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Article

Supplementation of Calcium Through Seed Enrichment Technique Enhances Germinability and Early Growth of Timothy (Phleum pratense L.) Under Salinity Conditions

by
Masahiro Akimoto
1,* and
Li Ma
2
1
Department of Agro-Environmental Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
2
School of Agriculture and Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1905; https://doi.org/10.3390/agronomy15081905
Submission received: 30 June 2025 / Revised: 31 July 2025 / Accepted: 6 August 2025 / Published: 7 August 2025
(This article belongs to the Special Issue Responses of Forage Crops to Environmental Stresses and Stress Alleviating Strategies)
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Abstract

Calcium ameliorates salt-related growth defects in plants. The objective of this study was to determine whether supplying calcium through a seed enrichment technique enhances the germinability and early growth of timothy (Phleum pratense L.) under saline conditions. For seed enrichment, timothy seeds were soaked in CaCl2 solutions at concentrations of 50 mM or 100 mM for 24 h at room temperature. Seeds treated with distilled water served as the control. Under distilled water conditions, germination rates among the seeds showed minimal variation, approximately 95% on average. However, in a 200 mM NaCl environment, the germination rate of the control seeds significantly decreased to 25%, while the germination rates of the Ca-enriched seeds remained high, exceeding 86%. Additionally, the Ca-enriched seeds germinated more quickly than the control seeds. When plants were grown with distilled water, the total dry matter weights did not differ significantly among the treatment types. However, under salt stress with 100 mM NaCl, the plants derived from Ca-enriched seeds thrived and exhibited higher dry matter weights compared to the control plants. The Ca-enriched seeds contained more soluble sugars and demonstrated higher catalase activity than the control seeds, and their corresponding plants accumulated less sodium under salt stress compared to the control plants. Seed enrichment is an effective technique for supplying calcium to timothy, and a concentration of 50 mM of CaCl2 in the treatment solution is sufficient to achieve salt tolerance.

