The experiment utilized alfalfa (Medicago sativa L.) seeds of the variety ‘Bara 416WET,’ obtained from Bailu (China). This high-yield, fibrous-root type cultivar is characterized by strong disease resistance, rapid regeneration, and tolerance to moisture stress.

Uniform and plump alfalfa seeds were selected and disinfected with a 1% sodium hypochlorite (NaClO) solution for 10 min, then rinsed thoroughly with distilled water and air-dried. The seeds were placed in 9 cm diameter Petri dishes lined with double-layer filter paper. Thirty seeds were evenly distributed in each dish. The filter paper was moistened with approximately 5 mL of the respective treatment solutions. The dishes were covered and incubated at 25°C under a photoperiod of 16 h light and 8 h dark. Each petri dish was considered one replicate, and four replicates were prepared for each treatment. Germination was monitored daily at 6 p.m., and water loss due to evaporation was compensated using the weighing method. For drought and calcium salt stress treatments, deionized water was used as the control, while for pH stress treatments, a medium with a pH of 7 served as the control.

Seed germination potential and germination rate were determined following the Chinese National Standard GB/T 2930.4–2001, using radicle emergence as the criterion:

Germination index: calculate the germination index on the 14th day of cultivation, with germination index (GI) = ∑ (Gt/Dt), where Gt is the number of sprouts on the t-th day; Dt is the corresponding germination day;

Vitality index (VI) = germination index x shoot length.

On the 14th day of cultivation, the embryo length and embryonic root length of alfalfa seedlings from each treatment were measured using a millimeter ruler with an accuracy of 0.1 cm.

For fresh and dry weight measurements, five seedlings were randomly selected from each Petri dish. Surface moisture was removed using filter paper. The fresh weight was measured using an analytical balance with a precision of 0.0001 g. The seedlings were then dried to constant weight in an oven at 80°C, and the dry weight was recorded.

Data were organized and plotted using Microsoft Excel 2021 and Origin 2022. Statistical analyses were performed using SPSS 27.0 software. One-way analysis of variance (ANOVA) was conducted, and Tukey’s multiple range test was used for comparisons among means at the 5% significance level (p < 0.05). Results were expressed as mean ± standard error (SE).

As shown in Table 1, compared with the control (0 mM CaCl₂), a calcium salt concentration of 20 mM significantly promoted germination rate (GR%) and germination potential (GP%), increasing them by 13.33 and 11.67%, respectively, while the vigor index (VI) increased by 10%. High concentrations of calcium salt (CaCl₂ concentration ≥ 80 mM) had a significant inhibitory effect on the germination index (GI). These results indicate that a calcium salt concentration of 20 mM is optimal for alfalfa seed germination.

PEG6000 was used to simulate drought stress at different osmotic potentials. As shown in Table 2, compared to the control (0% PEG6000), a PEG6000 concentration of 20% significantly inhibited all germination indicators. PEG6000 concentrations ≥10% significantly inhibited GI and VI, while concentrations ≥7% significantly inhibited VI. At a PEG6000 concentration of 5%, GR%, GP%, and GI reached their maximum values, increasing by 11.67, 9.17, and 0.99%, respectively, compared to the control. This suggests that a PEG6000 concentration of 5% is most conducive to seed germination.

As shown in Table 3, certain pH treatments promoted alfalfa seed germination. At pH 6, GR%, GP%, and GI were enhanced compared to the control (pH 7), although VI was slightly inhibited. At pH 8, all germination index values increased; compared to the control, GR% and GP% increased by 10%, GI increased by 1.99%, and VI increased by 6.47%. Therefore, pH 8 appears to be the optimal pH for seed germination.

Based on the analyses of the effects of calcium salt, drought, and pH treatments on alfalfa seed germination, it was determined that a 20 mM CaCl₂ solution is most beneficial for seed germination. Additionally, low concentrations of polyethylene glycol (5% PEG6000) and distilled water adjusted to pH 8 can effectively promote seed germination.

Figure 1 and Supplementary Table S1 show that calcium salt stress inhibits the elongation of both the shoot and the root. The root is more sensitive to calcium salt stress than the shoot, with high concentrations of calcium salt (80 mM) having a significant negative impact on radicle growth.

