Physiological and Biochemical Changes in the Seeds of Naturally Aged Wenling Medic (Medicago polymorpha) with Its Recovery of Viability

Abstract

Wenling Medic (Medicago polymorpha) is common in southern China and has long been utilized as a vegetable in eastern China, as well as a significant raw ingredient for livestock and pickled meals. As a legume, there is still a research vacuum, and the first problem Wenling Medic faces in production is the problem of seed germination. The germination percentage of Wenling Medic was low, according to production practice and laboratory tests performed in this work. Furthermore, after more than two years of storage, the germination percentage of Wenling Medic dropped sharply, and it lacked a long enough seed life. An attempt was made to restore the viability of the seeds using the polyethylene glycol (PEG) osmoregulation-mediated priming method, after the physiological and biochemical characteristics of the seeds were assessed using the anthrone method, Bradford assay, 3,5-dinitrosalicylic acid (DNS) method, and thiobarbituric acid (TBA) method. The findings showed that it has a relatively short storage age and, with a useable life of only two years under normal aging circumstances, is not deactivated soon after harvesting. In addition, whereas protein content and α-amylase concentration did not significantly correlate (p > 0.05) with storage life, the conductivity of exudate, malondialdehyde content, and carbohydrate content did (p < 0.05). The seed viability was not considerably increased by the saturation initiation mediated by osmoregulation, utilizing PEG. In conclusion, the decrease in Wenling Medic seeds’ germination ability was substantially connected with higher levels of lipid peroxidation and decreased carbohydrate levels, but not with protein concentrations or α-amylase activity. The timing of dehydration may need to be carefully controlled when using PEG osmoregulation to prime Wenling Medic seeds.

1. Introduction

Wenling Medic (Medicago polymorpha L.), with the common name “bur clover”, has a very wide distribution in China, and the Chinese name means a usable plant with golden flowers [1]. Also known as Nan-mu-xu (“南苜蓿”, Wade-Giles: nan-mu-hsü), Yang-cao(“秧草”, Wade-Giles: yang-tsao) and Cao-tou (“草头”, Wade-Giles: tsao-tou) [2], it is an annual leguminous forage with fast growth, easy reseeding, soil improvement, and high-quality forage characteristics for grazing livestock. In southern Australia, M. polymorpha is used with other annual clovers in grassland agricultural systems, in rotation with cereal crops [3].

The primary problem faced in M. polymorpha production is seed germination, which has also been considered a prerequisite in other studies. In a study on the germination of M. polymorpha seeds from different sources in China, Ren et al. [4] found that the germination of M. polymorpha seeds from different sources varied greatly in terms of hardness, and also found that M. polymorpha seeds from Wenling, Zhejiang Province of China, had the highest germination percentage at between 15 °C and 25 °C, and that breaking the hardness of the seed coat significantly increased the germination percentage of M. polymorpha seeds. M. polymorpha seeds usually germinate at 15 °C and 25 °C, and germination above 40 °C is very rare. [4]. Prior to this, storage of the seeds is also a great challenge, and De Vitis et al. [5] suggest that for orthodox species, seeds can be usually stored at −18 °C for more than 5 years. To achieve such a freezing environment for general experimental sites or production sites, is a rather difficult thing to do. Therefore, in practice, a storage environment of 4 °C is usually used in production. This is at the expense of the storage life of the seeds. It is obvious that the storage material, biological enzyme activity, and genetic material of the seeds are lost during this period, which in turn weakens the viability of the seeds themselves.

There are few studies on M. polymorpha seeds’ viability or germination. Kibinza et al. [6] noted that lipid peroxidation is a major contributor to membrane damage, and as a result, many studies aimed to assess the main byproduct of this reaction, namely malondialdehyde (MDA). This was noted in a study on the naturally occurring aging-related biochemical changes in the seeds of two legume varieties (Trifolium repens and Trifolium pratense), stored for 40 years by Cakmak et al. [7]. MDA is one of the main indicators of oxidative damage, and one of the byproducts generated during lipid peroxidation, according to Min et al. [8]. Therefore, it is conceivable to use plant malondialdehyde content to determine the level of seed age and lipid peroxidation. Furthermore, it was discovered, in a study on rice by Damaris et al. [9], that α-amylase, the main type of amylase with secondary carbohydrate binding sites, is an essential enzyme for angiosperm growth and development. It is clear that α-amylase plays a crucial role in plant morphogenesis. Additionally, conductivity is a widely used technique for determining the viability of seeds, and is well known for its straightforward operation. Prado et al. [10] used linear regression analysis to investigate the physiological potential of soybean seeds in legumes and its connection to conductivity. 2,3,5-Triphenyltetrazolium chloride (TTC) is used as a stain to identify seed viability, because of its convenient properties [11,12,13].

