Method for improving seed germination ability of alfalfa under saline-alkali stress

Abstract

The application discloses a method for improving seed germination ability of Medicago sativa under saline-alkali stress, which comprises the following steps: after seed disinfection, the seed is subjected to far-infrared pretreatment, room temperature stabilization treatment, and then is placed in a seed soaking treatment solution containing spermidine and astaxanthin for oscillation soaking. Further, a seed moisture content standardization adjustment step can be added before the far-infrared pretreatment, and a variable-speed oscillation in stages is adopted during seed soaking. The application realizes time sequence integration and three-stage cascade synergism of physical and chemical means by opening the seed coat absorption channel through far-infrared physical pretreatment, combining the chemical synergistic effect of spermidine stabilizing cell membrane and astaxanthin removing active oxygen, significantly improves the germination rate, germination potential and seedling growth quality of Medicago sativa seeds under saline-alkali stress, and simultaneously reduces the oxidative damage level, and is suitable for popularization and planting of Medicago sativa in saline soil areas.

Description

Technical Field

[0001] This invention relates to the field of forage cultivation technology. More specifically, this invention relates to a method for improving the seed germination ability of alfalfa under salt-alkali stress. Background Technology

[0002] Salt-alkali stress is one of the main abiotic stress factors limiting the widespread cultivation of alfalfa in saline-alkali soil areas. Salt-alkali stress inhibits seed imbibition, radicle breakthrough, and seedling formation in alfalfa through both osmotic stress and ion toxicity. Currently, the main technical means to alleviate the inhibition of alfalfa seed germination by salt-alkali stress include chemical initiation treatment and physical pretreatment.

[0003] In their paper "Study on the Relief of Alfalfa Growth Inhibition by Spermine Soaking" (Chinese Journal of Grassland Science, Vol. 41, No. 5, 2019), Xia Guanxueying et al. disclosed a method for alleviating salt-alkali stress by treating alfalfa seeds with spermidine soaking. This method used two concentrations of spermidine (0.5 mmol / L and 1 mmol / L) to treat Dongmu No. 1 alfalfa seeds. The treated seeds were then cultured in pots under saline-alkali soil conditions in a greenhouse. The results showed that spermidine soaking improved the germination rate and biomass of alfalfa seeds under salt-alkali stress, promoted the accumulation of soluble sugars, chlorophyll, and proline, and reduced malondialdehyde content. However, this method only involves the soaking of seeds with a single exogenous substance, spermidine. Under severe salt-alkali stress, the recovery of seed germination rate by spermidine treatment alone is limited. Furthermore, this method does not address systematic countermeasures against post-germination oxidative damage, nor does it consider the synergistic combination of physical pretreatment and chemical soaking.

[0004] Jiao Yang et al., in their paper "Effects of Exogenous Astaxanthin on Seed Germination and Seedling Growth of Radish under Salt Stress" (Shandong Agricultural Sciences), disclosed a method of using astaxanthin to treat plant seeds to alleviate salt stress. This method involved treating radish seeds with different concentrations of astaxanthin under 150 mmol / L NaCl stress. The results showed that soaking seeds with appropriate concentrations of astaxanthin could improve the germination rate, germination potential, and fresh weight of seedlings under salt stress. However, this method only treated radish seeds, not alfalfa seeds, and only involved the soaking treatment of astaxanthin as a single exogenous substance, without addressing the combined use of astaxanthin and spermidine.

[0005] In the aforementioned existing technologies, spermidine seed soaking technology relies solely on a single polyamine substance to alleviate salt-alkali stress, and astaxanthin seed soaking technology relies solely on a single carotenoid substance. Both are used independently and have never been combined, nor has any potential synergistic effect between them been considered. Furthermore, none of the aforementioned existing technologies involve introducing a physical pretreatment step before chemical seed soaking and establishing a temporal synergistic mechanism between physical pretreatment and chemical seed soaking. Therefore, the existing technologies lack a method for temporally integrating physical pretreatment with spermidine-astaxanthin chemical seed soaking to trigger a far-infrared-speridine-astaxanthin synergistic effect to improve the germination ability of alfalfa seeds under salt-alkali stress. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide a method for improving the germination ability of alfalfa seeds under salt-alkali stress. This method integrates far-infrared physical pretreatment with spermidine-astaxanthin chemical soaking to achieve synergistic effects among far-infrared treatment, spermidine, and astaxanthin, thereby improving the seed germination rate under salt-alkali stress while systematically reducing the level of oxidative damage.

[0008] To achieve these objectives and other advantages of the present invention, a method for improving the germination ability of alfalfa seeds under salt-alkali stress is provided, comprising the following steps: S1. After disinfecting alfalfa seeds, perform far-infrared pretreatment. The wavelength of far-infrared pretreatment is 300 μm, the temperature is 35~45℃, and the treatment time is 10~30 min. S2. After the far-infrared pretreatment, the alfalfa seeds were placed at 20-25℃ in the dark for 15-30 minutes to allow the seeds to complete the transition from the far-infrared thermal activation state to the physiological state suitable for absorbing exogenous substances at room temperature. S3. Add the alfalfa seeds that have been stabilized at room temperature to the soaking solution and soak them with shaking under constant temperature and dark conditions. The concentration of spermidine in the soaking solution is 0.1~2.0 mmol·L⁻¹. -1 The astaxanthin concentration is 0.05~1.0 g·L. -1 Soaking time is 4 hours.

[0009] Preferably, the far-infrared pretreatment temperature in S1 is 40°C and the treatment time is 20 min.

