Melatonin Priming Increases the Tolerance of Tartary Buckwheat Seeds to Abiotic Stress

Abstract

Increasing abiotic stress, particularly salinity, poses a significant threat to the germination and seedling development of Tartary buckwheat, thereby limiting its yield potential and broader cultivation. Given Tartary buckwheat’s rich nutritional profile and inherent stress adaptability, enhancing seed tolerance to abiotic stress is essential for ensuring food security and the development of functional food resources. To investigate the role of melatonin in mitigating abiotic stress, seeds of the cultivar ‘Jinqiaomai 2’ were primed with varying melatonin concentrations (with water as the control) at multiple time points. The effects of salt stress on germination and seedling quality were evaluated to determine optimal priming conditions. Subsequent analyses examined seed vigor and physiological and biochemical responses during storage under high temperature and humidity, room temperature, and low-temperature conditions. The results showed that a 3 h melatonin priming consistently resulted in high germination rates (98.7–100.0%). Notably, melatonin at 50 μmol·L⁻¹ was identified as the optimal concentration, significantly improving seedling growth under salinity stress, with increases of 61.1% in seedling length, 59.3% in root length, and 38.9% in root fresh weight compared with the control. Across all storage environments, melatonin-primed seeds exhibited superior vigor and enhanced antioxidant enzyme activity relative to water-primed controls. In conclusion, melatonin priming at an appropriate concentration and duration effectively enhanced the vigor of Tartary buckwheat seeds and alleviated the adverse effects of salinity on germination and storage resilience. However, improved seeds may possess a limited safe storage window and should be sown promptly rather than stored long-term.

1. Introduction

Buckwheat, also known as huamai or sanjiaomai, is a short-lived herbaceous dicotyledonous plant that belongs to the genus Fagopyrum within the family Polygonaceae [1]. This species is ecologically adapted to cool temperate climates [2,3]. Taxonomically, the genus includes two primary cultivated species: Fagopyrum esculentum Moench (common buckwheat) and F. tataricum Gaertn. (Tartary buckwheat) [4]. F. esculentum is predominantly grown in agricultural regions of northern China, whereas F. tataricum is more widely cultivated in the southwestern highlands [5]. As a minor crop with dual roles in nutrition and medicine, it is generally regarded as richer in bioactive substances than other widely distributed cereal crops, particularly because of its high content of bioflavonoids [2,6]. These bioactive substances exhibit notable hypoglycemic and hypolipidemic effects and have been shown to improve insulin resistance through specific metabolic pathways [7].

Seeds are widely recognized as a critical agricultural resource, and their quality directly determines crop productivity, underscoring the need for high-quality seed applications in modern agriculture [8]. Seed vigor is a principal indicator of seed quality and plays a pivotal role in ensuring optimal crop establishment and yield. Among the various agronomic strategies developed to enhance seed vigor, seed priming has emerged as a widely studied and effective physiological conditioning method. Priming techniques can be categorized by water uptake mechanisms into water priming, osmopriming, and matric priming and further functionally classified into biopriming, chemical priming, and hormonal priming [9]. These methods aim to break seed dormancy, enhance seed vigor, and maximize germination by modulating physiological and biochemical pathways crucial for early seedling development [10]. As a traditional and extensively applied technique, water priming involves pre-sowing soaking, and its efficacy has been well documented. Its renewed attention in recent years can be attributed to its ability to improve seed vigor across multiple species, including Elymus nutans, cowpea, corn, wheat, and spinach [11,12,13,14,15]. In addition to enhancing germination, seed priming can significantly increase resistance to abiotic stress, thereby mitigating seedling damage under adverse conditions [16,17,18,19,20,21]. For example, water priming improves vigor and low-temperature tolerance in conventional, waxy, and sweet corn [13]; polyethylene glycol (PEG) priming enhances germination in alfalfa and milkweed under cold and drought stress [16]; and salicylic acid treatment facilitates rice seed germination at low temperatures [17]. Similarly, drought resistance in cotton is improved by priming with 5 g·L⁻¹ KNO₃ or 100 mg·L⁻¹ betaine [18], and diethyl aminoethyl hexanoate (DA-6) treatment alleviates drought-induced damage in clover [19]. Recent advances have also introduced nanotechnology-based seed priming, which promotes rice seed germination and seedling growth [20] and improves both germination percentage and seedling development in chili under saline conditions [21].