The traditional and simple plant seed initiation approach works by osmotically controlling post-drying, to increase seed viability. Salicylic acid was employed by Bortolin et al. [14] to start Trifolium vesiculosum (annual) and Trifolium repens (perennial), which reduced aluminum oxidation, and enhanced the performance of seedlings of the two legumes when exposed to aluminum.

In this work, MDA and conductivity detection methods were used to attempt to evaluate the degree of aging of M. polymorpha seeds under the impact of peroxide. To further understand the connection between nutritional storage capacity and storage age of M. polymorpha seeds, the contents of protein and carbohydrates were also determined. Finally, the embryonic viability and biological enzymatic activities of goldenrod seeds were determined using the TTC staining technique and the 3,5-dinitrosalicylic acid (DNS) method. The 3 factors were brought together to thoroughly analyze the viability of M. polymorpha seeds under normal aging conditions and to offer theoretical support for better storage methods, and using the genetic resources for M. polymorpha, as well as the growth of the Wenling Medic market.

2. Materials and Methods

2.1. Seed Material Sources and Storage Conditions

Wenling Medic (M. polymorpha) comes in a limited number of commercial cultivars, and most of the local varieties are used in manufacturing. The artificially cultivated “Wenling Grandifoliate Tsao-tou (Wenling Medic)” material was produced in Wenling, Zhejiang Province, China (121.386° S, 28.372° N), where the average annual temperature is 16 °C to 23 °C, and the average daily maximum temperature in June is 30 °C; the average daily minimum temperature is 23 °C. In addition, according to a communiqué from the Taizhou Water Resources Bureau, the annual precipitation in the Wenling area is 1590.5 mm. The seeds used in this study were obtained from this area. In June of 2017–2022, the seed material was collected, and the seed pods were eliminated. The kidney-shaped seeds were left uncoated, sealed in aluminum–plastic composite bags with the air removed using a plastic sealer, and kept at a constant 4 °C temperature in a controlled atmosphere.

The samples were numbered, for ease of presentation (

Table 1. The experimental materials used in the experiment.

Harvested Year	Storage Age (Years)	Test Number	Location
2017	6	MP2017	Wenling, Zhejiang, China
2018	5	MP2018	Wenling, Zhejiang, China
2019	4	MP2019	Wenling, Zhejiang, China
2020	3	MP2020	Wenling, Zhejiang, China
2021	2	MP2021	Wenling, Zhejiang, China
2022	1	MP2022	Wenling, Zhejiang, China

).

2.2. Methodology of the Experiment

2.2.1. Germination Test and Seed Germination Capacity

Seeds were tested for germination using the method of germination on paper (top of papers, TP) given in the methodology of the Chinese National Standard GB/T 2930.4-2017 “Rules of seed testing for forage, turfgrass and other herbaceous plant—The germination test” and germinated at (25 °C/20 °C day/night) for 10 d, with a light intensity of 42,000 lm m⁻². Each replicate used 100 grains. The number of seeds germinated per day was recorded; ultra-pure water is sprayed every 2 days to keep the germination substratum high humidity.. Abnormal seedlings were excluded. Germination was carried out in an MGC-800BP-2L light incubator (Shanghai Yiheng, Shanghai, China), with an experimental water conductivity σ < 0.01 μS cm⁻¹. Seeds were weighed using a Sartorius^® analytical balance (Sartorius Beijing, Beijing, China).

2.2.2. Seed Conductivity Test

The methods described by Colete et al. [15] and Hopper et al. [16], as well as the approach described in the ISTA “Manual of Methods for Vigor Determination” were used to determine the conductivity of the seeds. Five 250 mL beakers in all were employed, with one serving as a blank control group and the other four serving as experimental groups. After the mass (g) was weighed beforehand, the experimental group’s samples were placed in each beaker with the appropriate number, the surface was cleaned, the water was wiped off with absorbent paper, and an equal amount of 100 mL of ultrapure water was added to each beaker (include the blank control group). Seed conductivity was calculated using Equations (1) and (2):

$${\sigma }_{\mathrm{seed}}=\frac{{\sigma }_n}{{\mathrm{W}}_n}$$

(1)

$${\sigma }_n=\sigma -{\sigma }_{CK}$$

(2)

where ${\sigma }_{seed}$ refers to the arithmetic mean of the four conductivity values measured for the n^th group of seeds, in μS cm⁻¹ g⁻¹; ${\sigma }_n$ refers to the conductivity value obtained for each beaker in the nth group (the conductivity of the seed leachate measured for each experimental group beaker in the same group of beakers, minus the conductivity of the blank control in the same group); ${\mathrm{W}}_n$ refers to the conductivity of the seeds in each beaker, measured for the n^th group of seeds; and ${\mathrm{W}}_n$ refers to the dry weight of the seeds in each beaker for the n^th group of seeds. Conductivity was determined using a DDS-309 + conductivity meter, manufactured by Chengdu Century Ark Technology Co., Ltd. (Chengdu, China), with an accuracy of ±0.5% FS and a minimum resolution of 0.001 μS cm⁻¹.

