A method for improving cold tolerance in alfalfa

Abstract

The present application relates to the field of agricultural ecology and crop cultivation technology, and particularly relates to a method for improving cold resistance of alfalfa, comprising the following steps: disinfecting alfalfa seeds, soaking the alfalfa seeds in a silicon solution for 20h-24h, then transferring the alfalfa seeds to a low-temperature environment to cultivate alfalfa seedlings, and adding 0.025mL-0.05mL of sterilized distilled water to each alfalfa plant every day during the cultivation of the alfalfa seedlings; the silicon solution is a Na2SiO3.9H2O solution; the concentration of the Na2SiO3.9H2O solution is 1mM-4mM; and the temperature of the low-temperature environment is 5-20℃. The present application shows that silicon-induced resistance can improve the germination rate of non-overwintering alfalfa varieties, promote the growth of seedling roots, and reduce the damage to the seedling plasma membrane, membrane lipid peroxidation and osmotic damage caused by low-temperature stress. The method of the present application provides a new way to improve the cold resistance of alfalfa.

Description

Technical Field

[0001] This invention relates to the fields of agricultural ecology and crop cultivation technology, specifically to a method for improving the cold resistance of alfalfa. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is an important legume forage crop globally, widely cultivated due to its high yield, high protein content, and nitrogen-fixing capacity. However, in cold northern regions, low winter temperatures often cause frost damage or even death to alfalfa plants, severely impacting their overwintering rate and the following year's yield. Low-temperature stress damages cell membrane structure, triggers oxidative stress, and interferes with photosynthesis and metabolic balance, thereby limiting alfalfa growth and development.

[0003] As the starting point for plant growth and development, the quality of seeds directly affects the establishment and stress resistance of alfalfa seedlings. Studies have shown that the germination rate, seedling vigor, and antioxidant system activity of alfalfa seeds under low-temperature stress are closely related to their cold tolerance. Currently, existing technologies use physical or chemical methods, such as low-temperature pre-incubation, hormone soaking, or osmotic regulation, to improve seed cold resistance, but the effects are mostly limited to the germination stage, with limited improvement on the overwintering ability of mature plants. In addition, exogenous application of plant growth regulators, such as abscisic acid and salicylic acid, or antioxidants, such as proline and betaine, can partially alleviate low-temperature damage, but there are drawbacks such as difficulty in standardizing application concentrations and strong environmental dependence.

[0004] Therefore, developing an efficient and stable seed treatment technology to enhance the cold resistance of plants is of great significance for solving the overwintering problem of alfalfa in northern regions. Summary of the Invention

[0005] To address the above problems, this invention provides a method for improving the cold resistance of alfalfa.

[0006] A method for improving the cold resistance of alfalfa includes the following steps:

[0007] Disinfect alfalfa seeds, soak them in a silica solution for 20-24 hours, and then transfer them to a low-temperature environment to cultivate alfalfa seedlings. During the cultivation period, add 0.025-0.05 mL of sterile distilled water to each alfalfa seedling daily.

[0008] The silicon solution is a Na2SiO3·9H2O solution; the concentration of the Na2SiO3·9H2O solution is 1mM~4mM.

[0009] The temperature of the low-temperature environment is 5℃~20℃.

[0010] Preferably, the disinfection method involves soaking in sodium hypochlorite solution for 8 to 10 minutes, followed by rinsing with water 3 to 5 times.

[0011] Preferably, the mass concentration of the sodium hypochlorite solution is 0.5% to 0.7%.

[0012] Preferably, the water is sterile distilled water.

[0013] Preferably, the culture time is 20 to 30 days.

[0014] Preferably, the culture process involves 16 hours of white light illumination and 8 hours of darkness daily, with the white light intensity being 52 μmol·m⁻¹. -2 s -1 .

[0015] Preferably, the alfalfa variety is Longmu 803.

[0016] Preferably, the alfalfa variety is Saidi 10.