1. Introduction

The establishment of artificial grasslands and forage production serves as the foundation for effective livestock farming operations. The stable production of forage crops on artificial grasslands plays a crucial role in achieving self-sufficiency in feedstuffs and in sustaining the agro-environment by preventing soil erosion, land degradation, and the decline of natural vegetation due to overgrazing [1]. However, in semi-arid regions where forage grasses are commonly cultivated, productivity has significantly declined in many areas, with salinity-related damage identified as one of the primary causes [2].
Excessive salt accumulation in the soil, which occurs in environments where the rate of water evapotranspiration exceeds that of rainfall, is one of the most detrimental abiotic stresses inhibiting crop growth [3]. Strategies to mitigate salinity-related damage and restore crop productivity include soil remediation, the introduction of salt-tolerant crops, and the enhancement of cultivation methods. While soil remediation through the use of soil-improvement materials [4,5] and green manure [6] has been implemented, it poses challenges in large fields and pastures due to the significant costs and labor involved [7]. Additionally, the introduction of salt-tolerant crops is unlikely to serve as a comprehensive solution, given the limited diversity of available salt-tolerant varieties [8,9]. In light of these challenges, it is crucial to establish effective cultivation methods that enable crops to thrive in salt-affected environments.
Research conducted to date has reported on the effects of various substances in enhancing the salt tolerance of crops. Inorganic salts, such as boron and calcium [10,11,12,13], as well as soluble organic compounds like polyamines, sugar alcohols, and salicylic acid [14,15,16], are effective in cellular osmotic regulation and sodium scavenging mechanisms. Therefore, supplying these substances to crops can increase their salt tolerance. Calcium is particularly advantageous due to its affordability, availability, and ease of handling. It is well-established that calcium promotes the appropriate regulation of cell osmolarity, effectively scavenges reactive oxygen species (ROS), and suppresses sodium overaccumulation. Research has demonstrated its effectiveness in enhancing crop growth under salinity stress [12,17,18,19,20,21]. However, when calcium is applied as a calcareous fertilizer, such as quicklime or slaked lime, it tends to form insoluble crystals under alkaline soil conditions and remains in the soil as calcium carbonate (CaCO3). Consequently, continuous or excessive application can lead to an accumulation of salts in the soil.
Seed enrichment is an alternative method for providing calcium to crops. This technique involves soaking seeds in a treatment solution that contains the desired substance, allowing the seeds to absorb the solution (in some references, this treatment is referred to as seed priming) [22,23]. By utilizing this method to provide essential nutrients [24,25,26,27,28,29] and hormonal substances [30,31,32,33,34,35], it is possible to regulate the growth and physiological state of the crops. The seed enrichment technique can supply calcium directly to crops, regardless of soil conditions, and is expected to enhance crop salt tolerance and productivity in saline areas. It has been reported that seed enrichment with a 50 mM CaCl2 solution enhances germinability and early seedling growth of wheat [36] and barley [37] seeds under 200 mM NaCl conditions. In tomato seeds, treatment with a 10 mM Ca(NO3)2 solution improved early seedling growth under 100 mM NaCl conditions [38]. These studies concluded that the enhanced salt tolerance was attributed to the ability of the supplied calcium to regulate cell osmolarity and sodium content, as well as to activate antioxidant enzymes. However, there is currently no information on the effectiveness of this technique in enhancing salt tolerance in temperate grass species.
Timothy (Phleum pratense L.) is a tall-statured temperate forage grass that is cultivated as a primary meadow grass in high-latitude temperate regions due to its excellent cold tolerance and suitability for livestock [39]. With the development of shorter, tillering varieties that are well-suited for grazing, timothy is also used to establish pastures with other forage grasses and legumes, thereby enhancing the reliability of forage production [40]. However, timothy exhibits lower salt tolerance compared to other temperate meadow grasses, such as tall fescue and orchard grass [41,42]. Wu et al. [43] reported that timothy experiences significant germination issues under salinity stress of 120 mM or higher, while tall fescue maintains its germination rate even under salt stress of 200 mM [44]. Soliman et al. [42] demonstrated that tall fescue grown hydroponically showed no significant growth impairment even under 200 mM NaCl, whereas timothy exhibited a marked reduction in plant size under 100 mM NaCl conditions and virtually no growth under 200 mM NaCl. This lower salt tolerance in timothy makes it susceptible to challenges in establishing grasslands and ensuring sustainable use in salt-affected areas [41].
In crops such as forage grasses, disturbances and damages caused by environmental stresses occur more frequently during the germination and juvenile growth stages, when individual plants are still small [45]. A similar trend is anticipated in timothy grass [41]. It can be hypothesized that supplying calcium to pre-sowing timothy seeds through seed enrichment may enhance their germinability and juvenile growth under salt-affected conditions, thereby contributing to the reliable forage production. The aim of this study is to verify whether calcium supply through seed enrichment improves the salt tolerance of timothy. We focused on the effect of calcium on the initial growth of timothy, which is crucial for assessing stress tolerance, and investigated whether the provision of calcium to the seeds enhances their germinability and juvenile growth under salt stress conditions.

2. Materials and Methods

2.1. Plant Materials

The timothy variety ‘Horizon’ (Yukijirushi Seed Cooperation, Sapporo, Japan) was utilized for all experiments. This variety is a widely used early timothy type for hay and silage production in Japan, known for its excellent overwintering and lodging resistance, as well as its stable productivity in high-latitude regions. The thousand-seed weight of this variety is approximately 350 g. Seeds were procured from a seed company in the year the experiments were conducted (2020).

2.2. Calcium Application Through Seed Enrichment

As a source of calcium for seed enrichment, anhydrous calcium chloride (CaCl2. Fujifilm Wako Pure Chemical Co., Osaka, Japan) was utilized. Since the optimal concentration of the treatment solution for timothy was unknown, two aqueous solutions of CaCl2 at concentrations of 50 mM and 100 mM were prepared based on previous studies conducted on other crops [23]. Twenty grams of timothy seeds were placed in a 9 cm-diameter Petri dish, and 20 mL of the treatment solution at either 50 mM or 100 mM was added to fully saturate the seeds. After allowing the Petri dishes to sit at room temperature (approximately 20 °C) for 24 h, the seeds were removed, rinsed with distilled water to eliminate surface calcium, and dried in a ventilated dryer (DSJ-7-1A, Shizuoka Seiki Co. Ltd., Fukuroi, Japan) set to 40 °C for 48 h. The dried seeds were then placed in sealed plastic bags and stored in a refrigerator until four days before the germination and plant growth tests (see Section 2.3 and Section 2.5) were conducted. For convenience, seeds treated with the 50 mM and 100 mM solutions will be referred to as 50 mM-Ca and 100 mM-Ca, respectively. Seeds treated with distilled water (seeds saturated with distilled water at 20 °C for 24 h and dried in a ventilated dryer set at 40 °C for 48 h) are designated as control.