[0010] Preferably, the soaking solution in S3 contains 1.0 mmol·L⁻¹ -1 spermidine, 0.1 g·L -1 Astaxanthin.

[0011] Preferably, in step S3, adding alfalfa seeds to the soaking solution and soaking them under constant temperature and light conditions specifically includes: transferring the seeds into an opaque container, adding the soaking solution, sealing the container opening with a microporous filter membrane, placing the opaque container in a shaker, and soaking the seeds under constant temperature and light conditions at 30°C.

[0012] Preferably, the shaking speed of the shaker is 100 rpm during the shaking soaking process.

[0013] Preferably, during the shaking soaking process, the first two hours are a high-speed shaking stage with the shaking table speed set to 120-150 rpm, and the next two hours are a low-speed shaking stage with the shaking table speed reduced to 60-80 rpm.

[0014] Preferably, the disinfection of alfalfa seeds in S1 specifically includes: disinfecting with 70% alcohol for 30 seconds, then disinfecting with 5% sodium hypochlorite solution for 3 minutes, and then rinsing with distilled water until no disinfectant residue remains.

[0015] Preferably, after step S3, a step of draining and passivating the residual treatment liquid on the seed surface after soaking is added. Specifically, the seeds after soaking are removed from the soaking treatment liquid and placed on a draining tray lined with double-layer sterile filter paper. They are drained for 12-18 minutes under light-proof conditions at 20-25°C, so that the residual amount of fluid on the seed surface is reduced to less than 0.25g per 100 seeds. During the draining process, the seeds are gently moved with sterile tweezers every 4 minutes to prevent adjacent seeds from agglomerating due to bridging of the surface liquid film, ensuring that the surface of each seed is drained evenly. After draining, the seed surface in contact with the sterile filter paper is rotated 90° and left to drain again for 3-5 minutes to eliminate the accumulation of liquid on the seed-filter paper contact surface.

[0016] Preferably, before the far-infrared pretreatment in S1, a seed moisture content standardization adjustment step is added. Specifically, the disinfected alfalfa seeds are placed in a constant temperature and humidity chamber and equilibrated at 25°C and 60% relative humidity for 6-12 hours to stabilize the seed moisture content within the standardized range of 8%-10%. After the equilibration treatment, the seed moisture content is sampled and tested batch by batch using a near-infrared moisture meter, with 50 seeds sampled per batch. When the sampling test results show that the moisture content of the batch of seeds exceeds the 8%-10% range, the equilibration treatment time is extended until the standard is met.

[0017] The present invention has at least the following beneficial effects: This invention constructs a three-level cascade synergistic mechanism of "physical pore formation - membrane stabilization - in-situ antioxidation" by sequentially integrating far-infrared physical pretreatment, room temperature stabilization transition and spermidine-astaxanthin chemical soaking, thereby realizing the functional coupling and synergistic amplification among far-infrared, spermidine and astaxanthin.

[0018] Compared with existing single or simple combination technologies, the present invention has the following outstanding advantages: First, far-infrared pretreatment induces the formation of uniform microcracks on the seed coat surface, providing an efficient transmembrane channel for exogenous substances and overcoming the problem of low absorption efficiency caused by the seed coat barrier in single chemical soaking.

[0019] Secondly, the combined use of spermidine and astaxanthin overcomes the limitations of single-substance action. Speridine establishes an intracellular protective network by stabilizing cell membrane structure and activating the expression of antioxidant enzyme genes, while astaxanthin uses its conjugated double bond structure to scavenge reactive oxygen species in situ in the lipid bilayer. The two complement and synergize in terms of their sites of action and duration of action, achieving a synergistic effect of "1+1>2".

[0020] Third, the precise timing between far-infrared pretreatment and chemical soaking is achieved through room temperature stabilization transition, avoiding energy conflicts between the physical thermal activation state and the chemical absorption process, and ensuring the full release of the synergistic effect of the two.

[0021] Fourth, the standardized adjustment of seed moisture content in the preferred scheme ensures the repeatability and stability of far-infrared pretreatment, while the staged variable-speed oscillation achieves efficient loading of molecules of different sizes based on the molecular weight differences of spermidine and astaxanthin, further amplifying the synergistic effect.

[0022] In summary, this invention can significantly improve the germination rate, germination potential, and seedling growth quality of alfalfa seeds under saline-alkali stress. At the same time, it systematically reduces oxidative damage indicators such as malondialdehyde, hydrogen peroxide, and superoxide anion, and enhances the activity of antioxidant enzymes such as peroxidase, catalase, and glutathione reductase, providing reliable technical support for the promotion and planting of alfalfa in saline-alkali soil areas.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the phenotypes of each group of seedlings in Example 3 of the present invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0027] <Example 1> This embodiment provides a method for screening the optimal synergistic concentration combination of spermidine (Sp) and astaxanthin (As) in the soaking treatment solution.

[0028] The seed soaking process is as follows: Select uniform and plump alfalfa seeds, then disinfect them with 70% alcohol for 30 seconds, followed by disinfection with 5% sodium hypochlorite solution for 3 minutes, and then rinse repeatedly with distilled water until no disinfectant residue remains.

[0029] The seed soaking solution used different concentrations of spermidine (Sp: 0.1, 0.5, 1.0, 2.0 mmol·L⁻¹). -1 ) and astaxanthin (As: 0.05, 0.1, 0.5, 1.0 g·L) -1 The samples were then paired up to form a total of 4 × 4 = 16 processing groups.