[0017] This invention is achieved through the following technical solution:

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides a method for improving the cold resistance of alfalfa, comprising the following steps: disinfecting alfalfa seeds, soaking them in a silica solution for 20-24 hours, and then transferring them to a low-temperature environment for cultivation into alfalfa seedlings. During the seedling cultivation period, 0.025 mL-0.05 mL of sterile distilled water is added to each alfalfa seedling daily. The silica solution is a Na₂SiO₃·9H₂O solution with a concentration of 1 mM-4 mM. The low-temperature environment is 5°C-20°C. This invention significantly improves the cold resistance of alfalfa seeds by treating them with different concentrations of Na₂SiO₃·9H₂O solution. Experiments show that silica solution treatment can effectively enhance the germination ability of seeds under low-temperature conditions, especially significantly improving the germination rate of the low-temperature sensitive variety, Saidi 10. During the seedling growth stage, silicon solution treatment significantly promoted root development, increasing root length, root surface area, and root volume. Among these, Longmu 803, treated with 1 mM silicon solution, showed outstanding performance at 15℃ and 5℃. Furthermore, silicon solution treatment enhanced cold resistance through multiple mechanisms: 4 mM silicon treatment significantly reduced membrane damage, protecting cell membrane integrity; simultaneously, it activated the antioxidant system, increasing SOD and POD activities by 344% and 67.5%, respectively, effectively alleviating oxidative stress damage. Silicon solution treatment also promoted the accumulation of osmotic regulators; seedlings treated with silicon solution showed a significant increase in soluble sugar and soluble protein content, enhancing cell freeze resistance. This invention not only significantly improved the overwintering ability of the cold-resistant LM variety but also enabled the previously cold-intolerant SD variety to acquire good low-temperature adaptability. It provides a simple, low-cost, and stable cold-resistant technology solution for alfalfa cultivation in cold northern regions, possessing significant value for widespread application. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This invention presents the dynamics of germination rate under different silicon solution concentrations. Figure 1In the table, A represents the germination rate dynamics of LM at 20℃ under different silicon solution concentrations; B represents the germination rate dynamics of LM at 15℃ under different silicon solution concentrations; C represents the germination rate dynamics of LM at 10℃ under different silicon solution concentrations; D represents the germination rate dynamics of LM at 5℃ under different silicon solution concentrations; E represents the germination rate dynamics of SD at 20℃ under different silicon solution concentrations; F represents the germination rate dynamics of SD at 15℃ under different silicon solution concentrations; G represents the germination rate dynamics of SD at 10℃ under different silicon solution concentrations; and H represents the germination rate dynamics of SD at 5℃ under different silicon solution concentrations.

[0022] Figure 2 The image shows the phenotypic results of the LM variety of this invention after silicon-induced resistance under four low-temperature conditions, Bar = 0.5 cm; Figure 2 In the diagram, A represents the phenotypic pattern of cultured at 20℃ without silicon-induced inhibition; B represents the phenotypic pattern of cultured at 20℃ with 1mM silicon solution induced inhibition; C represents the phenotypic pattern of cultured at 20℃ with 2.25mM silicon solution induced inhibition; D represents the phenotypic pattern of cultured at 20℃ with 4mM silicon solution induced inhibition; E represents the phenotypic pattern of cultured at 15℃ without induced inhibition; F represents the phenotypic pattern of cultured at 15℃ with 1mM silicon solution induced inhibition; G represents the phenotypic pattern of cultured at 15℃ with 2.25mM silicon solution induced inhibition; and H represents the phenotypic pattern of cultured at 15℃ with 4mM silicon solution induced inhibition. Phenotypic diagrams: I represents the phenotype of cultured at 10℃ without induction; J represents the phenotype of cultured at 10℃ with 1mM silica solution induction; K represents the phenotype of cultured at 10℃ with 2.25mM silica solution induction; L represents the phenotype of cultured at 10℃ with 4mM silica solution induction; M represents the phenotype of cultured at 5℃ without induction; N represents the phenotype of cultured at 5℃ with 1mM silica solution induction; O represents the phenotype of cultured at 5℃ with 2.25mM silica solution induction; P represents the phenotype of cultured at 5℃ with 4mM silica solution induction.

