Method for improving cold resistance of michelia figo

Abstract

The invention discloses a method for improving cold resistance of michelia figo, and belongs to the technical field of michelia figo cold resistance. A cold-resistant agent is prepared by adopting a gel matrix material to load inositol, proline and antifreeze protein, the cold-resistant agent is sprayed to michelia figo plants before late spring cold comes, the cold-resistant agent is gelatinized and attached to the body surfaces of the plants after being sprayed, and the michelia figo cold resistance is improved. Effective components such as inositol, proline and antifreeze protein are continuously released, the effective components improve the cold resistance of the plants by improving the enzyme activity in michelia figo plants and maintaining the stability of cell membranes, then the normal growth condition of the plants in the late spring cold period is maintained, the temperature rises after the late spring cold, the cold-resistant agent is solated and runs off from the surfaces of the plants, and the cold resistance of the michelia figo plants is improved. Normal growth of later plants cannot be influenced; the mixed liquid fertilizer is sprayed after late spring coldness to restore the good tree vigor of michelia figo plants and promote the good growth of michelia figo, and the method has important significance in promoting the development of the michelia figo industry.

Description

Technical Field

[0001] This invention relates to the field of Michelia champaca cold resistance technology, and more particularly to a method for improving the cold resistance of Michelia champaca. Background Technology

[0002] Michelia is a genus of evergreen shrubs or trees belonging to the family Magnoliaceae. In China, it is mainly produced from the southwest to the east. Plants in this genus have beautiful tree shapes, and most species have fragrant flowers, making them highly valuable for landscaping and ornamental purposes. Furthermore, their leaves, flowers, and fruits are rich in aromatic substances such as terpenes, alkenes, esters, and alcohols, giving them good medicinal value and broad application prospects.

[0003] In the introduction and cultivation of Michelia species in northern regions such as Xi'an, low-temperature freezing damage is a significant factor restricting the survival of these species. Many regions have conducted in-depth research on the physiology of this genus and carried out hybridization breeding to select varieties with strong frost resistance. These frost-resistant Michelia varieties can generally survive the winter by resisting frost damage on their own. However, if extreme weather such as a late spring frost occurs, some Michelia species, due to their early flowering period, have already begun bud swelling and bud formation. Since the buds and leaf buds are weak in cold resistance, sudden low temperatures can damage their cell structure, interfere with enzyme activity, and lead to metabolic disorders and inhibited photosynthesis, ultimately causing chilling injury. This results in slow growth, flower withering, and yellowing and shedding of leaves, limiting the further development of the Michelia industry in northern regions.

[0004] Therefore, it is currently necessary to find a way to improve the cold resistance of Michelia figo and solve the problems that Michelia figo plants are easily damaged by cold weather when they encounter cold snaps in spring, which leads to slow growth, flower withering, and yellowing and falling of leaves. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method to improve the cold resistance of Michelia figo, and to solve the problem that Michelia figo plants are easily damaged by cold weather when encountering cold snaps in spring, resulting in slow growth, withering flowers, and yellowing and falling leaves.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] A method for improving the cold resistance of Michelia champaca, the method comprising the following steps:

[0008] (1) According to the weather forecast, on the evening of the day before the arrival of the cold snap, spray the sol-state anti-cold agent evenly onto the surface of the Michelia figo plant at a rate of 0.1-2 kg / plant.

[0009] (2) Three days after the cold snap in spring, spray the mixed liquid fertilizer evenly on the surface of the Michelia figo plant at a rate of 0.1-1 kg / plant. Then, follow the conventional management method.

[0010] Furthermore, the cold-resistant agent in step (1) includes the following raw materials:

[0011] Gelatin, mannulic acid, glycerin, hyaluronic acid, methacrylic anhydride, N-isopropylacrylamide, ammonium persulfate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, antifreeze protein, proline, inositol.