Although the benefits of seed priming in enhancing seed quality are well established, limited attention has been paid to the extent of priming and post-treatment storability of primed seeds. This can directly constrain large-scale agricultural applications. One major challenge is the redrying process following priming because primed seeds generally exhibit reduced dehydration tolerance [22]. Further investigations should be conducted to support practical implementation of priming strategies for crop production. Empirical studies on Cucurbita ficifolia have emphasized the importance of precisely calibrating priming duration. Although extending priming beyond the optimal timeframe, even during early germination, did not demonstrate statistically significant adverse effects on germination or seedling growth, it notably reduced seed storability. Hence, seeds that have entered the radicle emergence phase should be sown immediately, as delayed sowing substantially diminishes germination capacity [23]. Additional research on wheat has examined the effects of post-germination redrying on its storage potential and resprouting ability. Although over germination is not lethal, it compromises seed viability and reduces agronomic value. Notably, the physiological advantages conferred by priming decline with storage time, and seeds subjected to excessive priming followed by redrying exhibit an increased rate of seedling deformities [24].

Melatonin was first isolated from the pineal glands of cattle in 1958 by a dermatologist at Yale University and was named for its ability to lighten darkened frog skin [25]. Chemically, melatonin is an indole derivative of tryptophan [26]. Multiple studies have confirmed that melatonin-treated seeds exhibit higher concentrations of osmotic regulators with significantly upregulated energy metabolism pathways and increased protein content, which may collectively enhance plant responses to abiotic stress [27]. Moreover, melatonin has also been identified as a novel plant growth regulator [28]. Exogenous application of melatonin has been shown to promote seed germination and root development, preserve chlorophyll to delay leaf senescence, and enhance plant tolerance to various abiotic stresses [29]. In particular, it mitigates drought-induced photodamage by supporting both the structural integrity and substrate availability of photosynthetic systems, thereby improving the photosynthetic efficiency of soybean under drought conditions [30]. However, whether melatonin priming can activate antioxidant defense mechanisms to mitigate stress effects requires further study.

Currently, limited research has addressed whether the extent of seed priming with melatonin is optimal. To explore the efficacy of melatonin priming on Tartary buckwheat seeds and the storage tolerance of redried seeds after melatonin treatment, the cultivar ‘Jinqiaomai 2’ (F. tataricum Gaertn.) was used as the experimental material to evaluate the effects of melatonin and water priming on seed resistance to salt stress. In addition, changes in seed vigor and physiological traits were assessed over extended storage durations following redrying to determine the effective duration of priming-induced benefits.

2. Materials and Methods

2.1. Geographical Location of the Study Area

The experiment was conducted from June to December 2023. The experimental materials were from the Research Center of Buckwheat Industry Technology of the School of Life Sciences, Guizhou Normal University, and were planted at the Anshun Experimental Base (26°17′ N, 106°63′ E) in Guizhou Province in the fall of 2022. The planting plots were 2 m in length with a row spacing of 0.35 m, and conventional field management practices were adopted. Seeds were harvested in November and stored at −20 °C. They were removed from cold storage one week before conducting the experiment. Plump, undamaged, and uniformly sized seeds of ‘Jinqiaomai 2’ were selected for this study.

2.2. Seed Priming Treatment and Standard Germination Test

Melatonin (2.325 mg) was weighed and dissolved in 10 mL of solution to prepare a 1 mM stock solution. This stock solution was then diluted to obtain the following concentrations: 0 (CK), 1 (T1), 5 (T2), 10 (T3), 50 (T4), and 100 (T5) μmol·L⁻¹. Seeds were immersed in these solutions and primed for 3, 6, 9, 12, or 24 h at 20 °C in the dark. Following priming, 50 seeds from each treatment were selected for germination tests under salt stress. Each treatment was repeated three times. The germination paper was initially moistened with 100 mmol/L NaCl solution, which was replenished daily to maintain constant salt stress. Germination was performed at 25 °C under a 16 h light/8 h dark photoperiod. On day 7, germination percentage (GP) was recorded, and ten seedlings from each treatment were randomly selected to measure seedling length (cm), root length (cm), and fresh root weight (g). These parameters were used to determine optimal priming duration and melatonin concentration.

2.3. Seed Storage and Germination Test

After identifying the optimal priming duration and melatonin concentration, the seeds of ‘Jinqiaomai 2’ were reselected and primed for 3 h at 20 °C in the dark, using either distilled water or 50 μmol·L⁻¹ melatonin. Following priming, the seeds were air-dried under natural conditions until they returned to their original moisture content and subsequently stored under three conditions: high temperature and humidity (40 °C, 100% relative humidity), room temperature, and low temperature (4 °C). Seeds stored at room temperature and low temperature were sampled on days 7, 14, 21, and 28, whereas seeds stored at high temperature and humidity were sampled on days 1, 2, 3, 4, and 5. All collected samples were subjected to germination and physiological assessments as described in Section 2.2. The daily germination counts were recorded, germination energy (GE) was measured on day 5, and GP and germination index (GI) were measured on day 7. On day 7, 10 samples per treatment were randomly selected to measure root length and shoot length using a ruler, as well as to determine seedling fresh and dry weights. All experiments were conducted in triplicate.