[0023] Figure 3 The image shows the phenotypic results of the SD variety of this invention after silicon-induced resistance under four low-temperature conditions, Bar = 0.5 cm. Figure 3In the diagram, A represents the phenotypic pattern of cultured at 20℃ without silicon-induced inhibition; B represents the phenotypic pattern of cultured at 20℃ with 1mM silicon solution induced inhibition; C represents the phenotypic pattern of cultured at 20℃ with 2.25mM silicon solution induced inhibition; D represents the phenotypic pattern of cultured at 20℃ with 4mM silicon solution induced inhibition; E represents the phenotypic pattern of cultured at 15℃ without induced inhibition; F represents the phenotypic pattern of cultured at 15℃ with 1mM silicon solution induced inhibition; G represents the phenotypic pattern of cultured at 15℃ with 2.25mM silicon solution induced inhibition; and H represents the phenotypic pattern of cultured at 15℃ with 4mM silicon solution induced inhibition. Phenotypic diagrams: I represents the phenotype of cultured at 10℃ without induction; J represents the phenotype of cultured at 10℃ with 1mM silica solution induction; K represents the phenotype of cultured at 10℃ with 2.25mM silica solution induction; L represents the phenotype of cultured at 10℃ with 4mM silica solution induction; M represents the phenotype of cultured at 5℃ without induction; N represents the phenotype of cultured at 5℃ with 1mM silica solution induction; O represents the phenotype of cultured at 5℃ with 2.25mM silica solution induction; P represents the phenotype of cultured at 5℃ with 4mM silica solution induction.

[0024] Figure 4 This invention relates to the effects of low-temperature stress and treatment with different concentrations of silicon solution on the fresh weight and dry weight of LM and SD seedlings; Figure 4 In the table, A represents the effect of low temperature stress and different concentrations of silicon solution treatment on the fresh weight of LM seedlings; B represents the effect of low temperature stress and different concentrations of silicon solution treatment on the fresh weight of SD seedlings; C represents the effect of low temperature stress and different concentrations of silicon solution treatment on the dry weight of LM seedlings; and D represents the effect of low temperature stress and different concentrations of silicon solution treatment on the dry weight of SD seedlings. Different letters indicate significant differences.

[0025] Figure 5 This invention relates to the effects of low-temperature stress and treatment with different concentrations of silicon solution on the root length and root diameter of LM and SD roots; Figure 5 In the figure, A represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root length of LM; B represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root length of SD; C represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root diameter of LM; and D represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root diameter of SD. Different letters indicate significant differences.

[0026] Figure 6 This invention relates to the effects of low-temperature stress and treatment with different concentrations of silicon solution on the root surface area and root volume of LM and SD roots; Figure 6 In the figure, A represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root surface area of ​​LM; B represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root surface area of ​​SD; C represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root volume of LM; and D represents the effect of low temperature stress and different concentrations of silicon solution treatment on the root volume of SD. Different letters indicate significant differences.

[0027] Figure 7 This invention investigates the effects of low-temperature stress and treatment with different concentrations of silicon solution on the degree of plasma membrane damage, superoxide dismutase activity, and peroxidase activity in LM and SD seedlings. Figure 7 In the table, A represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the degree of plasma membrane damage in LM seedlings; B represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the degree of plasma membrane damage in SD seedlings; C represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the activity of LM superoxide dismutase; D represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the activity of SD superoxide dismutase; E represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the activity of LM peroxidase; and F represents the effect of low-temperature stress and different concentrations of silicon solution treatment on the activity of SD peroxidase. Different letters indicate significant differences.

