Method for improving cold resistance of medicago sativa

Abstract

The invention relates to the technical field of agro-ecology and crop cultivation, in particular to a method for improving cold resistance of medicago sativa, which comprises the following steps: disinfecting medicago sativa seeds, soaking in a silicon solution for 20-24 hours, transferring to a low-temperature environment, culturing into medicago sativa seedlings, and in the time of culturing into the medicago sativa seedlings, transplanting the medicago sativa seedlings into a seedling culture medium. 0.025 mL to 0.05 mL of sterilized distilled water is added into each medicago sativa plant every day; the silicon solution is a Na2SiO3. 9H2O solution; the concentration of the Na2SiO3. 9H2O solution ranges from 1 mM to 4 mM; the temperature of the low-temperature environment is 5-20 DEG C. Experiments show that the silicon induced resistance can improve the germination rate of non-overwintering alfalfa varieties, promote growth of seedling roots and relieve seedling plasma membrane damage, membrane lipid peroxidation and permeation damage caused by low-temperature stress. The method provided by the invention provides a new way for improving 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: 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.

[0007] The silicon solution is a Na2SiO3·9H2O solution; the concentration of the Na2SiO3·9H2O solution is 1mM~4mM.

[0008] The temperature of the low-temperature environment is 5℃~20℃.

[0009] Preferably, the disinfection method involves soaking in sodium hypochlorite solution for 8 to 10 minutes, followed by rinsing with water 3 to 5 times.

[0010] Preferably, the mass concentration of the sodium hypochlorite solution is 0.5% to 0.7%.

[0011] Preferably, the water is sterilized distilled water.

[0012] Preferably, the culture time is 20 to 30 days.

[0013] Preferably, during the culture process, 16 hours of white light illumination and 8 hours of darkness are performed every day, the intensity of the white light illumination is 52 μmol·m-2s-1, and the intensity of the darkness is 0 μmol·m-2s-1. -2 s -1 .

[0014] Preferably, the alfalfa variety is Longmu No. 803.

[0015] Preferably, the alfalfa variety is Saide No. 10.

[0016] The present application is realized by the following technical scheme: Compared with the prior art, the present application has the following beneficial effects: The present application provides a method for improving cold resistance of alfalfa, comprising the following steps: disinfecting alfalfa seeds, soaking the alfalfa seeds in a silicon solution for 20 to 24 hours, and then transferring the alfalfa seeds to a low-temperature environment to culture alfalfa seedlings; during the culture of the alfalfa seedlings, 0.025 mL to 0.05 mL of sterilized distilled water is added to each alfalfa seedling every day; the silicon solution is a Na2SiO3·9H2O solution; the concentration of the Na2SiO3·9H2O solution is 1 mM to 4 mM; and the temperature of the low-temperature environment is 5 to 20℃. The present application significantly improves the cold resistance of alfalfa by soaking alfalfa seeds in a Na2SiO3·9H2O solution with different concentrations. Experiments show that the silicon solution treatment can effectively enhance the germination ability of seeds under low-temperature conditions, and especially significantly improves the germination rate of the low-temperature sensitive variety Saide No. 10. During the growth stage of the seedlings, the silicon solution treatment significantly promotes root development, increases root length, root surface area, and root volume, and the 1 mM silicon solution treatment of Longmu No. 803 performs outstandingly under 15℃ and 5℃. In addition, the silicon solution treatment enhances the cold resistance through multiple mechanisms: 4 mM silicon treatment can significantly reduce the degree of membrane damage and protect the integrity of the cell membrane; at the same time, the treatment activates the antioxidant system, and the activities of SOD and POD are increased by 344% and 67.5%, respectively, effectively alleviating oxidative stress damage. The silicon solution treatment also promotes the accumulation of osmotic adjustment substances, and the soluble sugar and soluble protein contents of the seedlings treated with the silicon solution are significantly increased, thereby enhancing the cell freezing resistance. The present application not only significantly improves the overwintering ability of the cold-resistant variety LM, but also makes the originally cold-intolerant SD variety obtain good low-temperature adaptability, thereby providing an anti-cold technical solution that is simple to operate, low in cost, and stable in effect for alfalfa planting in cold regions in the north, and has important popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0018] Figure 1 The germination rate dynamics of the LM under different silicon solution concentrations of the present application; Figure 1 In the figure, A is the germination rate dynamics of the LM under different silicon solution concentrations at 20℃; B is the germination rate dynamics of the LM under different silicon solution concentrations at 15℃; C is the germination rate dynamics of the LM under different silicon solution concentrations at 10℃; D is the germination rate dynamics of the LM under different silicon solution concentrations at 5℃; E is the germination rate dynamics of the SD under different silicon solution concentrations at 20℃; F is the germination rate dynamics of the SD under different silicon solution concentrations at 15℃; G is the germination rate dynamics of the SD under different silicon solution concentrations at 10℃; H is the germination rate dynamics of the SD under different silicon solution concentrations at 5℃.