2.4. Conductivity Measurement

The measurement was conducted based on the method described by Yao [31] with slight modifications. Fifteen seeds primed with either water or melatonin, redried, and stored under the three conditions were selected for the experiment. The seeds were rinsed three times with tap water and once with distilled water, surface-dried, and placed in a 50 mL centrifuge tube. Subsequently, 40 mL of distilled water was added, the tube was sealed, and the seeds were incubated in the dark at 25 °C for 24 h. After incubation and mixing, the electrical conductivity of the leachate was measured at room temperature using a DDS-307 conductivity meter (INASE Scientific Instrument Co., Ltd., Shanghai, China). Each measurement was performed in triplicate, and the average value was recorded for each group.

2.5. Measurement of SOD and APX Activities

The enzyme extract was prepared as follows. Seeds subjected to the two priming treatments were redried, stored under three different conditions, and then dehulled. Seed tissue (0.15 g) was weighed, and 4 mL of 0.05 M phosphate buffer (pH 7.0) was added. The mixture was then homogenized on ice. The homogenate was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 min in a 5424R centrifuge (Eppendorf, Hamburg, Germany). The resulting supernatant was collected in a separate centrifuge tube and stored at low temperature for subsequent analysis.

Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined according to the method described by Pérez-Rodríguez [32], with slight modifications. The 3 mL reaction mixture contained the following components: 0.3 mL of 130 mmol·L⁻¹ methionine (Met), 0.3 mL of 750 μmol·L⁻¹ Nitrotetrazolium Blue chloride (NBT), 0.3 mL of 100 μmol·L⁻¹ Ethylenediaminetetraacetic acid disodium salt (EDTA-Na₂), 0.3 mL of 20 μmol·L⁻¹ lactochrome, and 0.1 mL of enzyme extract. The final volume was adjusted using phosphate buffer. A blank control was prepared by replacing the enzyme extract with buffer. After mixing, the reaction was incubated under light at 25 °C and 4000 lx for 15 min and then immediately terminated in the dark. The zero was calibrated using the unlit tube, and the absorbance was measured at 560 nm using the NBT reaction solution as a blank control. One unit of SOD activity (U) was defined as the amount of enzyme required to inhibit 50% of NBT photoreduction and was expressed as U/g.

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined based on the method described by Bicalho [33] with slight modifications. The reaction mixture consisted of 0.1 mL of enzyme extract and 2700 μL of 0.25 mM phosphate buffer (pH 7.0, containing 2 mM EDTA), followed by the sequential addition of 100 μL of 7.5 mM ascorbic acid and 100 μL of 300 mM H₂O₂. The mixture was thoroughly shaken, and the absorbance at 290 nm was immediately recorded using a spectrophotometer. Measurements were performed every 30 s for 2 min to monitor the kinetics of the enzymatic reaction. The unit of APX activity (U) was defined as the amount of enzyme that oxidizes 1 nmol ascorbic acid per minute and was expressed as nmol·min⁻¹·g⁻¹ fresh weight (FW).

2.6. Statistical Analysis

The data were analyzed using SAS 9.4 software. Analysis of variance (ANOVA) was used to evaluate experimental data. The percentage data were subjected to an arcsine square root transformation y = arcsin[sqrt (x/100)] to stabilize the variance. Multiple comparisons were performed using Fisher’s least significant difference (LSD) test at a significance level of α = 0.05.

3. Results

3.1. Determination of Melatonin Priming Concentration and Duration

3.1.1. Effects of Melatonin Treatment on Tartary Buckwheat Seeds Under Salt Stress

As shown in

Table 1. Effect of melatonin priming duration on germination percentage of Tartary buckwheat seeds under salt stress.

Treatments (μmol·L−1)	Germination Percentage (%)
	3 h	6 h	9 h	12 h	24 h
CK (0)	100.0 ± 0.0 a	99.3 ± 1.2 a	99.3 ± 1.2 a	98.7 ± 1.2 a	99.3 ± 1.2 ab
T1(1)	100.0 ± 0.0 a	100.0 ± 0.0 a	100.0 ± 0.0 a	100.0 ± 0.0 a	98.7 ± 1.2 ab
T2 (5)	98.7 ± 2.3 a	100.0 ± 0.0 a	98.0 ± 3.5 a	97.3 ± 1.2 a	99.3 ± 1.2 ab
T3 (10)	100.0 ± 0.0 a	98.0 ± 3.5 a	99.3 ± 1.2 a	98.7 ± 2.3 a	100.0 ± 0.0 a
T4 (50)	98.7 ± 1.2 a	99.3 ± 1.2 a	98.7 ± 2.3 a	97.3 ± 3.1 a	98.7 ± 1.2 ab
T5 (100)	100.0 ± 0.0 a	99.3 ± 1.2 a	98.7 ± 1.2 a	98.7 ± 2.3 a	98.0 ± 0.0 b

Note: Different letters indicate significant differences between treatments with different melatonin concentrations at the same time (p < 0.05).