2.2.3. Seed Viability Test (Tetrazolium Test)

The viability of seeds was determined using the method of the Chinese National Standard GB/T2930.5-2017 “Rules of seed testing for forage, turfgrass and other herbaceous plant—Biochemical test for viability” using 2,3,5-triphenyltetrazolium chloride (TTC) as the stain. The seeds had no cutting before staining. Then, seeds were steeped in water at 20 °C for 22 h prior to TTC staining. After that, seeds were treated in TTC staining solution for 18 h, at a concentration of 0.01 g mL⁻¹. A dissecting needle and forceps were used to remove the seed coat, in order to reveal the cotyledons and embryonic roots for inspection. The purity of the reagents was greater than 98%, using products from the Shanghai Aladdin^® Biochemical Technology Co. Ltd. (Shanghai, China).

2.2.4. Determination of Seed Protein Content

The protein content was determined by the Bradford method, with reference to the method of Li et al. [17]. The standard curves were adopted from 0–100 μg mL⁻¹ and 0–1000 μg mL⁻¹, and 1000 μg mL⁻¹ bovine serum albumin (BSA) solution was used as the standard. Specifically, 200 mg of M. polymorpha seeds was accurately weighed in a mortar and crushed into a dry powder by adding liquid nitrogen (LN₂), then 5.0 mL of water was added and ground into a homogenate in an ice bath, transferred to a centrifuge tube, centrifuged at a relative centrifuge force (RCF) of 1825 g for 10 min, the supernatant was poured into a 10 mL volumetric flask, and then 2.0 mL of water was added to the residue. This was then suspended and centrifuged with RCF 1825 g for another 10 min, combined the supernatants, and the volume was set to the scale. Four centrifuge tubes were prepared, with caps, and 0.1 mL of the solution to be tested was aspirated into the tubes numbered 2–4, as 3 replicates. A volume of 5.0 mL of Coomassie brilliant blue was added to each of the centrifuge tubes, mixed thoroughly, and allowed to stand for 2 min, then Tube 1 of the standard curve was used as the blank, colorimetric analysis was performed at 595 nm, and the absorbance value recorded. Subsequently, the protein content of the samples was calculated using Equation (3):

$$\mathrm{PR}=\frac{\mathrm{A}\times {\mathrm{V}}_1}{{\mathrm{V}}_2\times \mathrm{W}}$$

(3)

where PR is the protein content of each replicate, in μg g⁻¹; A is the protein content found on the standard curve; $V_1$ is the total volume of the extract (mL); $V_2$ is the volume taken for the determination (mL); and W is the fresh weight of the sample (g). Reagents were obtained from the Shanghai Macklin^® Biochemical Technology Co., Ltd. (Shanghai, China), and all purities were greater than 98%.

2.2.5. Determination of Seed Carbohydrates Content

The amount of carbohydrates was determined using a modified anthrone method. Improvements were made to the defects of the classical method, such as lack of temperature control, reaction time control and interference of protein, which may lead to wrong results on the methods of Li et al. [18] and John et al. [19] were improved. The reference value was 100 g mL⁻¹ of glucose solution. To help with the investigation, 1 g of seeds was precisely weighed and put in a mill and pestle with LN₂. In a 50 mL centrifuge tube, 10 mL of water was added. This was swiftly placed in ice water to chill, after 15 min of being covered in a boiling water bath.

To precipitate the sample proteins, 2.5 mL of 0.1 g L⁻¹ lead acetate solution was added to each centrifuge tube. Then, after thoroughly mixing the process with a vortex mixer to remove any extra lead acetate, 0.5 g of potassium oxalate was added as well. The solution to be tested was then fixed in a 50 mL volumetric flask with ultrapure water, after being centrifuged for 2 min with RCF 16,421 g.

Five test tubes, with the numbers 0–4 were set up, and the solutions to be measured were added to tubes 1–4. Water was then added to each tube and mixed right away, and 5 mL of the anthrone reagent was added. The tubes were then shaken well and placed in a boiling water bath for 10 min, after which they were quickly cooled in an ice-water solution. The absorbance values were calculated successively, under 620 nm visible light, while using tube 0 as the reference solution. The horizontal coordinate for the curve was the sugar concentration, and the vertical coordinate was the absorbance value. The Shanghai Aladdin^® Biochemical Technology Company (Shanghai, China) provided the experimental reagents, which had purities of more than 98%.