[0028] Figure 8 The effects of low-temperature stress and treatment with different concentrations of silicon solution on the soluble sugar and soluble protein content of LM and SD seedlings were investigated in this invention. Figure 8 In the figure, A represents the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble sugar content of LM seedlings; B represents the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble sugar content of SD seedlings; C represents the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble protein content of LM seedlings; and D represents the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble protein content of SD seedlings. Different letters indicate significant differences. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0032] The Longmu 803 used in this invention was purchased from the Heilongjiang Provincial Animal Husbandry Research Institute; the Saidi 10 was purchased from Zhengzhou Huafeng Grass Industry Company.

[0033] Example 1: A method for improving the cold resistance of alfalfa

[0034] The alfalfa variety is Longmu 803, or LM for short, and it can survive in harsh winters.

[0035] Alfalfa seeds were soaked in a 0.6% sodium hypochlorite solution for 8 minutes, rinsed three times with distilled water that had been sterilized at 120℃, and then soaked in a 1mM Na2SiO3·9H2O solution for 20 hours.

[0036] Seeds treated with Na₂SiO₃·9H₂O solution were cultured in a 20℃ incubator, with 16 hours of white light and 8 hours of darkness per day during cultivation, at a light intensity of 52 μmol·m⁻¹. -2 s -1 .

[0037] Each morning, 1 mL of sterile distilled water was added to each petri dish containing 40 seeds, with two layers of filter paper placed in each dish. Each treatment consisted of three biological replicates. Alfalfa seedlings were harvested after 20 days for further analysis.

[0038] Example 2: A method for improving the cold resistance of alfalfa

[0039] The alfalfa variety is Longmu 803, or LM for short, and it can survive in harsh winters.

[0040] Alfalfa seeds were sterilized with a 0.5% sodium hypochlorite solution for 9 minutes, rinsed four times with distilled water that had been autoclaved at 120°C, and then soaked in a 1mM Na2SiO3·9H2O solution for 22 hours.

[0041] Seeds treated with Na₂SiO₃·9H₂O solution were placed in a 15℃ incubator for cultivation. During cultivation, the seeds were exposed to 16 hours of white light and 8 hours of darkness daily, with a light intensity of 52 μmol·m⁻¹. -2 s -1 .

[0042] Each morning, 1 mL of sterile distilled water was added to each petri dish containing 40 seeds, with two layers of filter paper placed in each dish. Each treatment consisted of three biological replicates. Alfalfa seedlings were harvested after 25 days for further analysis.

[0043] Example 3: A method for improving the cold resistance of alfalfa

[0044] The alfalfa variety is Longmu 803, or LM for short, and it can survive in harsh winters.

[0045] Alfalfa seeds were soaked in a 0.7% sodium hypochlorite solution for 10 minutes, rinsed five times with distilled water that had been sterilized at 120℃, and then soaked in a 1mM Na2SiO3·9H2O solution for 24 hours.

[0046] Seeds treated with Na₂SiO₃·9H₂O solution were placed in a 10℃ incubator for cultivation. During cultivation, the seeds were exposed to 16 hours of white light and 8 hours of darkness daily, with a light intensity of 52 μmol·m⁻¹. -2 s -1 .

[0047] Each morning, 2 mL of sterile distilled water was added to each petri dish containing 40 seeds, with two layers of filter paper placed in each dish. Each treatment consisted of three biological replicates. Alfalfa seedlings were harvested after 30 days for further analysis.

[0048] Example 4: A method for improving the cold resistance of alfalfa

[0049] The alfalfa variety is Longmu 803, or LM for short, and it can survive in harsh winters.

[0050] Alfalfa seeds were soaked in a 0.6% sodium hypochlorite solution for 10 minutes, rinsed five times with distilled water that had been sterilized at 120℃, and then soaked in a 1mM Na2SiO3·9H2O solution for 24 hours.