[0030] Three hundred uniformly sized, plump alfalfa seeds were selected from each treatment group and placed in 100 mL of the corresponding treatment solution in opaque glass bottles for soaking for 4 hours. During soaking, to promote sufficient contact between the seeds and Sp and As in the solution, the glass bottles were placed in a shaker at 30°C and shaken at 100 rpm in the dark to ensure adequate contact between the seeds and the solution. The bottle mouths were sealed with microporous membranes, which are breathable but impermeable to liquid, ensuring gas exchange between the inside and outside of the bottle during soaking (meeting the seeds' oxygen requirements) while effectively preventing external contaminants from entering and liquid from splashing out.

[0031] After soaking, remove the seeds from the soaking solution and place them on a draining tray lined with double-layered sterile filter paper. Drain at 22°C in the dark for 16 minutes to reduce the amount of liquid residue on the seed surface to less than 0.25g per 100 seeds. During the draining process, gently move the seeds with sterile tweezers every 4 minutes to prevent adjacent seeds from clumping together due to surface liquid film bridging, ensuring that each seed surface is evenly drained. After draining, rotate the seed surface in contact with the sterile filter paper 90° and let it stand to drain again for 3 minutes to eliminate liquid accumulation on the seed-filter paper contact surface.

[0032] The experimental groups were set up as follows: Normal control group (CK): After disinfection, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of distilled water was added to each petri dish to evenly moisten the filter paper.

[0033] Salt-alkali stress control group (SA): After seed disinfection, seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress solution (75 mmol·L⁻¹) was added to each petri dish to evenly moisten the filter paper. -1The pH was adjusted to 8.3 using NaCl:Na2SO4 = 9:4 and NaHCO3.

[0034] Seed soaking treatment group: After soaking, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of the corresponding salt-alkali stress soaking solution was added to each petri dish, meaning the corresponding soaking solution contained 75 mmol·L⁻¹. -1 The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0035] 100 seeds were sown in each dish. Each treatment was replicated three times.

[0036] The seed culture conditions are set as follows: Day and night temperature: 25°C (day) / 20°C (night); Photoperiod: 16 hours of light / 8 hours of darkness; Relative humidity: 75%; Germination was observed and recorded daily during the cultivation period, with the radicle breaking through the seed coat by 2 mm as the germination standard.

[0037] The methods for determining seed germination indices are as follows: The alfalfa seed germination and growth characteristics involved in this experiment included germination percentage (GP), germination energy (GE), germination index (GI), vigor index (VI), root length (RL), seedling length (SL), and fresh weight (FW). The calculation formulas are shown below: Germination rate (GP) = (Number of germinated seeds on day 8 / Number of seeds tested) × 100% Germination potential (GE) = (Number of germinated seeds on day 3 / Number of seeds tested) × 100% Method for determining root length (RL): Randomly select 10 healthy seedlings from each petri dish, measure the length from the root base to the root tip using an electronic digital caliper, and calculate the average value. Method for determining seedling length (SL): Randomly select 10 healthy seedlings from each petri dish, measure their vertical height from the base of the root to the top of the plant with an electronic digital caliper, and calculate the average value. Fresh weight (FW) determination method: Randomly select 10 healthy seedlings from each petri dish, then gently absorb the attached moisture on the surface of the seedlings with filter paper, weigh them on an analytical balance, and calculate the average value.

[0038] The test results are shown in Table 1 below: Table 1

[0039] Table 1 shows that under salt-alkali stress (SA group), the germination rate, germination potential, root length, seedling length, and fresh weight of alfalfa seeds were significantly lower than those of the normal control (CK group), indicating that the constructed salt-alkali stress system effectively inhibited seed germination and seedling growth. In 16 treatments with different concentrations of spermidine (Sp) and astaxanthin (As), the germination indices of each treatment group were superior to those of the SA group to varying degrees. Treatment No. 10 showed the most outstanding effect, with a germination rate of 56.33%, a germination potential of 51.33%, and root length, seedling length, and fresh weight of 47.11 mm, 6.59 mm, and 0.0308 g, respectively, representing increases of 43.2%, 87.9%, 128.2%, 88.8%, and 77.0% compared to the SA group. Furthermore, all indicators were superior to other concentration combinations, indicating that spermidine and astaxanthin had the best synergistic promoting effect at this ratio. Further analysis revealed that when the Sp concentration was fixed at 1.0 mmol·L⁻¹ -1 At that time, the germination rate increased with As concentration from 0.05 g·L⁻¹ -1 Increased to 0.1 g·L -1 And increase, but at 0.5 g·L -1 and 1.0 g·L -1 Instead, the concentration decreased, exhibiting a clear "low-promoting, high-inhibiting" dose-response effect; similarly, when the As concentration was fixed at 0.1 g·L⁻¹, the concentration decreased. -1 At that time, the Sp concentration was 0.1 mmol·L⁻¹. -1 Increased to 1.0 mmol·L -1 The time index continued to improve, but rose to 2.0 mmol·L⁻¹. -1 All subsequent indicators showed a significant decline. These results indicate that there is a strict optimal concentration window for the combination of spermidine and astaxanthin; excessively high levels of either component may induce antagonistic or toxic effects. Considering all indicators, the optimal synergistic concentration combination is Sp 1.0 mmol·L⁻¹. -1 With As 0.1 g·L -1 This combination provides the core chemical soaking parameters for the subsequent implementation of the present invention.

[0040] <Example 2> This embodiment provides a method for selecting the optimal parameters for far-infrared preprocessing.