, the seeds treated with different melatonin concentrations and primed for 3 h exhibited germination percentages that reached or approached 100.0%, demonstrating optimal stability. After 24 h of priming, the highest germination percentage was observed in the T3 treatment group (100.0%), which was significantly higher than that of the T5 group (98.0%). No significant differences were observed among the remaining treatment groups. These findings suggest that a 3 h priming duration is more favorable for seed germination, whereas prolonged priming with high melatonin concentrations may inhibit germination.

3.1.2. Effects of Melatonin Treatment on Tartary Buckwheat Seedlings Under Salt Stress

As shown in

Table 2. Effect of melatonin priming on seedling length, root length, and root fresh weight of Tartary buckwheat seedlings under salt stress.

Treatments (μmol·L−1)	Seedling Length (cm)	Root Length (cm)	Root Fresh Weight (g)
CK (0)	1.46 ± 0.13 c	6.68 ± 1.51 c	0.54 ± 0.03 bc
T1 (1)	1.40 ± 0.10 c	6.07 ± 0.53 c	0.50 ± 0.01 c
T2 (5)	1.45 ± 0.03 c	6.16 ± 0.97 c	0.51 ± 0.03 bc
T3 (10)	1.47 ± 0.16 c	6.49 ± 1.07 c	0.54 ± 0.04 bc
T4 (50)	2.36 ± 0.25 a	10.64 ± 0.38 a	0.75 ± 0.10 a
T5 (100)	1.85 ± 0.25 b	8.67 ± 1.12 b	0.60 ± 0.04 b

Note: Different letters indicate significant differences between the same column of data at different treatments (p < 0.05).

, seedling length, root length, and root fresh weight of buckwheat seedlings under salt stress exhibited a nonlinear trend with increasing melatonin concentration, which initially increased and then decreased. At concentrations of 50–100 μmol·L⁻¹, both seedling length and root length were significantly greater than those in CK. Specifically, at 50 μmol·L⁻¹, the seedling length reached 2.36 cm (61.6% higher than that of CK), and the root length reached 10.64 cm (59.3% increase). At 100 μmol·L⁻¹, the seedling and root lengths were 1.85 and 8.67 cm, respectively, corresponding to increases of 26.7% and 29.8% over CK. Among all treatments, 50 μmol·L⁻¹ melatonin exhibited the most pronounced growth-promoting effect. The root fresh weight also peaked at this concentration, reaching 0.75 g and showing a significant increase compared to CK (a 38.9% increase). Although the root fresh weight at 100 μmol·L⁻¹ was 11.1% higher than that of the CK, the difference was not statistically significant. These results indicated that melatonin at 50 μmol·L⁻¹ effectively mitigated the inhibitory effects of salt stress on seedling growth.

3.2. Seed Phenotypes Following Water Priming and Melatonin Priming for Storage Tolerance

As shown in Figure 1, Tartary buckwheat seeds primed with either water or melatonin, and subsequently redried, exhibited varying degrees of germination. After 5 d of storage under high temperature and humidity and 28 d at room temperature, the seeds in the CK (water-primed) group demonstrated superior germination performance compared to those treated with melatonin. However, after 28 d of storage at low temperatures, the melatonin-primed seeds exhibited longer shoot lengths, suggesting a sustained regulatory effect of melatonin on seed physiological metabolism under low temperature storage conditions.

3.3. Storage Tolerance of Redrying Seeds Under High Temperature and Humidity Conditions

As shown in

Table 3. Effects of 5-day storage under high temperature and humidity conditions on Tartary buckwheat seed germination and seedling growth.