2.2.6. Determination of Seed Amylase Activity

According to the protocol of Li et al. [17], the 3,5-dinitrosalicylic acid (DNS) method was employed to measure the amylase activity, and we modified the method for making DNS solution. The standard was a solution containing 1 mg mL⁻¹ of maltose. In a water bath at 45 °C, 1.00 g of DNS was dissolved in 20 mL of 1 mol L⁻¹ NaOH, 50 mL of distilled water was added, and 30.00 g of potassium sodium tartrate (Rochelle salt) was added, being careful not to allow the solution’s temperature rise over 48 °C. The mixture was then stirred until it was clear and transparent. After dissolving, it was diluted to 100 mL, then kept in a brown bottle that was tightly closed, to prevent the entry of carbon dioxide.

One gram of germinated seeds was weighed, this was combined with a tiny amount of 100 mesh quartz sand in a mortar, and ground into a homogenate before being transferred to a 50 mL centrifuge tube. A volume of 30 mL of water was added, thoroughly combined, and allowed to stand for 15–20 min at 20 °C, with periodic shaking. The supernatant was removed after 5 min of centrifuging at 1825 g of RCF. The enzyme dilution solution was created by aspirating 4 mL of the aforementioned amylase stock solution and diluting it to 100 mL with distilled water, in a volumetric flask (enzyme solution).

Four test tubes were numbered 1–4, with two serving as the controls and two as the experimental. Each test tube contained 1.0 mL of enzyme solution. The tubes were precisely heated for 15 min at 70 °C ± 0.5 °C, removed, and immediately cooled. Heat was used to inactivate α-amylase during this procedure.

Equal volumes of pH 5.6 citrate buffer (1.0 mL) were added to each tube. By initially adding 4.0 mL of 0.4 mol L⁻¹ sodium hydroxide solution to the control tube, the enzyme activity was stopped. The four test tubes were placed in a constant temperature water bath at 40 °C ± 0.5 °C for 10 min. Starch solution (2 mL), preheated at 40 °C, was added to each tube, mixed, and immediately placed in a constant temperature water bath at 40 °C ± 0.5 °C for 5 min. Then, 4.0 mL of 0.4 mol L⁻¹ sodium hydroxide solution was added to each tube immediately. After adding 2 mL of DNS solution to each tube, the enzyme was heated accurately in a boiling water bath for 5 min, removed, and cooled rapidly, diluted to 25 mL with distilled water, mixed well, and then the absorbance value of each tube was measured at the wavelength of 520 nm to find out the maltose content from the standard curve, and then the results were calculated according to Equation (4):

$$\mathsf{\alpha }-\mathrm{amylase}=\frac{\left(\mathrm{A}- {\mathrm{A}}^{\prime }\right)\times \mathrm{V}}{\mathrm{W}\times \mathrm{C}}$$

(4)

where A is the amount of maltose produced by α-amylase hydrolysis of starch, as found in the standard curve; $A^{\prime }$ is the amount of maltose in α-amylase control tubes, as found in the standard curve; V is the total volume of sample dilution; W is the sample mass (g); and C is the volume of sample liquid (mL) at colorimetry. The reagents used were from the Shanghai Aladdin^® Biochemical Technology (Shanghai, China), and the purities were > 98%.

2.2.7. Determination of Malondialdehyde Content of Seeds

The determination of the malondialdehyde content in the seeds was performed using the commonly utilized thiobarbituric acid (TBA) method, specifically, using TBA to produce a chromogenic reaction with malondialdehyde in tissues, by heating under acidic conditions to produce a reddish-brown trimethoprim (3,5,5-trimethyloxazole-2,4-dione), with a maximum absorption wavelength of 532 nm, referencing the method of Li et al. [17]. Naturally aged seeds were taken, 1 g from each year, ground in liquid nitrogen to a dry powder, and mixed with 2 mL of 0.1 g mL⁻¹ trichloroacetic acid solution (TCA) in a capped centrifuge tube, as a homogenate. Then, this was ground with 8 mL of 0.05 g mL TCA. The extract of malondialdehyde was made from the supernatant. A volume of 2 mL of the extraction solution was added to each tube, 2 mL of distilled water to the control tube, 2 mL of 0.6% TBA solution to each tube, and the tubes were shaken well. Four clean tubes were taken and numbered, three of which serves as samples (three technical replicates). The mixture was stirred for 15 min in a pot of boiling water, quickly cooled, then centrifuged for 10 min at RCF 1800 g. For the supernatants, the absorbance values were calculated at 450, 532, and 600 nm. These were used to compute the malondialdehyde concentration in the seeds after the concentration mistake was adjusted using Equation (5) [20].

$$c_{MDA}\left({\mathsf{\mu }\mathrm{mol} \mathrm{L}}^{-1}\right)=6.45\times \left({\mathrm{A}}_{532}-{\mathrm{A}}_{600}\right)-0.54\times {\mathrm{A}}_{450}$$

(5)

The Shanghai Aladdin^® Biochemical Technology Company (Shanghai, China) provided the experimental reagents, which each had a purity of more than 98%.