[0041] The far-infrared processing procedure is as follows: Uniform and plump alfalfa seeds were selected, then disinfected with 70% alcohol for 30 seconds, followed by disinfection with 5% sodium hypochlorite solution for 3 minutes, and then rinsed repeatedly with distilled water until no disinfectant residue remained. The disinfected seeds were then laid in a single layer on a heat-resistant tray and placed in a far-infrared treatment chamber with a wavelength of 300 μm. The temperature was set at three levels: 35℃, 40℃, and 45℃; the time was set at three levels: 10 min, 20 min, and 30 min. A 3×3 full factorial design was used to construct nine far-infrared treatment groups, numbered T1 to T9.

[0042] The experimental groups were set up as follows: Normal control group (CK): After disinfection, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of distilled water was added to each petri dish to evenly moisten the filter paper.

[0043] Salt-alkali stress control group (SA): After disinfection, seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress solution was added to each petri dish to evenly moisten the filter paper. The salt-alkali stress solution contained 75 mmol·L⁻¹. -1 The pH was adjusted to 8.3 using NaCl:Na2SO4 = 9:4 and NaHCO3.

[0044] Far-infrared treatment group: After far-infrared treatment, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress solution was added to each petri dish to evenly moisten the filter paper. The salt-alkali stress solution contained 75 mmol·L⁻¹. -1 The pH was adjusted to 8.3 using NaCl:Na2SO4 = 9:4 and NaHCO3.

[0045] The seed culture conditions were the same as in Example 1.

[0046] The method for determining seed germination indicators was the same as in Example 1, and the test results are shown in Table 2 below: Table 2

[0047] Table 2 shows that, without far-infrared pretreatment, the germination rate, germination potential, root length, seedling length, and fresh weight of the salt-alkali stress group (SA) were 38.00%, 27.33%, 20.76 mm, 3.47 mm, and 0.0206 g, respectively, which were significantly lower than those of the normal control (CK). This indicates that salt-alkali stress had a strong inhibitory effect on seed germination and seedling growth. Among the nine far-infrared pretreatment combinations with different temperatures and times, each treatment group alleviated the inhibitory effect of salt-alkali stress to some extent, but there were significant differences in the promoting effect among different parameter combinations. When the treatment time was 10 min, the germination rates at the three temperature levels (35℃, 40℃, and 45℃) were 41.67%, 43.33%, and 44.33%, respectively, showing limited improvement. When the treatment time was extended to 20 min, the germination indicators at all temperatures were further improved, with the 40℃-20 min (T5) treatment showing the most significant effect. Its germination rate reached 52.67%, and its germination potential reached 38.33%, which were 38.6% and 40.2% higher than those of the S group, respectively. The root length, seedling length, and fresh weight reached 37.37 mm, 4.73 mm, and 0.0303 g, respectively, which were 80.0%, 36.3%, and 47.1% higher than those of the SA group, respectively. When the treatment time was further extended to 30 min, all indicators of the 40℃ (T6) and 45℃ (T9) treatment groups showed a significant decline. The germination rate of the 45℃-30 min (T9) group was even lower than that of the SA group, at only 32.67%, indicating that excessively long-term or high-temperature far-infrared treatment can induce seed heat damage and produce negative effects. Considering the germination indicators of all treatment groups, the optimal parameters for far-infrared pretreatment were 40℃ for 20 min (T5). This condition effectively activates the seed coat microstructure to promote subsequent water and nutrient absorption without damaging embryo activity due to excessive heat accumulation. This provides the optimal physical pretreatment parameters for the subsequent synergistic implementation of this invention with chemical soaking.

[0048] <Example 3> Based on Examples 1 and 2, this embodiment provides a method to improve the germination ability of alfalfa seeds under salt-alkali stress. This method achieves synergistic effects of physical and chemical methods by integrating far-infrared physical pretreatment with spermidine-astaxanthin chemical soaking.

[0049] The method is as follows: Select uniform and plump alfalfa seeds, disinfect them with 70% alcohol for 30 seconds, then disinfect them with 5% sodium hypochlorite solution for 3 minutes, and then rinse them repeatedly with distilled water until no disinfectant residue remains. Spread the disinfected seeds in a single layer on a heat-resistant tray, and place the heat-resistant tray containing the seeds into a far-infrared treatment chamber. Pre-treat the seeds with far-infrared light at a wavelength of 300 μm and a temperature of 40℃ for 20 minutes. After the far-infrared pre-treatment, remove the seeds from the treatment chamber and let them stand at 22℃ in the dark for 25 minutes to complete the room temperature stabilization transition. After stabilization, transfer the seeds to an opaque glass bottle and add the soaking solution. The final concentration of spermidine in the soaking solution is 1.0 mmol·L⁻¹. -1 The final concentration of astaxanthin was 0.1 g·L⁻¹. -1 The glass bottle opening is sealed with a microporous filter membrane, which is breathable but impermeable to liquid. This ensures gas exchange between the inside and outside of the bottle during the 4-hour soaking period to meet the seeds' oxygen requirements, while preventing external contaminants from entering. The glass bottle is then placed in a shaker and soaked in the dark at a constant temperature of 30°C and 100 rpm.

[0050] The experimental groups were set up as follows: Normal control group (CK): After disinfection, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of distilled water was added to each petri dish to evenly moisten the filter paper.

[0051] Far-infrared control group (IR): After seed disinfection, the seeds were treated with far-infrared technology in the T5 group of Example 2, and then placed in a petri dish with two layers of sterile filter paper. 5 mL of distilled water was added to each petri dish to evenly wet the filter paper.

[0052] Salt-alkali stress control group (SA): Seeds were sterilized and placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress solution (75 mmol·L⁻¹) was added to each petri dish to evenly moisten the filter paper. -1 The pH was adjusted to 8.3 using NaCl:Na2SO4 = 9:4 and NaHCO3.