[0019] Figure 2 The phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; Figure 2 In the figure, A is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; B is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; C is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; D is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; E is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; F is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; G is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; H is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; I is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; J is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; K is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; L is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; M is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; N is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; O is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; P is the phenotype figure of the LM variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm.

[0020] Figure 3 The phenotype figure of the SD variety after silicon-induced resistance under 4 low-temperature conditions of the present application, Bar = 0.5 cm; Figure 3In the figure, A is the phenotype of LM seedlings cultured at 20℃ without silicon induction; B is the phenotype of LM seedlings cultured at 20℃ with 1 mM silicon solution induction; C is the phenotype of LM seedlings cultured at 20℃ with 2.25 mM silicon solution induction; D is the phenotype of LM seedlings cultured at 20℃ with 4 mM silicon solution induction; E is the phenotype of SD seedlings cultured at 15℃ without induction; F is the phenotype of SD seedlings cultured at 15℃ with 1 mM silicon solution induction; G is the phenotype of SD seedlings cultured at 15℃ with 2.25 mM silicon solution induction; H is the phenotype of SD seedlings cultured at 15℃ with 4 mM silicon solution induction; I is the phenotype of LM seedlings cultured at 10℃ without induction; J is the phenotype of LM seedlings cultured at 10℃ with 1 mM silicon solution induction; K is the phenotype of LM seedlings cultured at 10℃ with 2.25 mM silicon solution induction; L is the phenotype of LM seedlings cultured at 10℃ with 4 mM silicon solution induction; M is the phenotype of SD seedlings cultured at 5℃ without induction; N is the phenotype of SD seedlings cultured at 5℃ with 1 mM silicon solution induction; O is the phenotype of SD seedlings cultured at 5℃ with 2.25 mM silicon solution induction; P is the phenotype of SD seedlings cultured at 5℃ with 4 mM silicon solution induction.

[0021] Figure 4 Effects of low temperature stress and different concentrations of silicon solution treatment on the fresh weight of LM and SD seedlings, the dry weight of LM and SD seedlings of the present application; Figure 4 In the figure, A is the effect of low temperature stress and different concentrations of silicon solution treatment on the fresh weight of LM seedlings; B is the effect of low temperature stress and different concentrations of silicon solution treatment on the fresh weight of SD seedlings; C is the effect of low temperature stress and different concentrations of silicon solution treatment on the dry weight of LM seedlings; D is the effect of low temperature stress and different concentrations of silicon solution treatment on the dry weight of SD seedlings; wherein different letters represent significant difference.

[0022] Figure 5 Effects of low temperature stress and different concentrations of silicon solution treatment on the root length and root diameter of LM and SD of the present application; Figure 5 In the figure, A is the effect of low temperature stress and different concentrations of silicon solution treatment on the root length of LM; B is the effect of low temperature stress and different concentrations of silicon solution treatment on the root length of SD; C is the effect of low temperature stress and different concentrations of silicon solution treatment on the root diameter of LM; D is the effect of low temperature stress and different concentrations of silicon solution treatment on the root diameter of SD; wherein different letters represent significant difference.