2.2.8. Priming Based on Polyethylene Glycol (PEG) Osmoregulation

PEG-6000, which has a number-average molecular weight of 6000, was produced in three concentrations of solutions: 50 g L⁻¹, 100 g L⁻¹, and 150 g L⁻¹. Following the harvest year, four 50 mL plastic bottles with caps (with tiny holes drilled into the caps) were prepared for each seed lot. Each bottle contained one of three technical replicates of 8 g seeds from the same seed lot, along with 12 mL of the PEG-6000 solution. The bottles were then gently shaken, to ensure that the seeds were evenly coated with the water. The last plastic container contained 12 mL of water and 8 g of seeds. Each bottle was then left in the dark at 15 °C, for 24 h [21,22,23].

The seeds were taken out when the appropriate initiation time came, rinsed several times with distilled water, dried with absorbent paper, and then dehydrated in a ventilated environment, at 20–25 °C and 40–45% relative humidity, for 26 h, to make the seed dehydrated close to its original weight. For each treated seed, a control group was established for the germination test. Both the experimental and control groups were sampled and tested in accordance with the procedure in Section 2.2.1. Reagents were provided by the Shanghai Macklin^® Biochemical Technology Co., Ltd. (Shanghai, China), and the purities were greater than 98%.

2.2.9. Statistical Analysis and Plotting Methods

The experimental results were analyzed using the “ggplot2 3.4.0” analysis package [24] and the “ggpubr 0.5.0” enhancement package [25] with the “ggbreak 0.1.1” [26] package, in RStudio IDE 2021.09.0 (Posit Software, Boston, MA, USA), for statistical and graphical plotting of data using the R language editing commands. Non-parametric distribution tests, Pearson’s Correlation Analysis, and ANOVA tests for test data were performed using the IBM^® SPSS^® Statistics 27 (IBM, Armonk, NY, USA) software. The indices of seed germination were performed using an R package, which includes “SeedCalc 1.0.0” [27] and “germinationmetrics 0.1.7” [28].

3. Results

3.1. Age Strongly Correlated with Lipid Peroxidation and Cell Membrane Permeability

The seeds’ imbibition of water releases some solutes into the water, including some salt ions, free organic acids, sugars, and proteins. Seed conductivity and subsequently seed viability can serve as indicators of a seed’s ability to repair its membrane system. The MDA level is a crucial indicator of the body’s potential antioxidant capacity. It can also indirectly indicate the extent of tissue peroxidative damage, by providing information on the rate and intensity of the body’s lipid peroxidation.

Storage age and seed conductivity (mean value), and storage age and MDA content (mean value), were both found to be normally distributed, using the Kolmogorov–Smirnov statistic (K–S test). The MDA concentration and storage age were substantially associated at the 0.05 level, with an absolute value of 0.896 (p = 0.016 < 0.05), while the association between conductivity and storage age was not significant at the 0.05 level (Figure 1). Additionally, the significance of differences between many samples was examined using Duncan’s multiple range test, and it was discovered that the MDA content was similar in 2019 and 2020, the years in which MDA metabolism reached a plateau, and the content subsequently increased (Figure 1).

3.2. The Carbohydrate Content Decreases Significantly with Storage Age

The primary source of nutrient uptake during the initial stages of seed germination is seed storage material. Sugars, the primary source of cellular energy, are crucial to the germination of seeds.

The mean carbohydrate content of M. polymorpha seeds, which primarily consisted of soluble sugars, exhibited a very strong (Pearson’s Rho = −0.917) significant inverse correlation with storage age (p = 0.010 < 0.05), plateauing between the second and fourth years of storage, when the carbohydrate content also plateaued. The carbohydrate content was between 2.071 mg g⁻¹ for new seeds, and 0.470 mg g⁻¹ for sixth year seeds in storage (Figure 2).

3.3. Protein Content Was Not Significantly Correlated with Storage Time

Protein serves as the “bricks and mortar” for seeds to emerge from the ground and develop into seedlings. Protein is the primary substance that is dependent on the morphogenesis of seeds during germination.

However, it can be understood that protein degradation still occurs during seed storage, as the mean content decreased from 433.40 mg g⁻¹ in the first year of new seeds to 283.41 mg g⁻¹ in the sixth year, a decrease of 34.61%. The test results showed that the protein content (mean) was not significantly correlated with storage time (p = 0.323 > 0.05). Additionally, the protein level considerably dropped, starting in the second year of storage, and remained rather steady for the following four years, according to Duncan’s multiple range test (Figure 3).