As presented in Figure 2 and Supplementary Table S2, the growth of alfalfa shoots and roots is less affected by fluctuations in drought stress. Low concentrations of PEG6000 promote the growth of roots, while high concentrations have the opposite effect. When the PEG6000 concentration was 5 and 7%, root length increased by 17.5 and 8.75%, respectively, compared to the control. Higher concentration treatments had inhibitory effects on root growth, but the differences were not significant compared to the control.

Figure 3 and Supplementary Table S3 indicate that roots are more susceptible to pH changes. At pH 8, shoot growth was promoted, with a 0.68% increase in shoot length compared to the control (pH 7). At pH 6, root growth was more significantly promoted than with 5% PEG6000, showing a 27.76% increase compared to the control. The data show no significant difference in shoot length between the pH 4–6 and pH 8–9 groups compared to the control, nor in root length between the pH 5–6 and pH 8 groups.

Based on the effects of calcium salt stress, drought stress, and pH stress on the growth of alfalfa shoots and roots, we conclude that pH 8 promotes shoot growth, while low concentrations of PEG6000 (5 and 7%) and distilled water at pH 6 promote root growth, with pH 6 having a more significant effect on roots.

The changes in biomass and tissue moisture content of ‘Bara 416WET’ seedlings under different concentrations of calcium stress are shown in Figure 4. As the concentration of calcium stress increased, the fresh weight, dry weight, and tissue moisture content of alfalfa seedlings showed a trend of first decreasing, then increasing, and then decreasing again. Among the experimental groups, the fresh weight and dry weight reached their maximum values at 40 mM CaCl₂, increasing by 18.79 and 18.18%, respectively, compared to the control. The tissue moisture content decreased slightly by 0.17% compared to the control.

Figure 5 shows the changes in biomass and tissue moisture content of alfalfa seedlings under different levels of drought stress. Under various concentrations of PEG6000, the fresh weight and tissue moisture content were lower than the control, but the dry weight was slightly higher. At 10% PEG6000, the dry weight reached its maximum value, increasing by 17.24% compared to the control. Correspondingly, the fresh weight and tissue moisture content decreased by 25.43 and 2.96%, respectively.

Figure 6 illustrates the changes in seedling biomass and tissue moisture content under different pH levels. At pH 9, both dry weight and fresh weight reached their maximum increases of 5.26 and 15.54%, respectively, relative to the control. The corresponding tissue moisture content was relatively high, increasing by 0.72%, only 0.05% lower than the maximum value of 93.7% observed at pH 6. The fresh weight at pH 6 increased by 7.14% compared to the control (pH 7).

Figure 7 shows the correlation between alfalfa seed germination and various seedling indicators under calcium salt stress. CaCl₂ concentration was significantly negatively correlated with GI, VI, shoot length, and root length, which is consistent with the results shown in Table 1 and Figure 1. Additionally, there was no significant correlation between calcium concentration and GP%, GR%, tissue water content, fresh weight, and dry weight. The correlation analysis between drought stress and various indicators during the germination stage is shown in Figure 8. The PEG6000 concentration was significantly negatively correlated with GP%, GR%, GI, VI, fresh weight, tissue moisture content, and shoot length. Figure 9 presents the correlation analysis between germination and seedling growth indicators under pH stress. The pH was significantly positively correlated with GI, dry weight, and tissue moisture content; it was also significantly positively correlated with VI, fresh weight, and shoot length.

It is noteworthy that under calcium salt, drought, and pH stress, there was no significant correlation between root length and fresh weight or dry weight. In contrast, shoot length was highly or significantly correlated with fresh weight across all treatments and was significantly correlated with dry weight only under pH stress. These results confirm the consistency of the data analysis presented in Sections 3.2 and 3.3. The correlation matrix graphs indicate a certain degree of correlation between each indicator, resulting in overlapping growth effects. Therefore, evaluating the effects of different treatments on alfalfa seed germination and seedling growth should be based on multiple indicators to ensure reliability.