[0051] Seeds treated with Na₂SiO₃·9H₂O solution were placed in a 5℃ incubator for cultivation. During cultivation, the seeds were exposed to 16 hours of white light and 8 hours of darkness daily, with a light intensity of 52 μmol·m⁻¹. -2 s -1 .

[0052] Each morning, 2 mL of sterile distilled water was added to each petri dish containing 40 seeds, with two layers of filter paper placed in each dish. Each treatment consisted of three biological replicates. Alfalfa seedlings were harvested after 30 days for further analysis.

[0053] Example 5: A method for improving the cold resistance of alfalfa

[0054] Alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 1.

[0055] Example 6: A method for improving the cold resistance of alfalfa

[0056] Alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 2.

[0057] Example 7: A method for improving the cold resistance of alfalfa

[0058] Alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 3.

[0059] Example 8: A method for improving the cold resistance of alfalfa

[0060] Alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 4.

[0061] Example 9: A method for improving the cold resistance of alfalfa

[0062] Alfalfa seeds were soaked in a 4 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 1.

[0063] Example 10: A method for improving the cold resistance of alfalfa

[0064] Alfalfa seeds were soaked in a 4 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 2.

[0065] Example 11: A method for improving the cold resistance of alfalfa

[0066] Alfalfa seeds were soaked in a 4 mM Na2SiO3·9H2O solution, with all other conditions being exactly the same as in Example 3.

[0067] Example 12: A method for improving the cold resistance of alfalfa

[0068] Alfalfa seeds were soaked in a 4 mM Na2SiO3·9H2O solution, and the other conditions were exactly the same as in Example 4.

[0069] Example 13: A method for improving the cold resistance of alfalfa

[0070] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0071] The remaining steps are exactly the same as in Example 1.

[0072] Example 14: A method for improving the cold resistance of alfalfa

[0073] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0074] The remaining steps are exactly the same as in Example 2.

[0075] Example 15: A method for improving the cold resistance of alfalfa

[0076] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0077] The remaining steps are exactly the same as in Example 3.

[0078] Example 16: A method for improving the cold resistance of alfalfa

[0079] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0080] The remaining steps are exactly the same as in Example 4.

[0081] Example 17: A method for improving the cold resistance of alfalfa

[0082] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0083] The remaining steps are exactly the same as in Example 5.

[0084] Example 18: A method for improving the cold resistance of alfalfa

[0085] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0086] The remaining steps are exactly the same as in Example 6.

[0087] Example 19: A method for improving the cold resistance of alfalfa

[0088] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0089] The remaining steps are exactly the same as in Example 7.

[0090] Example 20: A method for improving the cold resistance of alfalfa

[0091] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0092] The remaining steps are exactly the same as in Example 8.

[0093] Example 21: A method for improving the cold resistance of alfalfa

[0094] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0095] The remaining steps are exactly the same as in Example 9.

[0096] Example 22: A method for improving the cold resistance of alfalfa

[0097] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0098] The remaining steps are exactly the same as in Example 10.

[0099] Example 23: A method for improving the cold resistance of alfalfa

[0100] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0101] The remaining steps are exactly the same as in Example 11.

[0102] Example 24: A method for improving the cold resistance of alfalfa

[0103] The alfalfa variety used was Saidi 10, abbreviated as SD, which is sensitive to low temperatures and cannot survive the winter.

[0104] The remaining steps are exactly the same as in Example 12.

[0105] Comparative Example 1

[0106] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, with the remaining steps being exactly the same as in Example 1. This group served as the control group in subsequent experiments.

[0107] Comparative Example 2

[0108] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 2. This group served as the control group in subsequent experiments.

[0109] Comparative Example 3

[0110] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 3. This group served as the control group in subsequent experiments.

[0111] Comparative Example 4

[0112] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 4. This group served as the control group in subsequent experiments.

[0113] Comparative Example 5

[0114] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 13. This group served as the control group in subsequent experiments.

[0115] Comparative Example 6

[0116] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 14. This group served as the control group in subsequent experiments.