2.3. Germination Tests

Two types of germination tests were conducted to assess the germinability of timothy seeds under salt stress and to evaluate the effect of calcium on this process.
Experiment 1: Two sheets of filter paper were placed in a 9 cm-diameter Petri dish, to which 1.5 mL of distilled water, a 100 mM NaCl solution, a 200 mM NaCl solution, or a 300 mM NaCl solution was added. Because timothy seeds are small and challenging to arrange uniformly, seventy to one hundred control seeds were spread evenly without overlapping on the filter paper in each Petri dish. These dishes were then placed in an incubator (MIR-253, Panasonic Co., Tokyo, Japan) set to a constant temperature of 17 °C under dark conditions. The number of germinated seeds was recorded every 12 h. In this series of germination tests, a seed was considered germinated if its taproot elongated by more than 1 mm. The experiment was terminated 14 days after sowing, and the number of seeds that had not germinated by the end of the experiment was recorded. Four Petri dishes were prepared for each solution per experiment, and four replicates were conducted.
Experiment 2: Two sheets of filter paper were placed in a 9 cm-diameter Petri dish, to which 1.5 mL of a 200 mM NaCl solution was added. The concentration of NaCl in this experiment was determined based on the results of Experiment 1, in which a salt concentration of 200 mM had a significant effect on germination (see Section 3.1). Seventy to one hundred control seeds, as well as 50 mM-Ca seeds and 100 mM-Ca seeds, were placed on the filter paper in the Petri dish. Additionally, Petri dishes using distilled water instead of the 200 mM NaCl solution were prepared, and the treated seeds were placed in the same manner. These Petri dishes were then placed in an incubator set to a constant temperature of 17 °C in the dark. The number of germinated and ungerminated seeds was recorded in the same manner as in Experiment 1. Four Petri dishes for each solution/seed combination were prepared per experiment, and four replicates were conducted.
Using the data obtained from the respective experiments, the germination rate (GR), mean germination time (MGT), coefficient of uniformity of germination (CUG), and germination index (GI) were calculated using the following methods [46,47].
GR was determined by dividing the cumulative number of seeds that had germinated until each survey by the total number of seeds.
M G T = ∑ t n / ∑ n ,
where t denotes the time elapsed since the start of the test, and n represents the number of newly germinated seeds at time t. Lower MGT values indicate that the seeds have germinated more quickly.
C U G = ∑ n / ∑ t ’ − t 2 n ,
where t and n represent the time elapsed since the start of the test and the number of newly germinated seeds at time t, respectively. The variable t’ denotes the average time required for germination, which is equivalent to the MGT. Higher values of CUG indicate that the seeds began germination more uniformly.
G I = ∑ ( n i   *   t 29 − i ) / ( t 28   *   T )
where ni represents the number of newly germinated seeds at the i-th observation, tj denotes the time from the start of the test to the j-th observation, and T is the total number of seeds used in the test. In this germination test, the observation period was set at 14 days, with observations conducted every 12 h, resulting in a total of 28 observations. Higher GI values indicate greater seed germinability.

2.4. Measurement of the Catalase Activity and Water-Soluble Sugar Content of the Seeds

To determine catalase (CAT) activity and soluble sugar content, the control, 50 mM-Ca, and 100 mM-Ca seeds were immersed in 200 mM-NaCl solution at 20 °C for 24 h and then rinsed three times with distilled water. For the extraction of crude enzymes, 50 mg of the dried seeds (approximately 150 seeds) were ground in a mortar containing 1.5 mL of 50 mM cooled potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 15,000× g at 4 °C for 10 min. The resulting supernatant was used to estimate catalase activity. CAT activity was determined according to a modified method by Farooq et al. [32]. Fifty microliters of the enzyme extract were added to 2.0 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM hydrogen peroxide. The decrease in absorbance at 240 nm over 10 min, from 1 to 11 min after the start of the reaction, was recorded. Enzyme activity was expressed in micromoles of H2O2 consumed per minute per gram of seeds. Eight measurements were replicated for each seed. For soluble sugar analysis, 100 mg of the ground seeds were mixed with 10 mL of distilled water and incubated for 1 h at 90 °C with gentle agitation. The filtrate of the mixture was used to determine soluble sugar content using the phenol-sulfuric method [48]. Eight measurements were replicated for each seed. All the chemicals used in the experiments were products of Fujifilm Wako Pure Chemical Co, Osaka, Japan.