Treatments		Germination Percentage (%)	Germination Energy (%)	Germination Index	Root Length (cm)	Shoot Length (cm)	SFW (g)	SDW (g)
1 d	CK	98.0 ± 3.5	98.0 ± 3.5	86.9 ± 3.3	4.8 ± 0.3	0.82 ± 0.04	1.51 ± 0.04	0.092 ± 0.003 *
	T	99.3 ± 1.2	99.3 ± 1.2	85.4 ± 2.6	4.9 ± 0.3	0.82 ± 0.03	1.45 ± 0.08	0.085 ± 0.003
2 d	CK	98.7 ± 2.3	98.7 ± 2.3	83.9 ± 2.9	4.5 ± 0.3	0.80 ± 0.01	1.52 ± 0.02	0.094 ± 0.002
	T	98.7 ± 2.3	98.7 ± 2.3	84.7 ± 2.1	4.4 ± 0.1	0.85 ± 0.01 **	1.56 ± 0.03	0.090 ± 0.002
3 d	CK	94.7 ± 3.1	94.0 ± 4.0	79.5 ± 3.7	4.3 ± 0.2	0.79 ± 0.01	1.38 ± 0.04	0.093 ± 0.002
	T	98.0 ± 0.0	98.0 ± 0.0	82.2 ± 1.2	4.8 ± 0.4	0.87 ± 0.10	1.52 ± 0.07 *	0.092 ± 0.003
4 d	CK	92.0 ± 7.2	91.3 ± 6.4	76.5 ± 5.4	1.5 ± 0.1	0.56 ± 0.05	1.40 ± 0.08	0.090 ± 0.004
	T	94.0 ± 4.0	94.0 ± 4.0	78.4 ± 4.7	1.5 ± 0.2	0.53 ± 0.02	1.38 ± 0.18	0.089 ± 0.003
5 d	CK	86.7 ± 5.0	86.0 ± 5.3	66.8 ± 4.6	1.5 ± 0.1 *	0.53 ± 0.04 *	1.42 ± 0.09	0.085 ± 0.003
	T	92.0 ± 2.0	92.0 ± 2.0	76.1 ± 1.3*	1.2 ± 0.1	0.39 ± 0.05	1.37 ± 0.10	0.087 ± 0.002

Note: * (p < 0.05), ** (p < 0.01). The units of fresh weight (SFW) and dry weight of seedlings (SDW) were g·10 plant⁻¹; the same was applied throughout this study.

, both treatment groups exhibited an overall decline in germination percentage, germination energy, and germination index during storage under high temperature and humidity conditions for 1–5 d. However, melatonin-treated seeds displayed a mitigating effect on germination loss during later stages of storage. After 5 d, the germination index of the melatonin-treated group reached 76.1, which was significantly higher than that of the CK group (66.8). Regarding seedling morphology, both root and shoot lengths generally decreased with prolonged storage. After 5 d, the CK group demonstrated significantly longer roots than the melatonin-treated group. Conversely, after 2 d of storage, the shoot length of the melatonin-treated group was significantly greater than that of the CK, whereas the trend reversed by day 5, with the CK group showing superior shoot length. The fresh and dry weights of the seedlings exhibited relatively minor variations. On day 3, the melatonin-treated group demonstrated significantly higher seedling fresh weight than the control, whereas on day 1, the CK group had a significantly greater dry weight. No significant differences in the dry weight were observed at any of the other time points. In summary, melatonin treatment under high temperature and humidity conditions delayed the decline in seed germination performance, whereas extended storage still led to reduced seedling growth potential.

As shown in Figure 2, electrical conductivity, SOD activity, and APX activity exhibited distinct trends over time in both treatment groups under high temperature and humidity conditions. Figure 2a illustrates that the electrical conductivity increased progressively with storage duration in both groups. However, from days 2 to 5, the conductivity of the CK group was significantly higher than that of the melatonin-treated group, indicating greater membrane damage. Figure 2b reveals that SOD activity in the melatonin group continuously increased, peaking at 180.09 U/mg after 5 d of storage. In contrast, SOD activity in the CK group peaked between days 3 and 4. Figure 2c shows a general decline in APX activity in both groups. On day 1, the APX activity was significantly higher in the CK group (1.22 nmol·min⁻¹·g⁻¹ FW) than in the melatonin group (1.09 nmol·min⁻¹·g⁻¹ FW), whereas by day 3, the melatonin-treated seeds exhibited the significantly higher APX activity.

3.4. Storage Tolerance of Redrying Seeds at Room Temperature

As shown in

Table 4. Effects of 28-day storage at room temperature on germination and seedling growth of Tartary buckwheat seeds.