[0023] Figure 6 Effects of low temperature stress and different concentrations of silicon solution treatment on the root surface area and root volume of LM and SD of the present application; Figure 6 In the figure, A is the effect of low temperature stress and different concentrations of silicon solution treatment on the root surface area of LM; B is the effect of low temperature stress and different concentrations of silicon solution treatment on the root surface area of SD; C is the effect of low temperature stress and different concentrations of silicon solution treatment on the root volume of LM; D is the effect of low temperature stress and different concentrations of silicon solution treatment on the root volume of SD; wherein different letters represent significant difference.

[0024] Figure 7 Effects of low temperature stress and different concentrations of silicon solution treatment on the membrane damage degree, superoxide dismutase activity and peroxidase activity of LM and SD seedlings; Figure 7 In the table, A is the effect of low temperature stress and different concentrations of silicon solution treatment on the membrane damage degree of LM seedlings; B is the effect of low temperature stress and different concentrations of silicon solution treatment on the membrane damage degree of SD seedlings; C is the effect of low temperature stress and different concentrations of silicon solution treatment on the superoxide dismutase activity of LM; D is the effect of low temperature stress and different concentrations of silicon solution treatment on the superoxide dismutase activity of SD; E is the effect of low temperature stress and different concentrations of silicon solution treatment on the peroxidase activity of LM; F is the effect of low temperature stress and different concentrations of silicon solution treatment on the peroxidase activity of SD; wherein different letters represent significant difference.

[0025] Figure 8 Effects of low temperature stress and different concentrations of silicon solution treatment on the soluble sugar content and soluble protein content of LM and SD seedlings; Figure 8 In the table, A is the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble sugar content of LM seedlings; B is the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble sugar content of SD seedlings; C is the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble protein content of LM; D is the effect of low temperature stress and different concentrations of silicon solution treatment on the soluble protein content of SD; wherein different letters represent significant difference. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms, and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0027] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the present application are only for the purpose of describing specific embodiments of the present application, and are not intended to limit the present application.

[0028] The beneficial effects of the present application will be illustrated below through specific examples.

[0029] Longmu 803 used in the present application is purchased from Heilongjiang Institute of Animal Husbandry; Saide 10 is purchased from Zhengzhou Huafeng Grass Industry Company.

[0030] Example 1, a method for improving cold resistance of alfalfa Alfalfa variety is Longmu 803, abbreviated as LM, which can survive in severe winter.

[0031] Alfalfa seeds were soaked in 0.6% sodium hypochlorite solution for 8 min, rinsed with distilled water sterilized by high pressure sterilization at 120°C for 3 times, and then soaked in 1 mM Na2SiO3·9H2O solution for 20 h.

[0032] The seeds treated by Na2SiO3·9H2O solution were placed in a constant temperature box at 20°C for culture, and 16 hours of white light illumination and 8 hours of darkness were performed every day during the culture process, and the light intensity was 52 μmol·m -2 s -1 .

[0033] In the morning every day, 1 mL of sterilized distilled water was added to the culture dish, 40 seeds per dish, and two layers of filter paper were placed in each culture dish. Each treatment contained 3 biological repeats. Alfalfa seedlings were harvested after 20 days for further analysis.

[0034] Example 2, a method for improving cold resistance of alfalfa Alfalfa variety is Longmu 803, abbreviated as LM, which can survive in severe winter.

[0035] Alfalfa seeds were sterilized with 0.5% sodium hypochlorite solution for 9 min, rinsed with distilled water sterilized by high pressure sterilization at 120°C for 4 times, and then soaked in 1 mM Na2SiO3·9H2O solution for 22 h.

[0036] The seeds treated by Na2SiO3·9H2O solution were placed in a constant temperature box at 15°C for culture, and 16 hours of white light illumination and 8 hours of darkness were performed every day during the culture process, and the light intensity was 52 μmol·m -2 s -1 .

[0037] In the morning every day, 1 mL of sterilized distilled water was added to the culture dish, 40 seeds per dish, and two layers of filter paper were placed in each culture dish. Each treatment contained 3 biological repeats. Alfalfa seedlings were harvested after 25 days for further analysis.