2.5. Plant Growth Test Under Salt Stress Conditions

The control, 50 mM-Ca, and 100 mM-Ca seeds were sown in 7.5 cm diameter pots filled with commercially available fertilizer-free soil. For each treatment, 35 pots were prepared. The pots were placed in a growth chamber (MIR-553, Panasonic Co., Osaka, Japan) maintained at a constant temperature of 17 °C with a 12 h light cycle, providing an irradiance of 26.3 μmol/m2/s. Timothy plants were grown using a 1/2 Hoagland solution, excluding the calcium component. On the fifth day of cultivation, the emerged plants were thinned to a density of four individuals per pot. On the 21st day of cultivation, five pots from each treatment group were randomly selected, and the plants were harvested. After washing the soil off the roots, the plants were separated into root and shoot components and weighed for dry matter (0 days after treatment, or 0 DAT). On this day, the remaining pots for each treatment were divided into two groups of 15 pots each. One group received 30 mL of distilled water daily, while the other group received 30 mL of a 100 mM NaCl solution daily. The concentration of NaCl in this experiment was determined based on the findings from our preliminary test using this variety. In that test, most plants withered within a week, regardless of the treatments applied, when a 200 mM NaCl solution was used. This made it impossible to collect reliable data. For NaCl-treated pots, 100 mL of distilled water was applied every 7 days from 0 DAT to leach out the NaCl accumulated in the soil. Five pots from each group were randomly selected on days 7 (7 DAT), 14 (14 DAT), and 21 (21 DAT) after the initiation of NaCl treatment, and the plants were collected to determine the dry matter weight of the roots and shoots. Relative growth rate (RGR) was calculated by dividing the difference in dry matter weight between each sampling by the number of days between concerning periods.
The calcium (Ca2+) content was determined for the plants collected at 0 DAT (before NaCl treatment). Dried plant samples were ground to a mesh size of 0.5 mm using a bead mill (Multi-beads Shocker MB601U, Yashui-Kikai, Osaka, Japan). A 10 mg portion of the ground sample was suspended in 500 μL of 100 mM hydrochloric acid (HCl, Sigma-Aldrich Japan K.K., Tokyo, Japan) and maintained at 80 °C for 3 h with gentle agitation. After the centrifugation at 15,000× g for 5 min, 300 μL of the supernatant was neutralized by adding 200 μL of 100 mM sodium hydroxide (NaOH, Fujifilm Wako Pure Chemical Co., Osaka, Japan), and the calcium content was measured using a calcium ion meter (B-751 Ca2+, Horiba, Tokyo, Japan). Five measurements were replicated for each treatment group. Sodium (Na+) content was determined for the plants collected at 21 DAT. A 10 mg ground sample, prepared in the same manner as for calcium content measurement, was suspended in 500 μL of deionized water and incubated at 80 °C for 3 h with gentle agitation. The sodium content was measured in the supernatant obtained after the centrifugation at 15,000× g for 5 min using a sodium ion meter (B-722 Na+, Horiba, Tokyo, Japan). Five measurements were replicated for each treatment group.

2.6. Statistical Analysis

In the text, the values of CAT activity, soluble sugar content, and calcium content represented the mean ± standard error. All data obtained from germination tests and plant growth experiments were subjected to analysis of variance (ANOVA), with the concentration of the CaCl2 solution used for seed enrichment and/or experimental treatment (salt stress) as the primary variable factor. In the absence of interaction, the variances were combined with the total error variance, and the effects of the main factors were re-evaluated. When ANOVA identified significant effects of the main factors, a Bonferroni post hoc comparison test at a 5% significance level was conducted to compare the means between seeds and/or treatments. Prior to the analysis, an arcsine transformation was applied to the germination rate data to ensure a normal distribution and equal variance. The software package STATA ver. 17TM (Lightstone, New York, NY, USA) was utilized for the analyses.

