salt condition.

Significant differences in photosynthetic pigment levels were noticed as a result of salt stress (Fig. 2). The outcomes demonstrated that the chlorophyll a level was substantially lower in salt-treated plants than in non-stressed plants. However, SA supplementation substantially increased the chlorophyll a level in SA2+salt-treated plants compared with that in salt-induced plants (Fig. 2A). Compared with non-stressed plants, salt-stressed plants presented substantially lower pigment levels. In contrast, compared with the salt-induced plants, the SA1+salt- and SA2+salt-stressed plants presented substantially increased chlorophyll b contents (Fig. 2B). The total chlorophyll content was also substantially affected by salt stress relative to non-stressed conditions (Fig. 2C). Compared with salt-treated plants, SA supplementation substantially increased the total chlorophyll content in the leaves of the SA1+salt-treated and SA2+salt-treated plants (Fig. 2C). Moreover, the carotenoid content was substantially lower in salt-induced plants than in untreated plants. Despite these findings, SA1 and SA2 spraying resulted in substantially greater contents of carotenoids in the leaves of the soybean plants under salt condition than in those of the salt-stressed plants (Fig. 2D).

We subsequently investigated the effects of SA supplementation on alleviating the oxidative damage induced by NaCl in soybean leaves by measuring H₂O₂ and MDA contents. The findings of the present study revealed that, compared with non-treated plants, salt-treated plants substantially increased the production of H₂O₂ (Fig. 3A). Conversely, SA supplementation of salt-induced plants hindered the onset of oxidative injury, as evidenced by substantially lower levels of H₂O₂ in SA1+salt- and SA2+salt-treated plants than in salt-stressed plants. (Fig. 3A). Under salt stress, the MDA content in the leaves of soybean plants significantly increased. In contrast, the SA1+salt- and SA2+salt-stressed plants presented considerably lower MDA levels (Fig. 3B). The upper results also revealed that SA2 supplementation raised the photosynthetic pigments and decreased the H₂O₂ and MDA contents much more than SA1 supplementation did under saline condition.

The average values of the studied parameters were employed to generate a heatmap and perform principal component analysis (PCA) (Fig. 4). Along the variable axis, two groups (Clusters a and b) were identified via hierarchical clustering (Fig. 4A). Cluster-a consists of the H₂O₂, RWL2, RWL1, MDA, and MGT parameters. The Cluster-a parameters tended to increase in the salt-stressed plants, with a downwards trend observed in the C, SA1, SA2, SA1+salt, and SA2+salt-stressed plants. Cluster b included the variables SVI, GI, FGP, SFW, RL, RDW, PH, SL, RWC2, RWC1, Caro, RFW, TChl, SDW, Chl b, GE, and Chl a. The Cluster b factors tended to increase in C, SA1, SA2, SA1+salt, and SA2+salt-stressed plants but decreased in salt-stressed plants. In addition, PCA was conducted to analyse the relationships among the studied parameters and the treatments (Fig. 4B). The PCA scores were divided into six treatments according to their PC1 and PC2 positive and negative values. PC1 together with PC2 collectively represented 97.32% of the data variability across the treatments and all of the soybean seedling parameters investigated. In this scenario, PC1 accounted for 93.79% of the data variability and separated C, SA1, and SA2 from salt and SA1+salt and SA2+salt treatments on the basis of their positive and negative PCA scores (Fig. 4B). Furthermore, PC2 showed only 3.53% data variability (Fig. 4B).