3.4. There Is No Discernible Relationship between Storage Age and Amylase Content

The endoglycosidase α-amylase mostly degrades maltose by randomly acting on the α-1,4-glycosidic link inside the starch chain. It is used by seed cells for cellular metabolism, following further hydrolysis, which can laterally represent seed vitality.

Storage age and α-amylase activity did not substantially correlate using the Pearson correlation test at the 0.05 level. The Duncan’s multiple range test revealed that the seed α-amylase activity was comparable after 3 and 6 years of storage (Figure 4).

3.5. PEG-6000 Seed Start Therapy Is Ineffective at Increasing Seed Viability

Using the osmoregulation technique, PEG, a polymeric organic chemical with significant water absorption, can be utilized to increase the internal humidity of seeds. PEG can absorb water from the air at room temperature.

The germination percentage of seeds did not significantly increase from the untreated control after osmoregulation with various concentrations of PEG-6000 solution; rather, PEG lowered the germination percentage by varying degrees (Figure 5). After more than 2 years of storage, the untreated M. polymorpha seeds lost their fundamental viability and displayed a significant decline in germination percentage until it reached 0 (

Table 2. Seed germination triggered by osmoregulation treatments with different concentrations of PEG-6000.

Samples	N	Mean ± SE * (p < 0.01)	95%CI †
			Upper	Lower
MP2017P1	3	0.33 ± 0.33 g	−1.10	1.77
MP2017P2	3	1.00 ± 0.58 g	−1.48	3.48
MP2017P3	3	0.33 ± 0.33 g	−1.10	1.77
MP2017CK1	4	1.00 ± 1.00 g	−2.18	4.18
MP2018P1	3	0.00 ± 0.00 g	0.00	0.00
MP2018P2	3	0.67 ± 0.33 g	−0.77	2.10
MP2018P3	3	0.33 ± 0.33 g	−1.10	1.77
MP2018CK2	4	2.00 ± 1.15 g	−1.67	5.67
MP2019P1	3	2.00 ± 1.15 g	−2.97	6.97
MP2019P2	3	0.33 ± 0.33 g	−1.10	1.77
MP2019P3	3	1.00 ± 0.58 g	−1.48	3.48
MP2019CK3	4	1.00 ± 1.00 g	−2.18	4.18
MP2020P1	3	1.00 ± 0.58 g	−1.48	3.48
MP2020P2	3	0.67 ± 0.33 g	−0.77	2.10
MP2020P3	3	0.33 ± 0.33 g	−1.10	1.77
MP2020CK4	4	2.00 ± 1.15 g	−1.67	5.67
MP2021P1	3	8.67 ± 0.33 f	7.23	10.10
MP2021P2	3	9.33 ± 0.33 f	7.90	10.77
MP2021P3	3	10.67 ± 0.33 f	9.23	12.10
MP2021CK5	4	24.00 ± 1.63 e	18.80	29.20
MP2022P1	3	70.67 ± 0.67 b	67.80	73.54
MP2022P2	3	51.00 ± 0.58 d	48.52	53.48
MP2022P3	3	60.33 ± 0.33 c	58.90	61.77
MP2022CK6	4	74.00 ± 2.00 a	67.64	80.36

P1, P2, and P3 materials correspond to the three concentrations of PEG-6000 solution in treatment columns T1, T2, and T3 at 50 g L⁻¹, 100 g L⁻¹, and 150 g L⁻¹, respectively. CK1–CK6 are 6 groups of untreated blanks for seeds, corresponding to the storage year. Lowercase letters a–g refer to the results of the analysis of the significance of differences between multiple samples, based on Duncan’s multiple range test, with a being the most significant and g being the least significant, at 0.05 level of significance. N refers to the sample size of each technical replicate. * SE refers to standard error. † CI refers to a statistically constructed 95% confidence interval.

and

Table 3. Table of indices of seed germination.

Samples	GSI * ± SE	CVG ‡ ± SE	CUG † ± SE
MP2017P	0.38 ± 0.20	35.00 ± 15.00	0.07 ± 0.03
MP2017	0.04 ± 0.00	100.00 ± 0.00	0.25 ± 0.25
MP2018P	0.11 ± 0.11	33.33 ± 0.00	0.06 ± 0.00
MP2018	0.20 ± 0.09	17.14 ± 2.86	0.09 ± 0.05
MP2019P	0.66 ± 0.37	25.40 ± 3.17	0.09 ± 0.02
MP2019	0.04 ± 0.00	50.00 ± 0.00	0.13 ± 0.13
MP2020P	0.46 ± 0.14	34.44 ± 8.68	0.07 ± 0.02
MP2020	0.04 ± 0.00	75.00 ± 25.00	0.38 ± 0.24
MP2021P	3.65 ± 0.38	37.79 ± 4.34	0.07 ± 0.02
MP2021	0.04 ± 0.00	49.36 ± 3.45	3.63 ± 0.25
MP2022P	25.56 ± 2.54	38.09 ± 0.15	0.05 ± 0.00
MP2022	0.06 ± 0.00	36.66 ± 2.58	10.82 ± 1.09