3. Results

3.1. Germinability of Timothy Under Salt Stress

The germination rate (GR) of the control seeds in NaCl solutions of varying concentrations is illustrated in Figure 1. In distilled water and a 100 mM NaCl solution, the seeds began to germinate approximately 50 h after the initiation of the experiment, with a rapid increase in GR observed after 80 h. In contrast, germination was significantly inhibited in 200 mM NaCl and 300 mM NaCl solutions, and no rapid increase in GR was noted throughout the testing period. Under the 100 mM NaCl condition, the final GR was relatively high at 91.1% (Table 1). However, germination was markedly suppressed under 200 mM NaCl (25.6%) and 300 mM NaCl (15.7%). The mean germination time (MGT) increased under salt stress conditions, with values of 108.8 h for 100 mM NaCl, 239.7 h for 200 mM NaCl, and 280.3 h for 300 mM NaCl. These times were approximately 15, 146, and 190 h longer than the MGT in distilled water (93.3 h), respectively. The coefficient of uniformity of germination (CUG) and germination index (GI) were significantly reduced under salt stress. Notably, in 200 mM NaCl (CUG: 0.27, GI: 0.27) and 300 mM NaCl (CUG: 0.34, GI: 0.006), the values were drastically lower compared to those in distilled water (CUG: 4.79, GI: 0.73) and 100 mM NaCl (CUG: 3.65, GI: 0.68).
Under the 200 mM NaCl condition, germinability was significantly reduced for all seed types compared to the distilled water condition (Figure 2, Table 2). In the distilled water condition, there was minimal variation in germinability among the seeds. Both the control seeds and Ca-enriched seeds germinated within 93 h (MGT: 93.1 h to 94.1 h) from the onset of the test, with final GRs exceeding 95%. CUG was higher in the 50 mM-Ca seed (5.70) compared to the control seed (4.79). GI ranged from 0.73 (control and 100 mM-Ca) to 0.74 (50 mM-Ca) with no significant differences among the seed types. In contrast, under the 200 mM NaCl condition, all seeds exhibited prolonged germination times (MGT: 177.0 h to 239.7 h), and the final GRs were significantly lower (GR: 25.6% to 87.9%). The uniformity of germination was also compromised (CUG: 0.34 and below), and the GIs were notably low, ranging from 0.27 (control) to 0.45 (100 mM-Ca). Nevertheless, under salt stress conditions, the Ca-enriched seeds initiated germination earlier than the control seeds, and their final GRs remained sufficiently high, exceeding 86%. Additionally, both CUG and GI were also higher in the Ca-enriched seeds, indicating that calcium application through seed enrichment effectively improves germinability under salt stress.

3.2. CAT Activity and Soluble Sugar Content of the Seeds

The antioxidant activity of CAT was higher in the Ca-enriched seeds (50 mM-Ca: 2.36 ± 0.20 unit, 100 mM-Ca: 2.40 ± 0.14 unit) compared to the control seeds (1.84 ± 0.11 unit) (Figure 3a). Calcium enrichment enhanced the seed’s ability to scavenge hydrogen peroxide under salt stress. No significant differences in the enzyme activity were observed between the seeds treated with varying calcium concentrations. The 50 mM-Ca and 100 mM-Ca contained significantly more water-soluble sugars (54.7 ± 1.1 mg/g and 55.0 ± 0.9 mg/g, respectively) than the control seeds (47.3 ± 0.6 mg/g) at the pre-seeding stage (Figure 3b). However, the soluble sugar content did not differ significantly between Ca-enriched seeds.

3.3. Calcium Content of the Plants

The calcium content in juvenile timothy plants at 0 DAT was higher in those derived from 50 mM-Ca (Root: 150.0 ± 2.4 μg/plant, Shoot: 147.0 ± 3.8 μg/plant) and 100 mM-Ca (Root: 154.5 ± 1.8 μg/plant, Shoot: 148.5 ± 2.8 μg/plant) compared to the control plants (Root: 135.0 ± 2.4 μg/plant, Shoot: 139.5 ± 6.1 μg/plant) (Figure 4). Overall, the calcium content in the entire plant was 1.1 times greater in the plants derived from Ca-enriched seeds than in the control-derived plants. Additionally, no significant difference in calcium content between the roots and shoots was observed in any of the plants.