[0053] Far-infrared + salt-alkali stress group (IR+SA): After the seeds were treated with far-infrared using the method of group T5 in Example 2, they were placed in a petri dish with two layers of sterile filter paper. 5 mL of salt-alkali stress solution was added to each petri dish to evenly wet the filter paper.

[0054] Spermine + salt-alkali stress group (Sp+SA): Seeds were treated with 1.0 mmol·L⁻¹ -1 After being treated with the same soaking method as in Example 1, spermidine solution was placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress-treated spermidine solution was added to each petri dish, meaning the spermidine solution contained 75 mmol·L⁻¹. -1The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0055] Astaxanthin + salt-alkali stress group (As+SA): seeds were subjected to 0.1 g·L⁻¹ -1 The astaxanthin solution, after being treated with the same soaking method as in Example 1, was placed in a petri dish lined with two layers of sterile filter paper. 5 mL of salt-alkali-stressed astaxanthin solution was added to each petri dish, meaning the astaxanthin solution contained 75 mmol·L⁻¹. -1 The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0056] Spermine + Astaxanthin + Salt-Alkali Stress Group (Sp+As+SA): After being treated with the same soaking method as Group 10 in Example 1, the seeds were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress soaking solution was added to each petri dish, meaning the soaking solution contained 75 mmol·L⁻¹. -1 The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0057] Far-infrared + spermidine + salt-alkali stress group (IR+Sp+SA): Seeds were treated with far-infrared radiation in the T5 group method of Example 2, and then left to stand at 22°C in the dark for 25 min. A 1.0 mmol·L⁻¹ solution was used. -1 After being treated with the same soaking method as in Example 1, the spermidine solution was placed in a petri dish lined with two layers of sterile filter paper. 5 mL of the saline-alkali stressed spermidine solution was added to each petri dish, meaning the spermidine solution contained 75 mmol·L⁻¹. -1 The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0058] Far-infrared + astaxanthin + salt-alkali stress group (IR+As+SA): Seeds were treated with far-infrared radiation in the T5 group method of Example 2, and then left to stand at 22°C in the dark for 25 min. 0.1 g·L⁻¹ of astaxanthin was used. -1 After being treated with the same soaking method as in Example 1, the astaxanthin solution was placed in a petri dish lined with two layers of sterile filter paper. 5 mL of salt-alkali-stressed astaxanthin solution was added to each petri dish, meaning the astaxanthin solution contained 75 mmol·L⁻¹. -1 The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0059] Far-infrared + spermidine + astaxanthin + salt-alkali stress group (IR+Sp+As+SA): After the seeds were treated as described above, they were placed in petri dishes lined with two layers of sterile filter paper. 5 mL of salt-alkali stress soaking solution was added to each petri dish, i.e., the soaking solution contained 75 mmol·L⁻¹. -1The ratio of NaCl:Na2SO4 was 9:4, and the pH was adjusted to 8.3 with NaHCO3.

[0060] The seed culture conditions were the same as in Example 1.

[0061] The method for determining seed germination indicators was the same as in Example 1, except that malondialdehyde (MDA) content, hydrogen peroxide (H2O2) content, and superoxide anion (O2) content were also added. - The content of glutathione reductase (GR) and its activities were determined.

[0062] Take an appropriate amount of germinating seeds, dry the surface moisture with absorbent paper, add an appropriate amount of extraction solution (or liquid nitrogen) to a pre-cooled mortar and grind thoroughly into a homogenate. Transfer the homogenate to a centrifuge tube and centrifuge at 8000~13000 g for 10~20 min at low temperature (4℃). After centrifugation, carefully aspirate the supernatant as the sample to be tested. The entire process should be carried out in an ice bath to prevent loss of enzyme activity.

[0063] MDA content was determined using the thiobarbituric acid (TBA) colorimetric method. The reaction system contained thiobarbituric acid. After reacting the sample with the reaction solution in a 100°C water bath for 60 minutes, the absorbance was measured at 532 nm and 600 nm, respectively. The MDA content was calculated based on ΔA (A532-A600). In practice, a commercially available kit (Solepro BC0025 kit) was used according to the manufacturer's instructions.

[0064] The H2O2 content was detected using the ferric sulfate colorimetric method. The reaction system contained ferric sulfate and other components. After the sample reacted with the reaction solution, the absorbance was measured at 415 nm, and the hydrogen peroxide content was calculated based on the standard reference. In practice, a commercially available kit (Solepro BC3595 kit) was used according to the instructions.

[0065] O2 - The content was determined using the hydroxylamine hydrochloride oxidative coupling method. The reaction system contained hydroxylamine hydrochloride, p-aminobenzenesulfonamide, and naphthylethylenediamine hydrochloride. After the sample and reaction solution were reacted in a 37°C water bath, the absorbance was measured at 530 nm. The superoxide anion content was calculated based on the sodium nitrite standard. Specifically, a commercially available kit (Solepro BC1295 kit) was used according to the instructions.

[0066] POD activity was detected using the guaiacol oxidation method. The reaction system contained guaiacol and hydrogen peroxide. The test sample was reacted with the reaction solution at 37°C (mammals) or 25°C (other species), and the rate of increase of absorbance at 470 nm (ΔA) was recorded. POD activity was calculated based on ΔA. In practice, a commercially available kit (Solepro BC0095 kit) was used according to the manufacturer's instructions.