[0012] Furthermore, the method for preparing the cold-resistant agent is as follows:

[0013] A: Add gelatin to 10 times its weight of water and stir to disperse. Add mannouric acid, mix well and adjust the pH to 7. Stir and mix at 100-200 r / min for 30 min, then heat to 50-60℃ and keep warm for 10-20 min. Then add glycerin, stir and mix well, and let cool naturally to room temperature to obtain gelatin gel.

[0014] B: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Add methacrylic anhydride to the hyaluronic acid solution, mix well, and adjust the pH to 8. Stir the reaction at room temperature for 6-8 hours. After the reaction is complete, cool naturally to room temperature to obtain the hyaluronic acid-methacrylic anhydride complex, which is ready for use.

[0015] C: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Adjust the pH of the hyaluronic acid solution to 8, add N-isopropylacrylamide and mix well. Then add ammonium persulfate and mix well. Heat to 60-70℃ and stir for 4-6 hours. After the reaction is complete, cool naturally to room temperature to obtain hyaluronic acid-poly(N-isopropylacrylamide) composite gel, which is ready for use.

[0016] D: Add the hyaluronic acid-methacrylic anhydride complex to the hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35℃ and keep constant temperature, add 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and crosslink under ultraviolet light with a wavelength of 365nm and an intensity of 10-20mW / cm² for 20-30 minutes to obtain a double composite gel, and keep it at 35℃ for later use;

[0017] E: Add proline, inositol, and antifreeze protein to water and stir to disperse. Then add the gelatin gelling agent, and then add the heat-insulating double composite gel and stir to mix evenly to obtain the antifreeze agent. Heat to 25°C to obtain the sol-state antifreeze agent, which can then be sprayed on Michelia figo.

[0018] This invention uses a gelatin gelling agent loaded with proline, inositol, and antifreeze proteins to prepare a cold-resistant agent. The agent is then evenly sprayed onto the surface of the Michelia figo plant. Due to the thermal reversibility of the gelatin gelling agent, under lower evening temperatures, the sol-state cold-resistant agent gels and adheres evenly and firmly to the plant surface, continuously releasing the loaded proline and inositol. These substances enter the Michelia figo plant and regulate its osmotic pressure to maintain water balance in low-temperature environments. Simultaneously, they regulate enzyme activity, maintain cell membrane stability, and enhance the plant's antioxidant capacity, thereby ensuring the normal physiological state of the Michelia figo plant under the cold-weather conditions of late spring frost and preventing chilling injury.

[0019] During a cold snap in spring, if the temperature drops below the freezing point of water, water inside plant cells will crystallize, forming ice crystals that pierce the cells and cause physical damage, leading to frost damage. Simultaneously, the gelatin matrix of the cold-resistant agent has strong water absorption and retention properties, and the retained water is also prone to forming ice crystals, causing physical damage to plant cells. Therefore, this invention adds glycerol during the preparation of the gelatin gel to lower the freezing point of the water within the gel, preventing water crystal formation and damage to plant cells. Additionally, antifreeze proteins are added and loaded into the gelatin gel. These antifreeze proteins, once inside the plant, not only lower the freezing point and inhibit ice crystal growth, thus suppressing frost damage, but also scavenge free radicals to reduce oxidative damage to Michelia figo at low temperatures, maintaining cell membrane stability. Furthermore, the gelatin gel, along with its loaded and released inositol, proline, and antifreeze proteins, maintain cell membrane stability and ensure internal enzyme activity under the low-temperature environment of a cold snap, thereby ensuring the normal growth of Michelia figo and preventing cold damage that could lead to slow growth and flower withering.