3.4. Plant Growth Under Salt Stress

Under distilled water conditions, the dry matter weights of the root, shoot, and whole body did not differ significantly between seed types during the period from 0 DAT to 14 DAT (Figure 5). At 21 DAT, plants derived from 50 mM-Ca exhibited higher root dry matter weight compared to control plants, resulting in an increased whole-body weight. When subjected to salt stress with 100 mM NaCl, the control plants demonstrated growth similar to that of non-salt-stressed counterparts from 7 DAT to 14 DAT; however, by 21 DAT, their growth declined, and dry matter weight was lower than that of plants without salt stress. In contrast, even under salt stress conditions, the plants derived from Ca-enriched seeds thrived, consistently exhibiting higher dry matter weights than the control plants throughout the experimental period. At 21 DAT, the plants derived from Ca-enriched seeds tended to have higher dry matter weights for both shoot and root compared to their non-salt-stressed counterparts, although the difference was not statistically significant.
The relative growth rate (RGR) did not differ significantly among the plants grown in distilled water; in all cases the values tended to increase gradually over time from the start of the treatment (Figure 6a–c). Plants grown in saltwater exhibited relatively constant shoot RGR, while the root RGR decreased during the period of 7–14 DAT. The whole-body RGRs were higher for the plants derived from Ca-enriched seeds compared to the control plants throughout the experimental period (Figure 6d–f).
Plants grown in distilled water collected at 21 DAT exhibited no significant differences in sodium content in both the roots and shoots (Figure 7). Under salt stress conditions of 100 mM NaCl, sodium content significantly increased in all plants compared to their counterparts in distilled water. However, the degree of sodium accumulation was lower in the plants derived from Ca-enriched seeds. Sodium accumulation was more noticeably alleviated in the shoots than in the roots, with the ratio of accumulation in Ca-enriched plants compared to controls being 0.65 in the roots and 0.80 in the shoots.

4. Discussion

The impaired growth of crops under saline conditions is primarily attributed to osmotic stress resulting from increased soil water potential, the overproduction of reactive oxygen species (ROS) due to the oxidative damage, and the accumulation of specific ions, such as sodium [36,49,50]. Calcium plays a crucial role in the metabolism of various soluble organic substances within the cytoplasm and promotes the production of compounds such as amino acids and water-soluble carbohydrates, which function as regulators of osmolarity in response to environmental stress [19]. It is well established that calcium can enhance the activity of alpha-amylase, an enzyme that degrades seed-storage starch into smaller soluble sugars [36,51]. Adequate calcium levels facilitate the proper regulation of cell osmolarity and prevent water leakage from cell membranes under saline conditions. Cytosolic calcium acts as a second messenger, responsible for activating specific transcription factors associated with antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), and CAT [51,52]. Under calcium-rich conditions, plants can develop an enzymatic antioxidant defense system to combat the overproduction of ROS. Furthermore, calcium at the cell membrane can mitigate sodium (Na+) toxicity by inhibiting non-selective cation channels, which serve as the primary pathway for Na+ influx in plants [52]. Free calcium in the cytosol activates salt overly sensitive (SOS) proteins that are responsible for expelling excess Na+ from the cell. Therefore, calcium supplementation is essential for reducing Na+ overaccumulation in saline conditions.