[0067] CAT activity was detected using the hydrogen peroxide UV absorption method. The reaction system contained hydrogen peroxide. The test sample and reaction solution were reacted at 37°C (mammals) or 25°C (other species), and the rate of decrease in absorbance at 240 nm (ΔA) was recorded. CAT activity was calculated based on ΔA. In practice, a commercially available kit (Solepro BC0205 kit) was used according to the manufacturer's instructions.

[0068] Glutathione reductase activity was detected using the NADPH-coupled enzyme method. The reaction system contained oxidized glutathione (GSSG) and NADPH. GR catalyzes the reduction of GSSG to GSH by NADPH, while NADPH is dehydrogenated to NADP⁺. NADPH has a characteristic absorption peak at 340 nm, while NADP⁺ has no absorption peak at this wavelength. GR activity was calculated by recording the rate of decrease (ΔA) of absorbance at 340 nm. In practice, a commercially available kit (Solepro BC1160 kit) was used according to the manufacturer's instructions.

[0069] The test results are shown in Tables 3 and 4 below: Table 3

[0070] Table 4

[0071] Tables 3-4 show that under salt-alkali stress (SA group), the germination rate, germination potential, root length, seedling length, and fresh weight of alfalfa seeds were significantly lower than those of the normal control (CK). Simultaneously, malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2) levels were also significantly increased. - The significant increase in the content of reactive oxygen species indicates that salt-alkali stress has triggered severe oxidative damage; while the activities of peroxidase (POD), catalase (CAT) and glutathione reductase (GR) have increased, they are still insufficient to completely remove the excess reactive oxygen species.

[0072] Compared with the SA group, far-infrared pretreatment alone (IR+SA), spermidine soaking alone (Sp+SA), and astaxanthin soaking alone (As+SA) all improved germination indicators and reduced oxidative damage levels to some extent. Astaxanthin alone showed a better effect on root length promotion (36.03 mm) than spermidine alone (31.03 mm), while spermidine alone improved germination rate (46.67%) better than astaxanthin alone (44.33%), indicating that each treatment had its own focus. After soaking in a combination of spermidine and astaxanthin (Sp+As+SA), all germination indicators were further improved (germination rate 54.67%, root length 43.67 mm), and the contents of MDA, H2O2, and O2⁻ further decreased. The activities of POD, CAT, and GR were significantly enhanced compared to the single treatment groups, indicating a synergistic effect between spermidine and astaxanthin at the chemical level.

[0073] In the combined physical-chemical treatment, far-infrared + spermidine (IR+Sp+SA) and far-infrared + astaxanthin (IR+As+SA) were both superior to their respective single chemical treatment groups, indicating that far-infrared pretreatment can enhance the absorption efficiency of exogenous substances. The combined treatment of all three (IR+Sp+As+SA) showed the most outstanding effect: its germination rate reached 60.67%, germination potential reached 53.33%, and root length, seedling length, and fresh weight reached 51.60 mm, 5.77 mm, and 0.3653 g, respectively, representing increases of 61.2%, 88.3%, 167.4%, and 80.3% compared to the SA group, and a significant increase. Simultaneously, MDA, H2O2, and O2 were also significantly increased. - The concentrations decreased to 122.01 nmol / g, 10.24 μmol / g, and 6.70 μmol / g, the lowest among all saline-alkali treatment groups, while the activities of POD, CAT, and GR increased to 869.55 U·g. -1 8.77 U·g -1 and 2.18 U·g -1 The result was the highest among all salt-alkali treatment groups. These results clearly demonstrate that the sequential integration of far-infrared physical pretreatment and spermidine-astaxanthin chemical soaking can trigger a synergistic effect among the three. By opening absorption channels through physical means and establishing a cell protection network through chemical means, germination is promoted while oxidative damage is systematically reduced, thereby significantly improving the germination ability of alfalfa seeds and the early growth quality of seedlings under salt-alkali stress.

[0074] <Example 4> Based on the optimal treatment parameters of Example 3 (far-infrared pretreatment at 40℃ for 20 min, room temperature stabilization for 25 min, and soaking solution containing 1.0 mmol·L⁻¹ spermidine and 0.1 g·L⁻¹ astaxanthin), this embodiment further optimizes the oscillation soaking method by changing the constant rotation speed (100 rpm) throughout the process to staged variable speed oscillation to verify the effect of high-speed-low-speed segmented oscillation on improving the synergistic absorption efficiency of spermidine and astaxanthin.

[0075] Seed soaking treatment method: After the alfalfa seeds have been stabilized at room temperature, they are transferred to an opaque glass bottle, and a seed soaking solution (containing 1.0 mmol·L⁻¹) is added. -1 Spermine and 0.1 g·L -1 Astaxanthin), the bottle opening is sealed with a microporous filter membrane. The glass bottle is placed in a shaker and subjected to staged shaking soaking under constant temperature and light conditions of 30℃, for a total time of 4 hours, specifically divided into two stages: 1) First 2 hours (high-speed oscillation stage): The shaker speed is set to 135 rpm. This high-speed stage utilizes the seed coat microcracks and cell membrane channels opened by far-infrared pretreatment to drive the preferential transmembrane absorption of small molecule spermidine (molecular weight 145.25 Da) under high hydrodynamic mass transfer efficiency, so that it completes intracellular enrichment in the first half of the soaking process.

[0076] 2) After 2 hours (low-speed oscillation stage): The shaking speed is reduced to 70 rpm. After the speed is reduced, the fluid shear force is weakened, which is conducive to the full embedding of large molecular astaxanthin (molecular weight 596.84 Da) into the lipid bilayer that has been stabilized by spermidine, and to the in-situ blocking scavenging of reactive oxygen species by means of its conjugated double bond structure.