[0020] This invention uses gelatin gel loaded with inositol, proline, and antifreeze proteins. Based on the thermal reversibility of gelatin gel, it can be heated to its melting point to form a uniform sol solution, facilitating even spraying. However, gelatin has a high melting point, typically requiring heating to at least 30°C to form a uniform sol solution. Plant watering temperatures are generally limited to 15°C to 25°C; higher temperatures can easily scorch leaves and cause stomatal closure. Therefore, this invention uses mannouronic acid to treat the gelatin, adjusting the internal charge density to increase the inter-chain molecular density. The repulsive effect reduces the stability of gelatin in forming a gel, thus enabling the formation of a uniform sol solution at lower temperatures. This facilitates even spraying onto the plant surface, allowing it to form a uniformly adhered solid gel for the continuous release of substances such as inositol and proline. At the same time, the low-temperature spraying of the cold-resistant agent will not adversely affect the plant. Furthermore, when the temperature rises after the late spring frost, the gelatin, due to its thermal reversibility, will revert to a liquid sol after reaching its melting point and then run off the plant surface, preventing it from adhering to the plant surface for a long time and affecting the plant's normal growth later on.

[0021] Since cold-resistant agents form a solid gel after being sprayed onto the plant surface, if the late spring frost lasts for a long time, the cold-resistant agent adhering to the plant surface for a long time will block the stomata of the leaves, affecting the entry and exit of physiological metabolic gases. Based on this, this invention prepares a dual composite gel mixed with gelatin as the gel matrix material. Specifically, the dual composite gel is prepared by mixing hyaluronic acid-methacrylic anhydride complex and hyaluronic acid-poly(N-isopropylacrylamide) composite gel at 35°C and then adding a photoinitiator for crosslinking. Because the hyaluronic acid-poly(N-isopropylacrylamide) composite gel is thermosensitive, it hydrophobically polymerizes at 35°C to form a solid gel. At this time, after adding the photoinitiator, the hyaluronic acid-methacrylic anhydride... Acrylic anhydride crosslinks under ultraviolet light to form a permanent crosslinked network. When combined with gelatin gelling agent to prepare a cold-resistant agent, it is sprayed onto the plant surface. In the low-temperature environment of late spring frost, the hyaluronic acid-poly(N-isopropylacrylamide) composite gel in the dual composite gel transforms into a soluble sol state and is lost, while the photocrosslinked gel network of hyaluronic acid-methacrylic anhydride remains stable. As a result, the sites where the hyaluronic acid-poly(N-isopropylacrylamide) gel dissolves and is lost form a porous structure, resulting in a porous hyaluronic acid-methacrylic anhydride gel skeleton. These porous channels can ensure the normal entry and exit of metabolic gases, thereby eliminating the obstruction of physiological metabolic gas entry and exit by the cold-resistant agent adhering to the plant surface after gelation.

[0022] Furthermore, in step A, the mass ratio of gelatin, mannouronic acid, and glycerol is (2-3):(0.1-0.2):(1-2).

[0023] Furthermore, in step B, the mass ratio of hyaluronic acid solution to methacrylic anhydride is (0.6-1.2):(0.018-0.036).

[0024] Furthermore, in step C, the mass ratio of hyaluronic acid solution, N-isopropylacrylamide, and ammonium persulfate is (1.5-3):(0.015-0.03):(0.0002-0.003).

[0025] Furthermore, in step D, the mass ratio of hyaluronic acid-methacrylic anhydride complex, hyaluronic acid-poly(N-isopropylacrylamide) composite gel, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is (0.6-1.2):(1.5-3):(0.001-0.002).

[0026] Furthermore, in step E, the mass ratio of gelatin gelling agent, proline, inositol, antifreeze protein, and dual composite gel is (20-30): (1-2): (1-2): (1.5-3): (2-4).

[0027] Furthermore, the method for preparing the mixed liquid fertilizer in step (2) is as follows:

[0028] Mix urea and potassium dihydrogen phosphate, then add 200-300 times their weight of water and stir to dissolve. Then add carbendazim and stir to mix evenly to obtain a mixed liquid fertilizer.

[0029] Furthermore, the mass ratio of urea, potassium dihydrogen phosphate, and carbendazim in the mixed fertilizer solution is 50:20:1.