[0038] Example 3, a method for improving cold resistance of alfalfa Alfalfa variety is Longmu 803, abbreviated as LM, which can survive in severe winter.

[0039] Alfalfa seeds were soaked in 0.7% sodium hypochlorite solution for 10 min, rinsed with distilled water sterilized by high pressure sterilization at 120°C for 5 times, and then soaked in 1 mM Na2SiO3·9H2O solution for 24 h.

[0040] The seeds treated with Na2SiO3·9H2O solution were placed in a 10 °C incubator for cultivation, during which 16 hours of white light illumination and 8 hours of darkness were performed every day, and the illumination intensity was 52 μmol·m -2 s -1 .

[0041] Every morning, 2 mL of sterilized distilled water was added to the culture dish, 40 seeds per dish, and two layers of filter paper were placed in each culture dish. Each treatment contained 3 biological replicates. After 30 days, alfalfa seedlings were harvested for further analysis.

[0042] Example 4, a method for improving cold resistance of alfalfa The alfalfa variety was Longmu No. 803, abbreviated as LM, which could survive in severe winter.

[0043] The alfalfa seeds were soaked in a 0.6% sodium hypochlorite solution for 10 min, rinsed 5 times with distilled water sterilized by high pressure sterilization at 120 °C, and then soaked in a 1 mM Na2SiO3·9H2O solution for 24 h.

[0044] The seeds treated with Na2SiO3·9H2O solution were placed in a 5 °C incubator for cultivation, during which 16 hours of white light illumination and 8 hours of darkness were performed every day, and the illumination intensity was 52 μmol·m -2 s -1 .

[0045] Every morning, 2 mL of sterilized distilled water was added to the culture dish, 40 seeds per dish, and two layers of filter paper were placed in each culture dish. Each treatment contained 3 biological replicates. After 30 days, alfalfa seedlings were harvested for further analysis.

[0046] Example 5, a method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, and the rest of the conditions were exactly the same as in Example 1.

[0047] Example 6, a method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, and the rest of the conditions were exactly the same as in Example 2.

[0048] Example 7, a method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 2.25 mM Na2SiO3·9H2O solution, and the rest of the conditions were exactly the same as in Example 3.

[0049] Example 8, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 2.25 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0050] Example 9, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 1.

[0051] Example 10, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 2.

[0052] Example 11, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 3.

[0053] Example 12, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0054] Example 13, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0055] The remaining steps were identical to those of Example 1.

[0056] Example 14, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0057] The remaining steps were identical to those of Example 2.

[0058] Example 15, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0059] The remaining steps were identical to those of Example 3.

[0060] Example 16, A method for improving cold resistance of alfalfa The alfalfa seeds were soaked in a 4 mM Na2SiO3-9H2O solution, and the remaining conditions were identical to those of Example 4.

[0061] The remaining steps are identical to Example 4.

[0062] Example 17. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0063] The remaining steps are identical to Example 5.

[0064] Example 18. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0065] The remaining steps are identical to Example 6.

[0066] Example 19. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0067] The remaining steps are identical to Example 7.

[0068] Example 20. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0069] The remaining steps are identical to Example 8.

[0070] Example 21. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0071] The remaining steps are identical to Example 9.

[0072] Example 22. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0073] The remaining steps are identical to Example 10.

[0074] Example 23. A method for increasing cold tolerance in alfalfa The alfalfa variety used is Sarge 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0075] The remaining steps are identical to Example 11.

[0076] Example 24, A method for improving cold tolerance of alfalfa The alfalfa variety used was Sady 10, abbreviated as SD, which is sensitive to low temperature and cannot survive in winter.

[0077] The remaining steps were exactly the same as Example 12.

[0078] Comparative Example 1 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 1, which was used as a control group in the subsequent experiments, i.e., the control group.

[0079] Comparative Example 2 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 2, which was used as a control group in the subsequent experiments, i.e., the control group.

[0080] Comparative Example 3 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 3, which was used as a control group in the subsequent experiments, i.e., the control group.

[0081] Comparative Example 4 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 4, which was used as a control group in the subsequent experiments, i.e., the control group.