[0077] Experimental group settings: Control group CK: Normal culture (distilled water, no salt or alkali stress).

[0078] Salt-alkali stress control group SA: only salt-alkali stress solution, no other treatment.

[0079] Constant speed group IR+Sp+As+SA (constant): The IR+Sp+As+SA treatment in Example 3 is adopted, namely far-infrared pretreatment + room temperature stabilization + soaking (1.0 mM Sp + 0.1 g / L As), and the oscillation condition is a constant speed of 100 rpm throughout.

[0080] Staged rotation speed group IR+Sp+As+SA (variable speed): far-infrared pretreatment + room temperature stabilization + same soaking solution, but the oscillation conditions are 135 rpm for the first 2 hours and 70 rpm for the last 2 hours.

[0081] Each group was replicated in 3 times, and 100 seeds were sown in each dish. The salt and alkali stress conditions, culture conditions, and germination index determination methods were the same as in Examples 1-3.

[0082] Determination of spermidine content in seeds: After soaking, seeds from each group were rinsed three times with distilled water, dried, ground with liquid nitrogen, and then determined by high performance liquid chromatography (HPLC). The determination was performed by pre-column derivatization with dansyl chloride, separation on a C18 column, detection with a fluorescence detector, and quantification by external standard method.

[0083] Determination of astaxanthin content in seeds: Soaked seeds were ground in liquid nitrogen and extracted with acetone-methanol (7:3). The astaxanthin content was determined by HPLC, separated by a C30 column, detected by a visible light detector (476 nm), and quantified by external standard method.

[0084] The test results are shown in Tables 5 and 6 below: Table 5

[0085] Table 6

[0086] As shown in Tables 5-6, the spermidine content in the seeds of the staged variable-speed oscillation group was 39.3% higher than that of the constant-speed group, and the astaxanthin content was 48.4% higher. This confirms that the high-speed stage promoted the rapid transmembrane absorption of small-molecule spermidine, while the low-speed stage facilitated the effective intercalation of large-molecule astaxanthin. Correspondingly, the antioxidant enzyme activities (POD, CAT, GR) in the variable-speed group were significantly higher than those in the constant-speed group, while MDA, H2O2, and O2 were significantly higher. - The content was significantly reduced, indicating that the more balanced intracellular spermidine enrichment and membrane lipid astaxanthin intercalation produced a stronger synergistic antioxidant effect. Ultimately, the germination rate, germination potential, root length, and seedling length of the variable-speed group were significantly better than those of the constant-speed group, demonstrating that the staged oscillation strategy, through molecular size-adapted mass transfer kinetics optimization, further amplified the far-infrared-spermine-astaxanthin three-level synergistic effect, providing a more efficient physical-chemical joint regulation scheme for alfalfa seed germination under salt-alkali stress.

[0087] <Example 5> Based on the optimal scheme in Example 4, this embodiment adds a seed moisture content standardization adjustment step after the disinfection step and before the far-infrared pretreatment to verify the effect of seed moisture content on the far-infrared pretreatment effect and the subsequent physical-chemical cascade synergistic effect.

[0088] Solution: First, the initial moisture content of each batch was determined. Seeds with an initial moisture content in the range of 8% to 10% were used as the CK group and SA group (without any treatment) and as the baseline control for subsequent experiments. Seeds from the same batch with a lower initial moisture content (7.2% in this case) were randomly divided into two groups: the natural moisture content group (N group, without moisture content adjustment) and the standardized moisture content group (S group, with moisture content adjustment) to eliminate the interference of seed genetic background differences on the experimental results.

[0089] Subsequently, both groups of seeds (S and N) underwent further treatment according to the optimal parameters of Example 4: far-infrared pretreatment (wavelength 300 μm, temperature 40℃, time 20 min), room temperature stabilization (22℃, in the dark, 25 min), and staged shaking soaking (135 rpm for the first 2 h, 70 rpm for the next 2 h, soaking solution containing 1.0 mmol·L⁻¹). -1 Sp + 0.1 g·L -1 After soaking, the seeds were drained and sown under salt-alkali stress conditions (75 mmol·L⁻¹). -1 (NaCl:Na2SO4 = 9:4, pH 8.3). A normal control (CK) without any treatment and a salt-alkali stress control (SA) were also set up.

[0090] The experimental groups are set up as shown in Table 7 below: Table 7

[0091] Each group was replicated 3 times, with 100 seeds sown in each dish, and the culture conditions were the same as in Example 1.

[0092] Detection method: Seed moisture content determination: A near-infrared moisture meter (model: FOSS Infratec) was used. TM 1241) The seeds after equilibration treatment were tested batch by batch, with 50 seeds in each batch, and the average value was taken.

[0093] Seed coat microcracks observation: After far-infrared pretreatment and before soaking, 10 seeds from each group were taken, stained with 2% safranin solution for 5 min, rinsed with distilled water, and the stained area on the seed coat surface was observed under a stereomicroscope (the dye penetration area reflects the microcrack density), and the percentage of stained area was counted using ImageJ software.

[0094] Common germination indicators: germination rate, germination potential, root length, seedling length, and fresh weight, measured using the same methods as in Example 1.

[0095] Oxidative damage and antioxidant enzyme indicators: MDA, H2O2, O2 - The content and activities of POD, CAT, and GR were determined using the same methods as in Example 3.

[0096] Spermine and astaxanthin content in seeds: After soaking, seeds from each group were taken and determined by HPLC according to the method in Example 4.