[0117] Comparative Example 7

[0118] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 15. This group served as the control group in subsequent experiments.

[0119] Comparative Example 8

[0120] Alfalfa seeds were treated with distilled water that had been autoclaved at 120°C for 24 hours, and the remaining steps were exactly the same as in Example 16. This group served as the control group in subsequent experiments.

[0121] Seed germination rate, seedling growth, model damage (MI), enzyme activity, soluble sugar, and soluble protein were measured in Examples 1-24 and Comparative Examples 1-8, respectively. The specific measurement methods and results are as follows:

[0122] I. Methods

[0123] 1. Germination Indicators

[0124] Germination test: The germination standard for seeds is when the radicle breaks through the seed coat and is more than 2 mm in length. Record the number of germinated seeds for each variety within 7 days. The germination percentage, or GP, is calculated as GP = n / N × 100%, where n is the number of germinated seeds and N is the total number of seeds tested.

[0125] 2. Measurement of seedling growth

[0126] After 20 days of treatment at different temperatures, 10 seedlings were randomly selected from 40 seedlings in each treatment group and analyzed using an Epson Expression 10000 XL (Epson, Long Beach, California, USA). The WinRHIZO program was used on the instrument, manufactured by Regent Instruments, Quebec City, Quebec, Canada. The resulting images were digitized, and root length (RL), root surface area (RSA), root diameter (RD), and root volume (RV) were measured. The fresh weight of the seedlings was measured after scanning. These samples were then dried in an oven at 45°C until a stable mass was achieved; the dry weight was used to determine physiological properties.

[0127] 3. Determination of plasm damage and enzyme activity

[0128] Electrolyte leakage (MI) was measured. Five seedlings were washed with deionized water, and the samples were immersed in 10 mL of deionized water in 15 mL centrifuge tubes and kept at 20 °C for 1 h. The conductivity of the exudate, R1, was then measured. The tissue samples were placed in a boiling water bath for 30 min and cooled to 20 °C, and the conductivity of the exudate, R2, was measured again. MI was evaluated using the formula MI = R1 / R2 × ​​100%. To determine antioxidant enzyme activity, 0.1 g of fresh seedlings were placed in 2 mL test tubes, frozen in liquid nitrogen, and then homogenized in 1.8 mL of 50 mM phosphate buffer (pH 7.8) using a benchtop ball mill at 50 Hz for 30 s. The homogenate was centrifuged at 12000 rpm for 15 min at 4 °C, and the resulting supernatant was used for superoxide dismutase (SOD) and peroxidase (POD) assays. Superoxide dismutase activity was determined using the nitroblue tetrazolium method (NBT) described by Giannopolitis and Ries. One unit of SOD was defined as the amount of enzyme required to induce 50% NBT reduction inhibition, as measured at 560 nm. POD activity was determined by assessing the rate of guaiacol oxidation in the presence of H₂O₂. One unit of POD was defined as the increase in absorbance per minute at 470 nm.

[0129] 4. Determination of soluble sugars and soluble proteins

[0130] Soluble sugar content was measured according to the method described. 20 mg of dried seedling material was extracted in 15 mL ddH₂O and boiled for 20 min. Then, 5 mL of anthrone reagent was added to 1 mL of the extract, incubated at 95 °C for 20 min, and cooled to room temperature. The absorbance was measured at 620 nm using a spectrophotometer. Soluble protein content was measured according to the method described. 20 mg of dried seedling material was extracted in 5 mL ddH₂O and shaken for 20 min. Then, it was centrifuged at 3000 rpm for 10 min. Then, 5 mL of Coomassie Brilliant Blue was added to 1 mL of the extract and allowed to stand for 2 min. The absorbance was measured at 595 nm using a spectrophotometer.