The germinability of timothy significantly declined under NaCl concentrations exceeding 200 mM, a condition in which many plant species tend to exhibit stunted growth [9]. However, by enriching the seeds with calcium, the germination index, which represents the degree of germinability, remained high even under salinity stress. In this study, timothy seeds were shown to increase soluble sugar content and catalase activity by supplying calcium. This enhancement may facilitate proper osmolarity regulation and effective scavenging of ROS generated during the germination process, resulting in higher germinability under saline conditions. Additionally, the RGR of the control plants significantly decreased under salinity stress of 100 mM NaCl, while those of the Ca-enriched plants remained elevated. Timothy plants derived from Ca-enriched seeds accumulated less sodium than control plants when grown in saline conditions. The calcium supplied to the seeds may improve ion homeostasis by inhibiting overaccumulation of Na+ and mitigating sodium toxicity, which contributes to the enhanced initial growth potential of the plants.
Ca-enriched seeds exhibited a germination rate exceeding 85% even under 200 mM NaCl stress. In the plant growth test, the plants were subjected to a lower level of salinity stress (100 mM) compared to the germination tests. This decision was based on the results of a preliminary test, in which even Ca-enriched plants nearly withered when exposed to a 200 mM NaCl solution. No research has been conducted to determine whether seedlings that germinated in a 200 mM NaCl solution can survive afterwards, although they were observed to develop healthy coleoptiles and taproots. Anyway, given that the germination rate in distilled water was approximately 95%, it can be inferred that the Ca seed enrichment, combined with a seeding rate 1.1 to 1.2 times higher than conventional methods, can ensure a comparable number of timothy seedlings in salt-affected areas with a NaCl concentration of 200 mM as in non-affected areas. On the other hand, the coefficient of uniformity of germination (CUG) decreased under salinity stress, even in the Ca-enriched seeds (Table 2). This indicates that the calcium supplied through seed enrichment does not enhance the consistency of germination in timothy under salt stress.
The calcium-induced improvement in growth was notable in younger seedlings shortly after germination. The differences in RGRs between plants grown with a 100 mM NaCl solution were most pronounced during the period of 0–7 DAT, with the RGRs of the Ca-enriched plants being 2.4 times higher in the roots and 2.5 times higher in the shoots compared to the control plants. The beneficial effect of calcium in alleviating salt injury was particularly pronounced in the roots. The root-to-shoot dry matter weight ratio of the control plants at 21 DAT was 0.54 under the distilled water (H2O) condition, while the ratio under the NaCl condition was lower, at 0.49. Impaired growth due to salt stress is believed to affect roots more severely than the shoots. However, Ca-enriched plants maintained their root-to-shoot ratios even when grown in a 100 mM NaCl solution (50 mM-Ca-derived plants: in H2O = 0.53, in 100 mM-NaCl = 0.58; 100 mM-Ca derived plants: in H2O = 0.54; in 100 mM-NaCl = 0.60).
The results of this study showed that seed enrichment is an effective technique for supplying calcium to fodder grass seeds and enhancing salt tolerance. Plants enriched with a 50 mM CaCl2 solution or a 100 mM CaCl2 solution contained approximately 25 μg more calcium in their whole bodies compared to untreated plants (Figure 4). Based on simple calculations, the plants acquired this amount by absorbing approximately 12 μL of the 50 mM CaCl2 solution or 6 μL of the 100 mM CaCl2 solution. Doubling the concentration of the treatment solution from 50 to 100 mM did not result in a significant difference in calcium content in the plant body. Both treated seeds exhibited no excess symptoms, indicating that 50 mM is a sufficient concentration for providing the maximum amount of calcium that timothy seeds can absorb. In fact, the same degree of improvement in germinability and early growth ability was obtained with a calcium concentration of either 50 or 100 mM. Despite some statistical ambiguity, the dry matter weight of H2O-grown plants at 21 DAT tended to be higher in the Ca-enriched plants compared to the control plants. Proper application of calcium, without causing excess symptoms, has been reported to promote early crop growth [51,53,54]. The application of calcium to seeds through the technique of seed enrichment could enhance crop growth and help establish a robust sward even in typical, non-salt-affected fields.