SE refers to the standard error. * refers to germination speed index being statistically significant at the 0.01 level; † refers to coefficient of uniformity of germination being statistically significant at the 0.01 level; ‡ refers to velocity of germination coefficient being statistically significant at the 0.01 level.

).

3.6. TTC Staining Test for Seed Viability

To check the vitality of seeds, we frequently utilize the lipid-soluble photosensitive compound 2,3,5-triphenyltetrazolium chloride (TTC). It functions as a proton receptor for the respiratory chain’s pyridine-nucleoside-structured enzyme system, which causes normal tissues to turn red when it interacts with dehydrogenase. Seed cells that are dead, or not viable, fail to stain or stain more lightly, because dehydrogenase in living cells of viable seed germ tissue can convert TTC to the insoluble, red, stable 1,3,5-triphenylformazan (TTF).

The majority of M. polymorpha seeds would lose viability after more than 2 years of seed storage, according to the staining studies (Figure 6). There were also some fresh seeds that may germinate, that had a hard seed rate between 10% and 20%.

4. Discussion

During natural aging, the germination percentage and viability decline significantly. However, the mechanism behind this decline has not been fully elucidated so far. Naturally aged M. polymorpha seeds, that have been stored for more than 2 years, have almost lost their ability to germinate (Table 2, Figure 5). An empirical explanation was proposed by Chen et al. [29], who noted that the enzymes that participate in glycerolipid metabolism and fatty acid degradation probably lead to the degradation of oil bodies (TAG) and membrane lipids (PC, PE, PS, PI, PG) and, ultimately, destroy the structure, causing a decline in seed vigor during natural seed ageing. While other researchers report that the quality of the messenger RNAs, stored during embryo maturation on the mother plant, is a factor in seed germination success. Moreover, the germination phenotype is greatly influenced by proteostasis and DNA integrity [30].

It is well known that lipid peroxidation is increased in aged seeds, and free radical scavenging capacity is reduced. Aging inhibits seed germination and increases lipid peroxidation, with a higher temperature making matters worse. Moreover, seeds harvested from spring crops are more susceptible to aging than seeds harvested from fall crops [31]. Malondialdehyde (MDA) is one of the best investigated products of lipid peroxidation [32]. The reaction of 3,5-dinitrosalicylic acid with MDA clearly shows the different degrees of natural aging, allowing the lipid peroxidation within M. polymorpha seeds to be revealed. The findings revealed a substantial association between storage time (p < 0.05) and MDA. Lipid peroxidation in seed cells deteriorated with longer storage times, demonstrating that MDA increased with longer storage times (Figure 1b). At the same time, the result that EC increased with increasing seed storage age (Figure 1a) (p < 0.05) [10], could also strongly indicate the condition of the damaged seed membrane system [33,34], even when the leakage of the seed coat is affected by hard seed, it could still significantly indicate the situation. There are three phases to the water absorption by seeds (imbibition) prior to germination, with the second phase being particularly crucial. A DNA repair event in the “pregerminative metabolism” is started by the living seed during the second phase of imbibition, which is stimulated by water absorption during imbibition. Base and nucleotide excision repairs, important DNA repair processes, are turned on early in the seed imbibition process, to preserve genome integrity.

Carbohydrates have an important effect on the germination process of seeds. A study by Huang et al. [35] indicated that seed germination was promoted by increasing the biosynthesis of seaweed sugars in cucumber (Cucumis sativus L.). However, endophytic bacteria are commonly present in legume seeds, and during long-term storage, it is possible that endophytes may use the sugars stored inside the seeds for metabolism and thus deplete the seed storage material. A study by Zhang et al. [36] mentioned that the ratio of cottonseed sugar to sucrose increased significantly with seed aging, in both endophyte-free and endophyte-infected embryonic tissues, whereas in dissected seeds the concentration of alginate detected in the tissues dissected from endophytic seeds was significantly lower, regardless of the status of the endophytes. This may explain why the carbohydrate content stored within the M. polymorpha seeds decreased significantly during long-term storage (Figure 2).