[0131] 5. Data Analysis

[0132] Statistical analysis was performed using SPSS 21.0 software. One-way ANOVA was conducted to determine significant treatment effects. p When the variance was 0.05, Duncan's multiple range test was used to compare the means among varieties. Multivariate analysis of variance (MANOVA) was employed to analyze germination rate, biomass, root morphology, and physiological parameters. The data are the mean of three biological replicates, i.e., the standard error. Excel 2010 was used for graphical representation of the data.

[0133] II. Results

[0134] 1. Effects of low temperature stress, silica solution treatment and their interaction on germination rate and seedling growth

[0135] Analysis of variance showed that low temperature stress significantly affected the seed GP and seedling growth of both varieties. p <0.0001. Silica solution treatment had no significant effect on the final seed germination rate and seedling biomass, but it significantly affected root morphology. The interaction between them significantly influenced root morphology.

[0136] Table 1. Analysis of variance (p-value) on the effects of various experimental factors and their interactions on germination rate, biomass, and root morphology.

[0137]

[0138] Note: p <0.0001: The difference is statistically significant; p >0.05: No statistically significant difference; low temperature stress was 5℃, 10℃, 15℃ and 20℃. The concentrations of silicon solution treated were 1mM, 2.25mM and 4mM.

[0139] 2. Effects of low temperature stress, silicon solution and their interaction on physiological indicators

[0140] Strong interactions exist between low-temperature stress, seedling MI, soluble sugars, and soluble proteins. The concentration of silica solution significantly affects seedling MI in varietal LM and POD activity in both varieties, as shown in Table 2. The interactions between these factors significantly influence soluble sugars and seedling MI and POD activity in varietal SD.

[0141] Table 2. Analysis of variance on the effects of different treatment factors and their interactions on the membrane damage index, enzyme activity, and soluble substance content of wheat seedlings. p value

[0142]

[0143] Note: p <0.0001: The difference is statistically significant; p >0.05: No statistically significant difference. Low temperature stresses were 5℃, 10℃, 15℃, and 20℃. Silicon solution treatment concentrations were 0, 1mM, 2.25mM, and 4mM.

[0144] 3. Effects of low temperature stress and silica solution treatment on germination rate

[0145] The effects of different silica solution concentrations on the germination rate (GP) of two alfalfa varieties under different low-temperature stresses are as follows: Figure 1 As shown in the figure. Under different low-temperature conditions, the GP value of the LM variety was higher than that of the SD variety with and without silicon. The GP of LM grown at 5℃ and 10℃ was slightly lower than that at 20℃ and 15℃. In particular, the GP of LM grown at 5℃ with the addition of 2.25mM silicon solution was significantly higher than that of the control. The GP of SD variety grown at 15℃ and 5℃ with the addition of 2.25mM or 4mM silicon solution was significantly increased.

[0146] 4. Effects of low temperature stress and silica solution treatment on seedling growth

[0147] from Figure 2 and Figure 3 The phenotypes of different alfalfa varieties LM and SD after 20 days of growth at 20℃, 15℃, 10℃ and 5℃ can be visually observed. It was found that silicon-induced resistance can improve the root growth of non-wintering alfalfa variety SD seedlings.

[0148] like Figure 4As shown, there were no significant differences in biomass accumulation between LM and SD of the same variety at 20℃, 15℃, and 10℃. However, at 5℃, both varieties showed a significant decrease in fresh weight and dry weight accumulation. Simultaneously, at 5℃, LM seedlings treated with silica solution grew faster than the control, while SD seedlings treated with 1mM silica solution grew slightly faster than the control. Overall, SD biomass accumulation was greater than LM under both higher and lower temperature conditions. Furthermore, RL, RSA, and RV of both varieties decreased significantly at 5℃, but RD remained at its highest level at 5℃. LM treated with 1mM silica solution showed significant increases in RL, RSA, and RV at both 15℃ and 5℃. Figure 5 A in Figure 6 As shown in A and C. The RD, RSA, and RV of SD treated with 1 mM silicon solution increased under 10 °C low-temperature stress, as... Figure 5 D in Figure 6 As shown in B and D in the diagram.