5. Conclusions

The area of salt-affected land has been gradually expanding on a global scale. Seed size in many pasture species, including timothy, is extremely small compared to common crops. Therefore, they are more susceptible to salt stress during the process of initial growth. Calcium has the effect of ameliorating salt damage, and supplying timothy seeds with calcium by the technique of seed enrichment improved germinability and early growth ability under salt stress. The findings of this study were obtained from experiments conducted under controlled conditions. It is necessary to verify the efficacy of this technique in actual salt-affected soils. Additionally, the effectiveness of this technique should be tested on forage legumes, which are commonly mixed with timothy.

Author Contributions

Conceptualization and methodology, M.A. and L.M.; formal analysis, M.A.; investigation and data curation, M.A. and L.M.; writing—original draft preparation, M.A. and L.M.; writing—review and editing, M.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors gratefully acknowledge Takahiro Terado in National Livestock Breeding Center and Masaaki Hanada in Obihiro University of Agriculture and Veterinary Medicine for helping in the plant growth test.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Changing of the germination rate of the control seeds sown in the germination media of varying NaCl concentrations.
Figure 1. Changing of the germination rate of the control seeds sown in the germination media of varying NaCl concentrations.
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Figure 2. Changing of the germination rate in the control and Ca-enriched seeds sown in (a) distilled water and (b) 200 mM NaCl solution.
Figure 2. Changing of the germination rate in the control and Ca-enriched seeds sown in (a) distilled water and (b) 200 mM NaCl solution.
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Figure 3. (a) Catalase activity and (b) soluble sugar content in the control and Ca-enriched seeds. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
Figure 3. (a) Catalase activity and (b) soluble sugar content in the control and Ca-enriched seeds. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
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Figure 4. Calcium contents of the (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
Figure 4. Calcium contents of the (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
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Figure 5. Changing of the dry matter weight of (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds grown in distilled water and 100 mM NaCl solution. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test. DAT stands for “ Days After Treatmants”.
Figure 5. Changing of the dry matter weight of (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds grown in distilled water and 100 mM NaCl solution. Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test. DAT stands for “ Days After Treatmants”.
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Figure 6. Relative growth rate expressed as the increase in dry matter weight per day in the plants grown in distilled water ((a): root, (b): shoot, and (c): whole body) and 100 mM NaCl solution ((d): root, (e): shoot, and (f): whole body). Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
Figure 6. Relative growth rate expressed as the increase in dry matter weight per day in the plants grown in distilled water ((a): root, (b): shoot, and (c): whole body) and 100 mM NaCl solution ((d): root, (e): shoot, and (f): whole body). Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
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Figure 7. Sodium contents of the (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds at the termination of the test (21 DAT). Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
Figure 7. Sodium contents of the (a) root, (b) shoot, and (c) whole body in the plants derived from the control and Ca-enriched seeds at the termination of the test (21 DAT). Values with different letters are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
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Table 1. Germinability indices for the control seeds sown in the germination media of varying NaCl concentrations. The values represent the mean ± standard error.
Table 1. Germinability indices for the control seeds sown in the germination media of varying NaCl concentrations. The values represent the mean ± standard error.
Germination MediaGR (%)MGT (Hours)CUGGI
Distilled Water96.2 ± 1.0a93.3 ± 1.6d4.79 ± 0.38a0.73 ± 0.01a
100 mM NaCl91.1 ± 0.5b108.8 ± 2.0c3.65 ± 0.63b0.68 ± 0.01b
200 mM NaCl25.6 ± 4.7c239.7 ± 1.9b0.27 ± 0.03c0.27 ± 0.03c
300 mM NaCl15.7 ± 0.3d280.3 ± 3.5a0.34 ± 0.04c0.06 ± 0.01d
GR: germination rate, MGT: mean germination time, CUG: coefficient of uniformity of germination, GI: germination index. Values followed by different letters within columns are significantly different at p = 0.05 according to the Bonferroni post hoc comparison test.
Table 2. Germinability indices for the control and Ca-enriched seeds sown in H2O and 200 mM NaCl solutions. The values represent the mean ± standard error.
Table 2. Germinability indices for the control and Ca-enriched seeds sown in H2O and 200 mM NaCl solutions. The values represent the mean ± standard error.
GR (%)MGT (Hours)CUGGI
Distilled Water
Control96.2 ± 1.0a93.3 ± 1.6c4.79 ± 0.38b0.73 ± 0.01a
50 mM-Ca95.6 ± 1.3a94.1 ± 3.1c5.70 ± 0.80a0.74 ± 0.01a
100 mM-Ca95.6 ± 0.6a93.1 ± 1.1c5.05 ± 0.47b0.73 ± 0.02a
200 mM-NaCl
Control25.6 ± 4.7c239.7 ± 1.9a0.27 ± 0.03d0.27 ± 0.03c
50 mM-Ca86.8 ± 1.7b183.5 ± 11.0b0.31 ± 0.02c0.42 ± 0.02b
100 mM-Ca87.9 ± 2.7b177.0 ± 9.3b0.34 ± 0.01c0.45 ± 0.02b
Values followed by different letters within columns are significantly different at p = 0.05 according to Bonferroni post hoc comparison test.
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Akimoto, M.; Ma, L. Supplementation of Calcium Through Seed Enrichment Technique Enhances Germinability and Early Growth of Timothy (Phleum pratense L.) Under Salinity Conditions. Agronomy 2025, 15, 1905. https://doi.org/10.3390/agronomy15081905

AMA Style

Akimoto M, Ma L. Supplementation of Calcium Through Seed Enrichment Technique Enhances Germinability and Early Growth of Timothy (Phleum pratense L.) Under Salinity Conditions. Agronomy. 2025; 15(8):1905. https://doi.org/10.3390/agronomy15081905

Chicago/Turabian Style

Akimoto, Masahiro, and Li Ma. 2025. "Supplementation of Calcium Through Seed Enrichment Technique Enhances Germinability and Early Growth of Timothy (Phleum pratense L.) Under Salinity Conditions" Agronomy 15, no. 8: 1905. https://doi.org/10.3390/agronomy15081905

APA Style

Akimoto, M., & Ma, L. (2025). Supplementation of Calcium Through Seed Enrichment Technique Enhances Germinability and Early Growth of Timothy (Phleum pratense L.) Under Salinity Conditions. Agronomy, 15(8), 1905. https://doi.org/10.3390/agronomy15081905

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