[0082] Comparative Example 5 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 13, which was used as a control group in the subsequent experiments, i.e., the control group.

[0083] Comparative Example 6 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 14, which was used as a control group in the subsequent experiments, i.e., the control group.

[0084] Comparative Example 7 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 15, which was used as a control group in the subsequent experiments, i.e., the control group.

[0085] Comparative Example 8 The alfalfa seeds were treated with distilled water sterilized at 120°C for 24 hours only, and the remaining steps were exactly the same as Example 16, which was used as a control group in the subsequent experiments, i.e., the control group.

[0086] Seed germination, seedling growth, the measurement of the model injury, i.e. MI and enzyme activities, soluble sugars and soluble proteins were determined for the seeds of Examples 1 to 24, Comparative Examples 1 to 8, respectively, in the following specific methods and results: I. Methods 1. Germination index Germination test: The germination criterion for seeds was the radicle breaking through the seed coat with a length of more than 2 mm. The number of germinated seeds was recorded for each variety within 7 days. The germination percentage, i.e. GP was calculated as GP = n / N x 100%, where n is the number of germinated seeds and N is the total number of seeds tested.

[0087] 2. Determination of seedling growth After 20 days of temperature treatment, 10 seedlings were randomly selected from 40 seedlings of each treatment group and scanned using an Epson Expression 10000 XL, purchased from Epson America Inc., Long Beach, CA, USA. The resulting images were digitized using the WinRHIZO program, which is manufactured by Regent Instruments Inc., Quebec, QC, Canada. Root length, i.e. RL, root surface area, i.e. RSA, root diameter, i.e. RD and root volume, i.e. RV were determined. After scanning, the fresh weight of the seedlings was measured. These samples were then dried in an oven at 45°C until a stable mass was achieved, and the dry mass was used to determine the physiological attributes.

[0088] 3. Measurement of model injury and enzyme activities Model injury, i.e. MI, was measured by electrolyte leakage. Five seedlings were washed with deionized water, the samples were immersed in 10 mL of deionized water in a 15 mL centrifuge tube and kept at 20°C for 1 h. The conductivity of the exudate was then measured, R1. The tissue samples were placed in a boiling water bath for 30 min and cooled to 20°C, and the conductivity of the exudate was measured again, R2. The formula MI = R1 / R2 x 100% was used to evaluate MI. To determine antioxidant enzyme activities, 0.1 g of fresh seedlings were packed into 2 mL tubes, frozen in liquid nitrogen and then homogenized in 1.8 mL of 50 mM phosphate buffer, pH 7.8 using a table 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, i.e. SOD and peroxidase, i.e. POD determination. Superoxide dismutase activity was determined using the nitro blue tetrazolium, i.e. NBT assay described by Giannopolitis and Ries. One unit of SOD was defined as the amount of enzyme required to cause a 50% inhibition of NBT reduction as determined 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.

[0089] 4. Determination of soluble sugars and soluble proteins 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.

[0090] 5. Data Analysis 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.

[0091] II. Results 1. Effects of low temperature stress, silica solution treatment and their interaction on germination rate and seedling growth 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.

[0092] Table 1. Analysis of variance (p-value) on the effects of various experimental factors and their interactions on germination rate, biomass, and root morphology.

[0093] 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.

[0094] 2. Effects of low temperature stress, silicon solution and their interaction on physiological indicators 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.

[0095] 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

[0096] 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.

[0097] 3. Effects of low temperature stress and silica solution treatment on germination rate 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.

[0098] 4. Effects of low temperature stress and silica solution treatment on seedling growth 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.

[0099] like Figure 4 As 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.

[0100] 5. Effects of low temperature stress and silica solution treatment on seedling membrane damage and enzyme activity 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.

[0101] 6. Effects of low temperature stress and silica solution treatment on soluble sugars and soluble proteins in seedlings 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 8 As 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.

[0102] 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.

[0103] Any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations are described, however, as long as there is no conflict, any combination of the technical features should be considered within the scope of the present disclosure.

[0104] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. For ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be subject to 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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