Treatments		Germination Percentage (%)	Germination Energy (%)	Germination Index	Root Length (cm)	Shoot Length (cm)	SFW (g)	SDW (g)
7 d	CK	100.0 ± 0.0	100.0 ± 0.0	92.7 ± 0.6	4.2 ± 0.3	0.85 ± 0.03	1.25 ± 0.12	0.093 ± 0.003
	T	100.0 ± 0.0	100.0 ± 0.0	93.0 ± 1.0	4.6 ± 0.1 *	0.92 ± 0.02 *	1.35 ± 0.02	0.095 ± 0.002
14 d	CK	99.3 ± 1.2	99.3 ± 1.2	85.0 ± 1.9	2.6 ± 0.2	0.65 ± 0.04	1.18 ± 0.07	0.089 ± 0.002
	T	100.0 ± 0.0	100.0 ± 0.0	87.8 ± 1.4	3.3 ± 0.2 *	0.77 ± 0.06 *	1.25 ± 0.03	0.089 ± 0.005
21 d	CK	99.3 ± 1.2	97.3 ± 1.2	92.2 ± 3.5	1.0 ± 0.1	0.44 ± 0.06	1.52 ± 0.09	0.086 ± 0.005
	T	98.0 ± 2.0	98.0 ± 2.0	93.0 ± 2.9	1.0 ± 0.1	0.47 ± 0.04	1.65 ± 0.04	0.095 ± 0.011
28 d	CK	95.3 ± 3.1	92.7 ± 7.6	84.1 ± 6.5	0.7 ± 0.0	0.29 ± 0.02 *	1.27 ± 0.05	0.091 ± 0.004
	T	97.3 ± 1.2	96.7 ± 1.2	89.0 ± 0.7	0.4 ± 0.1	0.20 ± 0.05	1.39 ± 0.14	0.092 ± 0.005

Note: * (p < 0.05).

, the germination percentage, germination energy, and germination index gradually declined with prolonged storage in both treatment groups. However, the melatonin-treated group maintained higher values than the CK group throughout the storage period. In terms of seedling morphology, the melatonin-treated group exhibited significantly greater root and shoot lengths during days 7–14 of storage. Specifically, after 7 d, the melatonin-treated group had root and shoot lengths of 4.6 and 0.92 cm, respectively, which were significantly greater than those of the CK group. After 14 d, the melatonin-treated group still had longer roots (3.3 cm) and shoots (0.77 cm) than the CK group. However, after 28 d, the CK group showed superior shoot length, with a significant difference observed. Although there was little variation in the fresh and dry weights of the seedlings, the melatonin-treated group showed a slight advantage over the CK group. Overall, melatonin treatment under room temperature storage conditions effectively maintained seed germination and promoted early seedling growth. However, optimizing the storage cycle may be necessary to balance seed vigor preservation and seedling development.

As shown in Figure 3, the conductivity, SOD activity, and APX activity of seeds in both treatment groups exhibited different trends under room temperature storage as the storage time increased. Figure 3a illustrates that, except after 14 d of storage when the melatonin-treated group showed higher electrical conductivity than the CK group, the CK group had a higher conductivity at all time points, with a highly significant difference observed after 7 d. Figure 3b indicates that after 21 d of storage, SOD activity in the melatonin-treated seeds (241.13 U/mg) was significantly higher than that in the CK group, reflecting an antioxidant advantage. No significant differences in SOD activity were observed at any other time point. Figure 3c shows that APX activity initially decreased in both groups, then increased, and subsequently declined again. After 21 d, APX activity peaked at 0.931 nmol·min⁻¹·g⁻¹ FW in the melatonin-treated group and 0.922 nmol·min⁻¹·g⁻¹ FW in the CK group, but the difference was not significant.

3.5. Storage Tolerance of Redrying Seeds at Low Temperature

As shown in

Table 5. Effects of 28-day low-temperature storage on germination and seedling growth of Tartary buckwheat seeds.

Treatments		Germination Percentage (%)	Germination Energy (%)	Germination Index	Root Length (cm)	Shoot Length (cm)	SFW (g)	SDW (g)
7 d	CK	98.7 ± 1.2	98.0 ± 2.0	87.7 ± 1.1	5.0 ± 0.2	0.80 ± 0.01	1.21 ± 0.05	0.086 ± 0.002
	T	99.3 ± 1.2	99.3 ± 1.2	90.9 ± 1.7	4.6 ± 0.2	0.84 ± 0.03	1.28 ± 0.01	0.091 ± 0.003
14 d	CK	95.3 ± 1.2	95.3 ± 1.2	81.6 ± 1.9	2.8 ± 0.2	0.63 ± 0.02	1.14 ± 0.04	0.088 ± 0.002
	T	96.7 ± 1.2	96.0 ± 0.0	81.0 ± 0.9	3.4 ± 0.3	0.82 ± 0.03 **	1.18 ± 0.02	0.091 ± 0.002
21 d	CK	98.0 ± 2.0	97.3 ± 1.2	83.1 ± 2.6	1.3 ± 0.1	0.53 ± 0.03	1.37 ± 0.12	0.093 ± 0.004
	T	97.3 ± 1.2	96.7 ± 1.2	86.9 ± 1.6	1.2 ± 0.1	0.53 ± 0.04	1.50 ± 0.09	0.087 ± 0.003
28 d	CK	97.3 ± 1.2	96.0 ± 2.0	83.5 ± 2.7	0.5 ± 0.1	0.21 ± 0.01	1.34 ± 0.06	0.091 ± 0.006
	T	96.7 ± 1.2	95.3 ± 3.1	83.4 ± 0.3	1.0 ± 0.2 **	0.40 ± 0.06 **	1.32 ± 0.13	0.091 ± 0.007

Note: ** (p < 0.01).