An appropriate concentration of CaCl₂ solution can significantly improve seed germination rate, germination vigor, and vigor index, positively impacting seed germination quality. Our results showed that germination indicators of alfalfa first increased and then decreased with the rise in calcium ion concentration. This may be because low salt concentrations promote water absorption to some extent, accelerating the seed germination process (Cornacchione and Suarez, 2017). However, when the salt concentration exceeds a certain threshold, osmotic stress occurs, making it difficult for cells to absorb water. Excessive ions entering the cells can cause toxicity, leading to membrane damage, decreased seed vitality, and even cell death (Carillo et al., 2019).

The experimental results indicated that calcium salt stress inhibited the growth of both the aboveground parts and root systems of alfalfa, with a significantly greater effect on root length than on seedling height. Wang et al. (2023) also found in their studies on salt tolerance evaluation of alfalfa varieties during germination that embryonic root length was more sensitive to salt stress than shoot length. Under moderate calcium stress (40 mM), the biomass and tissue water content of alfalfa reached their peak, while other calcium stress concentrations caused a relatively small decrease in these parameters, with no significant difference compared to the control (Supplementary Table S4). This suggests that this alfalfa variety can maintain a high growth rate and biomass accumulation in calcium-rich environments, demonstrating a strong tolerance to calcium salts. Zhou et al. (2023) reported that varieties with stronger salt tolerance might possess better salt regulation mechanisms, such as more efficient salt excretion or ion balance regulation.

The ability of plants to maintain good water status and effectively utilize available resources is crucial for their growth and survival in water-scarce environments. Our study revealed that alfalfa seeds showed no significant difference in germination rate and vigor under PEG6000 concentrations ranging from 5 to 15%. However, at a PEG6000 concentration of 20%, the germination rate significantly decreased, consistent with the findings of Liu et al. (2018). The germination index and vigor index under mild drought (5% PEG6000) did not differ significantly from those under adequate water conditions. In fact, the germination rate under 5 to 10% PEG6000 was higher than that under the control treatment, aligning with the research of Yu et al. on alfalfa germination under certain drought stress (Yu et al., 2012). Slight differences in drought resistance among different varieties were observed.

Our study also showed that drought stress reduced the growth of alfalfa shoots and had a “low promotion and high inhibition” effect on root growth. Low concentrations of drought stress (5 and 7% PEG6000) promoted root growth, while higher concentrations inhibited it. Although root growth was not significantly affected compared to the control, root length decreased with increasing PEG6000 concentration (Supplementary Table S2). This may be due to the enhanced absorption capacity of the root system under water deficit conditions (Huang et al., 2024), leading to an increased root-to-shoot ratio (Liu et al., 2004; Turner and Begg, 1981). Han Zhishun et al. also found in their study on the effects of drought stress on the morphological and physiological characteristics of different alfalfa varieties that root length first increased and then decreased (Han et al., 2020). Interestingly, under water-deficient conditions, the fresh weight and tissue moisture content of alfalfa seedlings were lower than those under adequate water, but the dry weight increased. This indicates that while the overall growth of alfalfa seedlings is inhibited under drought stress, they exhibit strong tolerance to water-deficient environments.

Our results indicate that extreme pH values inhibit alfalfa germination, possibly due to high acidity or alkalinity damaging cell membranes, causing cellular system disorders, and inhibiting germination. Alfalfa seeds exhibited good tolerance to both acidic and alkaline conditions, with distilled water at pH 6 and pH 8 promoting germination. We found that pH 4–9 had no significant effect on the aboveground parts of seedlings, while the roots were more sensitive to pH changes. Weakly acidic conditions (pH 6) were beneficial for root elongation, whereas weakly alkaline conditions (pH 8) slightly inhibited root growth.

Alkaline stress had no significant effect on the fresh weight, dry weight, and tissue moisture content of alfalfa seedlings. The key for plants to cope with high pH lies in the regulatory role of the root system, mainly reflected in the synthesis and secretion of organic acids. Plants synthesize organic acids by absorbing large amounts of inorganic anions (Cl⁻, SO₄²⁻, NO₃⁻) to balance cation accumulation, maintaining ion balance and pH stability (Liu and Shi, 2010). Organic acids serve as precursors for the synthesis of many compounds and are important intermediates in substance and energy metabolism, playing crucial roles in plant responses to environmental changes (José et al., 2000). Additionally, research has shown that the regulation of organic acid metabolism may play different roles under different types of stress (Chen et al., 2011).