[0149] 5. Effects of low temperature stress and silica solution treatment on seedling membrane damage and enzyme activity

[0150] Under various low-temperature conditions, the membrane damage in LM seedlings treated with silica solution was lower than that in the control. Clearly, compared to the control, LM seedlings treated with 4 mM silica solution showed the lowest root membrane damage at 20°C, 15°C, and 5°C. Furthermore, SD seedlings treated with 4 mM silica solution showed the lowest damage at 5°C. Figure 7 As shown in Figure A. At 5℃, the SOD activity of LM was consistent with that of SD, indicating that silica solution treatment strongly stimulated SOD activity in seedlings. At 15℃ and 10℃, the POD activity of seedlings treated with 4mM silica solution increased by 344% and 67.5% compared to the control, respectively, while the POD activity of seedlings treated with 1mM silica solution increased by 406%, 46.4%, and 323% at 15℃, 10℃, and 5℃, respectively.

[0151] 6. Effects of low temperature stress and silica solution treatment on soluble sugars and soluble proteins in seedlings

[0152] Overall, low-temperature stress and silica solution treatment promoted the accumulation of soluble sugars and soluble proteins in both varieties. Figure 8 As shown, at 20℃ and 15℃, the LM varieties with and without silicon treatment accumulated more soluble sugars than the SD variety; at 10℃ and 5℃, the soluble sugars of LM treated with 1mM silicon solution or 2.25mM silicon solution increased significantly by 157% and 52% compared to the control, respectively. Figure 8 As shown in A in the figure. At 10℃, a 2.25 mM silica solution significantly increased the soluble sugar content of variety SD by 21%, as shown in Figure A. Figure 8As shown in B. In the case of the cultivar LM, application of 2.25 mM and 1 mM silica solutions at 20°C and 5°C resulted in a significant increase of 43% and 21% in soluble protein content, respectively. Figure 6 As shown in C. Under SD conditions at 20°C and 10°C, application of 1 mM and 2.25 mM silicon solutions resulted in a significant increase of 95% and 22% in soluble protein content, respectively. Figure 8 As shown in D in the diagram.

[0153] This invention demonstrates that silicon-induced resistance can improve the germination rate of non-wintering alfalfa varieties, promote seedling root growth, and reduce seedling plasma membrane damage, membrane lipid peroxidation, and osmotic damage caused by low-temperature stress. The method of this invention provides a new approach to improving the cold resistance of alfalfa.

[0154] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0155] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for improving the cold resistance of alfalfa, characterized in that, Includes the following steps: Disinfect alfalfa seeds, soak them in a silica solution for 20-24 hours, and then transfer them to a low-temperature environment to cultivate alfalfa seedlings. During the cultivation period, add 0.025 mL to 0.05 mL of sterile distilled water to each alfalfa seedling daily. The silicon solution is a Na2SiO3·9H2O solution; the concentration of the Na2SiO3·9H2O solution is 1mM~4mM; The temperature of the low-temperature environment is 5℃~20℃.

2. The method according to claim 1, characterized in that, The disinfection method involves soaking in sodium hypochlorite solution for 8 to 10 minutes, followed by rinsing with water 3 to 5 times.

3. The method according to claim 2, characterized in that, The sodium hypochlorite solution has a mass concentration of 0.5% to 0.7%.

4. The method according to claim 2, characterized in that, The water mentioned is sterile distilled water.

5. The method according to claim 1, characterized in that, The cultivation time is 20 to 30 days.

6. The method according to claim 1, characterized in that, The culture process involves 16 hours of white light illumination and 8 hours of darkness daily, with the white light intensity being 52 μmol·m⁻¹. -2 s -1 .

7. The method according to claim 1, characterized in that, The alfalfa variety mentioned is Longmu 803.

8. The method according to claim 1, characterized in that, The alfalfa variety mentioned is Saidi 10.

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