, the germination percentage and germination energy of seeds in the melatonin-treated group remained relatively stable under low-temperature conditions, with no significant differences observed compared with the CK group during 7 to 14 d of storage. The germination index exhibited slight fluctuations but remained higher in the melatonin group than in the CK group after both 7 and 21 d of storage. Regarding seedling morphology, the melatonin-treated group showed a significantly greater shoot length (0.82 cm) after 14 d of storage than the CK group. After 28 d, both root length (1.0 cm) and shoot length (0.40 cm) were significantly higher in the melatonin group. Overall, the fresh and dry weights of seedlings tended to be higher in the melatonin group, although these differences were not statistically significant. In conclusion, melatonin treatment under low-temperature storage conditions effectively mitigated the inhibitory effects of long-term cold storage on seedling growth, although it had a limited effect on promoting germination rate and biomass accumulation.

As shown in Figure 4, conductivity, SOD activity, and APX activity of the seeds in both treatment groups under low-temperature storage exhibited distinct trends over time. Figure 4a shows that, except after 14 d of storage, the electrical conductivity of seeds in the CK group was significantly higher than that in the melatonin group. Between days 14 and 21, seed vigor declined rapidly in both groups. Figure 4b shows that SOD activity initially decreased before increasing, with no significant differences observed across storage times. Figure 4c demonstrates that APX activity initially decreased in both groups, followed by an increase and then another decrease, with no significant difference between them.

4. Discussion

4.1. Melatonin Priming Increases Salt Tolerance of Seeds

Previous studies have demonstrated that Tartary buckwheat seeds treated with melatonin exhibit significantly improved salt tolerance compared to CK [34]. Treatment with 10 and 20 μmol·L⁻¹ melatonin significantly enhances both the germination energy and percentage of cotton seeds under salt stress [35]. Optimal germination of melon seeds under salt stress was observed at melatonin concentrations of 10–50 μmol·L⁻¹ [36]. Similarly, melatonin application promoted germination and seedling growth in soybean under salt stress while reducing the relative salt damage index [37]. The germination rates of aged seeds of rice, barley, and sorghum have been shown to improve with melatonin priming [38]. In this study, a 3 h melatonin priming treatment maintained a germination percentage of 98.7–100.0% in Tartary buckwheat seeds, indicating high and stable viability. Furthermore, melatonin not only improved germination parameters but also promoted seedling growth by regulating physiological metabolic processes. Specifically, melatonin stimulates cell division and elongation, enhances water and nutrient uptake by roots, and consequently promotes both seedling and root growth, leading to a higher fresh root weight [39]. In Cyperus esculentus L., exogenous melatonin at low concentrations has been shown to enhance seedling germination and growth under combined salt and drought stress, particularly by increasing coleoptile length and plant fresh biomass [40]. These findings are consistent with those of previous studies, confirming that melatonin priming at 50 μmol·L⁻¹ can significantly increase seedling length, root length, and root fresh weight in salt-stressed Tartary buckwheat seedlings.

4.2. Vigor and Storability of Redrying Seeds After Priming

Seed vigor and storage tolerance are critical indicators of seed quality, with substantial implications for agriculture. Seeds undergoing priming followed by redrying can germinate quickly and uniformly [41]. Proper drying after priming restores or even enhances seed vigor [9], making it an effective strategy for improving seed quality and storability. A study on sorghum seeds showed that priming with PEG for 12 h followed by drying to 7%, or priming for 36 h and drying to 15%, could provide optimal germination conditions and improve storability under both constant 25 °C and fluctuating natural temperatures [42]. In this study, melatonin-primed Tartary buckwheat seeds demonstrated superior germination and seedling growth compared with the CK group under various storage conditions and durations. These findings suggest that melatonin priming delays the decline in seed vigor and enhances storability.