For legumes, the intra-plant variability in single seed weight and protein content is large, and these variations are thought to be related to the source/sink ratio within the plant [37], which contributes to the variation in protein storage of legume seeds at the initial stage of entry into the storage process. The seeds involved in the experiment were harvested in June of each year, which seems to indicate that the viability of the seeds is inherently lower during this time. Similar to the case of naturally-aged sweet corn, which showed no or little difference in total protein content [38], M. polymorpha seeds also showed statistically insignificant differences (Figure 3). It is speculated that, in macroscopic significance of content, protein changes are not sensitive during long-term storage.

Surprisingly, the alpha-amylase activity of M. polymorpha seeds, an important carbohydrate utilizer, after 24 h of infiltration and swelling, did not significantly correlate with storage age. This is different from previous studies on sweet corn and wheat [39,40,41,42]. Admittedly, the results of the experiment (Figure 4) do not exclude that the results differ due to differences in seed storage conditions, production systems, and species.

TTC staining, which in principle uses the red MTT formazan generated by the reaction to identify and characterize the activity of cells, was first developed and first used by Lakon [43] to assess the viability of single seeds. It is still widely used today and is a reliable validation method [11,12,13]. Since hard seed is a form of physical dormancy in plants, which is prevalent in legumes, the usual determination of germination and field emergence is not valid for seeds with dormancy and hard seeds. Therefore, the method using TTC staining is a reasonable choice and has a high reference value. It is clear from the visual results, that the sampled seeds exhibit loss of viability after more than two years of storage. Obviously, even under good storage conditions, oxides or other toxic substances accumulated by natural aging can still be the first element to be addressed for long-term storage of M. polymorpha seeds.

Ren’s study [4], pointed out that the temperature at the time of germination and the hard seed condition of the seeds themselves have an effect on the germination of M. polymorpha seeds. Therefore, a good control of the incubator temperature and photoperiod is required during experiments. Banerjee et al. [44] found that seed initiation and foliar fertilization affected the yield of relay grass pea (Lathyrus sativus L.), by accelerating the efficiency of photosynthetically active radiation (PAR) use. Osmopriming is a commonly adopted priming technique, and offers a highly attractive solution for improving seed germination performance and crop stand establishment [45,46,47]. Additionally, PEG is typically thought to have low biological toxicity [48]. So, it is possible to assume that non-significant priming findings could be influenced by other factors, such as PEG concentration and priming duration. Moreover, seed priming with PEG resulted in earlier and synchronized seed germination in various crops such as alfalfa, cereal, turfgrasses, and vegetable crops, upon exposure to low or high temperature stress [47].

However, trials on M. polymorpha showed no significant improvement in seed vigor, and even led to a decrease in seed germination, which could possibly be related to the high hardness of 67.67% of M. polymorpha seeds in Wenling [4]. In addition, it has been suggested [49] that, under artificial hydration–dehydration cycles, the seeds suffered more damage and more rapid loss of vigor due to unsaturated swelling after 2 h of moisture absorption, compared to dehydration after more than 4 h of saturated swelling. It is likely that the seeds themselves failed to fully utilize the antioxidant mechanism because of the unsaturated post-dehydration treatment. Therefore, it cannot be assumed that the PEG osmoregulation-mediated seed initiation technique does not act as a viability enhancer for M. polymorpha seeds, and further discussion on the timing of dehydration is recommended. Previous studies on legumes concluded that a seed priming duration of 6 h resulted in faster and improved emergence and a higher grain yield of soybean [50]. It is generally accepted that seed longevity depends on genetic characteristics, seed coat structure, seed chemical composition, and environmental conditions [51]. Moreover, legumes are generally considered to be long-lived seeds, that can still guarantee a certain amount of germination after more than five years of storage [52]. A germination percentage of 86.3% has been reported for white lupine seeds after 26 years of storage, at −14 °C [53]; even, historically, the legume arctic tundra lupine (Lupinus arcticus) was found to have a storage life of 10,000 years, which is currently the longest-lived seed in the world [54]. For M. polymorpha, a legume that is mostly sown in summer and autumn in the middle and lower reaches of the Yangtze River in China [55], it needs further discussion whether the ambient temperature at harvest time causes a decrease in seed viability, as mentioned by Wang et. al. [38].

5. Conclusions

The results indicate that lipid peroxidation, which was exhibited by a rise in exudate, as well as an increase in electrical conductivity and MDA value, during the process of water absorption, reduced the ability of cells to repair themselves. The amount of carbohydrates in the sample dramatically decreased as storage years increased, however there was no discernible relationship between the protein content, amylase activity, or M. polymorpha seed activity. The PEG priming procedure was then utilized to rejuvenate the seeds. In M. polymorpha, it is important to investigate the right amount of concentration and priming time. It is suggested that, in order to establish the ideal preservation circumstances, M. polymorpha preservation research should further validate the DNA-level modifications under various storage environments. Moreover, the development of artificial aging is a research tool.