[0097] The test results are shown in Tables 8 and 9 below: Table 8

[0098] Table 9

[0099] Table 8 shows that the initial moisture content of the natural moisture content group (N group) was 7.2%, lower than the standardization lower limit of 8%; while the moisture content of the standardized group (S group) after equilibrium treatment was 9.3%, within the preferred range of 8% to 10%. After far-infrared pretreatment, the seed coat staining area of ​​group S (28.6%) was significantly larger than that of group N (12.8%), indicating that under standardized moisture content conditions, far-infrared radiation energy is more effectively converted into a dielectric heating effect, inducing more uniformly distributed microcracks in the seed coat, providing a more sufficient physical channel for the subsequent transmembrane absorption of spermidine and astaxanthin.

[0100] Correspondingly, the content of spermine and astaxanthin in the seeds of group S after soaking increased by 34.9% and 37.6% respectively compared with group N, indicating that standardized moisture content significantly enhanced the loading efficiency of exogenous substances by optimizing seed coat permeability. Under saline-alkali stress, the germination rate, germination potential, root length, seedling length, and fresh weight of group S increased by 20.1%, 26.6%, 27.8%, 26.8%, and 12.3% respectively compared with group N, while MDA, H2O2, and O2 were lower. - The contents decreased by 18.4%, 27.7%, and 24.2%, respectively; while the activities of antioxidant enzymes POD, CAT, and GR increased by 8.7%, 14.4%, and 23.6%, respectively. These results clearly demonstrate that standardized regulation of seed moisture content is a crucial preliminary step in triggering the synergistic effect of the far-infrared-spermine-astaxanthin cascade: when the moisture content is too low (<8%), the far-infrared heat conversion efficiency is insufficient, and the formation of seed coat microcracks is limited, leading to insufficient subsequent chemical absorption and antioxidant network activation; when the moisture content is too high (>10%), it may cause local hotspot damage (no high moisture content was observed in this experiment). Precisely controlling the seed moisture content within the range of 8%–10% ensures that far-infrared pretreatment exerts the optimal seed coat porosification effect, thereby maximizing the synergistic gain of physical pretreatment and chemical soaking, ultimately achieving a significant improvement in the germination ability of alfalfa seeds under salt-alkali stress.

[0101] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for improving the germination ability of alfalfa seeds under salt-alkali stress, characterized in that, Includes the following steps: S1. After disinfecting alfalfa seeds, perform far-infrared pretreatment. The wavelength of far-infrared pretreatment is 300 μm, the temperature is 35~45℃, and the treatment time is 10~30 min. S2. After the far-infrared pretreatment, the alfalfa seeds were placed at 20-25℃ in the dark for 15-30 minutes to allow the seeds to complete the transition from the far-infrared thermal activation state to the physiological state suitable for absorbing exogenous substances at room temperature. S3. Add the alfalfa seeds that have been stabilized at room temperature to the soaking solution and soak them with shaking under constant temperature and dark conditions. The concentration of spermidine in the soaking solution is 0.1~2.0 mmol·L⁻¹. -1 The astaxanthin concentration is 0.05~1.0 g·L. -1 Soaking time is 4 hours.

2. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, The far-infrared pretreatment temperature in S1 is 40℃, and the treatment time is 20min.

3. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, The soaking solution in S3 contains 1.0 mmol·L⁻¹ -1 spermidine, 0.1 g·L -1 Astaxanthin.

4. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, In S3, alfalfa seeds are added to a soaking solution and soaked under constant temperature and light conditions by shaking. Specifically, the process involves: transferring the seeds into an opaque container, adding the soaking solution, sealing the container opening with a microporous filter membrane, placing the opaque container in a shaker, and soaking the seeds under constant temperature and light conditions at 30°C by shaking.

5. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 4, characterized in that, The shaking speed of the shaker during seed soaking is 100 rpm.

6. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 4, characterized in that, During the shaking soaking process, the first two hours are the high-speed shaking stage, with the shaking table speed set to 120-150 rpm. The next two hours are the low-speed shaking stage, with the shaking table speed reduced to 60-80 rpm.

7. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, The disinfection of alfalfa seeds in S1 specifically includes: disinfecting with 70% alcohol for 30 seconds, then disinfecting with 5% sodium hypochlorite solution for 3 minutes, and then rinsing with distilled water until no disinfectant residue remains.

8. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, S3 adds a step of draining and passivating the surface of the seeds after soaking. Specifically, the seeds are removed from the soaking solution and placed on a draining tray lined with double-layer sterile filter paper. They are drained at 20-25°C in the dark for 12-18 minutes to reduce the amount of liquid remaining on the seed surface to less than 0.25 g per 100 seeds. During the draining process, the seeds are gently moved with sterile tweezers every 4 minutes to prevent adjacent seeds from agglomerating due to bridging of the surface liquid film and to ensure that each seed is drained evenly. After draining, the seed surface in contact with the sterile filter paper is rotated 90° and left to drain again for 3-5 minutes to eliminate liquid accumulation on the seed-filter paper contact surface.

9. The method for improving the germination ability of alfalfa seeds under salt-alkali stress as described in claim 1, characterized in that, Before the far-infrared pretreatment in S1, a standardized adjustment step for seed moisture content is added. Specifically, the disinfected alfalfa seeds are placed in a constant temperature and humidity chamber and equilibrated at 25℃ and 60% relative humidity for 6-12 hours to stabilize the seed moisture content within the standardized range of 8%-10%. After the equilibration treatment, the seed moisture content is sampled and tested batch by batch using a near-infrared moisture meter, with 50 seeds sampled per batch. If the sampling test results show that the moisture content of the batch of seeds exceeds the 8%-10% range, the equilibration treatment time is extended until the standard is met.

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