Seed aging, mechanical damage, and suboptimal storage conditions can compromise the integrity of cellular membranes, leading to increased electrical conductivity due to electrolyte leakage and nucleotide leakage into the soaking medium. Thus, electrolyte conductivity has been identified as a critical biochemical index for assessing seed vigor and decay extent [43]. In this study, the electrical conductivity of melatonin-treated seeds was lower than that of the CK group, except in the groups exposed to high temperature and humidity for 1 d and those stored at room temperature and low temperature for 14 d. This suggests that melatonin effectively mitigated membrane damage caused by long-term storage and preserved seed vigor. The results indicated that melatonin helped reduce seed deterioration and maintain vigor. These findings are consistent with those reported by Nie [44] and Huang [45], supporting the idea that melatonin can reduce electrolyte loss in plants under stress.

Due to stress, the accumulation of reactive oxygen species in plant cells results in reduced seed viability and inhibited growth [46]. Plants can regulate the reactive oxygen species (ROS) levels through a complex antioxidant defense system, which includes both enzymatic antioxidants (SOD, catalase (CAT; EC 1.11.1.6), and APX) and non-enzymatic antioxidants (ASA (EC 3.1.6.8) and GSH (EC 1.1.1.284)) [47]. This system maintains redox balance by scavenging free radicals and inhibiting lipid peroxidation [48,49,50]. Castanares and Bouzo [36] and Khan et al. [51] demonstrated the effectiveness of melatonin as a potent ROS scavenger that protects critical biomolecules from oxidative damage. In seeds, germination and early seedling development under salt stress are inhibited by three main factors: osmotic stress, ionic toxicity, and oxidative damage [52]. Zeng et al. [53] reported that melatonin modulated plant stress responses by directly suppressing the accumulation of ROS and reactive nitrogen species (RNS) and indirectly affecting stress signaling pathways. Melatonin is known to regulate the ROS/RNS network by upregulating the expression of antioxidant enzymes [54,55]. Previous studies have shown that priming corn seeds with 1000 μM melatonin can significantly promote the activities of SOD, POD (EC 1.11.1.7), and APX [56]. Similarly, melatonin priming can significantly enhance the activities of SOD, POD, CAT, and APX in quinoa under drought stress [57]. In Gentiana rigescens seeds, redrying after priming increases POD activity, effectively preventing lipid peroxidation of membranes [58]. Studies have shown that melatonin application can significantly enhance antioxidant enzyme activities in wheat and reduce oxidative stress, thereby supporting wheat growth and development in harsh environments [59]. In addition, in studies of triticale ‘Raritet’ and rye ‘Pamyat Khudoerka’ seeds, researchers found that melatonin increased the activity of SOD and CAT in both crops [60]. In this study, melatonin-treated seeds exhibited higher SOD activity than the CK group after 2 and 5 d of high temperature and humidity exposure, although the difference was not significant. Similarly, APX activity was significantly higher in melatonin-treated seeds than that in the CK group after only 3 d of aging. This may be attributed to the relatively short storage duration in this study, which limited the full effects of melatonin treatment. Under both room temperature and low-temperature conditions, SOD activity in melatonin-treated seeds was significantly higher than in the CK group after 21 d of storage. However, no significant differences in APX activity were observed between the groups, which could be due to variations in seed physiological status and insufficient melatonin concentration to fully activate antioxidant metabolic pathways. Apart from the continuous increase in SOD activity observed in melatonin-treated seeds under high temperature and humidity, the other treatment groups exhibited a general pattern of initial decreases in SOD and APX activities during storage, followed by a transient rebound. This rebound occurred after 4–5 d under high temperature and humidity and between 21 and 28 d under room and low-temperature conditions. This phenomenon could be attributed to enhanced cellular stress responses that counteract the accumulation of reactive oxygen species under increasing oxidative stress. However, with prolonged storage, the capacity of the antioxidant system is eventually exhausted, leading to a decrease in antioxidant enzyme activities.

5. Conclusions

Priming Tartary buckwheat seeds with 50 μmol·L⁻¹ melatonin for 3 h mitigated the decline in seed vigor induced by salt stress. This treatment enhanced germination percentage, seedling and root length, and root fresh weight. Additionally, it alleviated membrane damage caused by long-term storage, increased antioxidant enzyme activity, and improved storage tolerance. However, the safe storage period was limited, suggesting that timely sowing after priming is recommended to avoid loss of treatment benefits due to prolonged storage. In summary, melatonin seed priming is a practical and promising technology with strong theoretical foundations and substantial application potential. When applied at an appropriate concentration, melatonin pretreatment significantly enhances seed antioxidant capacity and germination rate under suboptimal storage conditions. Its inherent biosafety, low cost, and ease of application make it well suited for large-scale adoption in stress-prone agricultural systems. Further validation under field conditions, such as saline soils and long-term storage, is required. Additionally, molecular marker or transcriptomic analyses should be conducted to elucidate the regulatory mechanisms of melatonin-induced antioxidant pathways and stress memory imprinting, thereby supporting the development of standardized implementation protocols.