[0030] The beneficial effects of this invention are as follows:

[0031] 1. This invention uses a gel matrix material loaded with inositol, proline, and antifreeze protein to prepare a cold-resistant agent, which is then sprayed onto Michelia figo plants. The cold-resistant agent adheres to the plant surface, forming a uniform gel and continuously releasing inositol, proline, and antifreeze protein to maintain enzyme activity and cell membrane stability within the plant, improve the antioxidant capacity of Michelia figo, and enhance the cold resistance of Michelia figo plants. This ensures that the plants can effectively resist cold damage or freezing damage during late spring frosts and maintain normal physiological state.

[0032] 2. Spraying cold-resistant agents ensures that the plants can safely survive the late spring frost. After the frost, spraying mixed liquid fertilizer on the leaves restores the plant's growth vigor and promotes the healthy growth of Michelia figo plants. This is of great significance to the development of the Michelia figo industry. Attached Figure Description

[0033] Figure 1 : These are pictures of the growth status of Michelia figo plants in experimental group 1 of this invention under natural outdoor conditions;

[0034] Figure 2 : This is a picture showing the leaf drop status of the Michelia figo plant in the blank control group of the present invention. Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0036] Example 1: Preparation of Cold Resistance Agent

[0037] A: Add 2.4 kg of gelatin to 10 times its weight of water and stir to disperse. Add 0.15 kg of mannuronic acid, mix well, and adjust the pH to 7. Stir and mix at 150 r / min for 30 min, then heat to 55℃ and keep warm for 15 min. Then add 1.5 kg of glycerin, stir and mix well, and let cool naturally to room temperature to obtain gelatin gel.

[0038] B: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Add 0.027kg of methacrylic anhydride to 0.9kg of hyaluronic acid solution, mix well, and adjust the pH to 8. Stir at room temperature for 7 hours. After the reaction is complete, cool naturally to room temperature to obtain a hyaluronic acid-methacrylic anhydride complex for later use.

[0039] C: Dissolve hyaluronic acid in water to prepare 2.2 kg of 1 wt% hyaluronic acid solution. Adjust the pH of the hyaluronic acid solution to 8, then add 0.022 kg of N-isopropylacrylamide and mix well. Then add 0.00025 kg of ammonium persulfate and mix well. Heat to 65℃ and stir for 5 h. After the reaction is complete, cool naturally to room temperature to obtain hyaluronic acid-poly(N-isopropylacrylamide) composite gel for later use.

[0040] D: Add 0.9 kg of hyaluronic acid-methacrylic anhydride complex to 2.2 kg of hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35℃ and maintain constant temperature, add 0.0015 kg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and crosslink under ultraviolet light irradiation at a wavelength of 365 nm and an intensity of 15 mW / cm² for 25 min to obtain a double composite gel, and keep it at 35℃ for later use;

[0041] E: Add 1.5kg of proline, 1.5kg of inositol and 2kg of antifreeze protein to 5kg of water and stir to disperse. Then add 24kg of gelatin gelling agent and 3kg of heat-insulating double composite gel to obtain the cold-resistant agent.

[0042] Example 2: Preparation of Cold Resistance Agent II

[0043] A: Add 2 kg of gelatin to 10 times its weight of water and stir to disperse. Add 0.1 kg of mannuronic acid, mix well and adjust the pH to 7. Stir and mix at 100 r / min for 30 min, then heat to 50℃ and keep warm for 20 min. Then add 1 kg of glycerin, stir and mix well, and let cool naturally to room temperature to obtain gelatin gel.

[0044] B: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Add 0.018kg of methacrylic anhydride to 0.6kg of hyaluronic acid solution, mix well, adjust the pH to 8, stir at room temperature for 6h, and allow to cool naturally to room temperature after the reaction is complete to obtain a hyaluronic acid-methacrylic anhydride complex for later use.

[0045] C: Dissolve hyaluronic acid in water to prepare 1.5 kg of 1 wt% hyaluronic acid solution. Adjust the pH of the hyaluronic acid solution to 8, then add 0.015 kg of N-isopropylacrylamide and mix well. Then add 0.0002 kg of ammonium persulfate and mix well. Heat to 60℃ and stir for 6 hours. After the reaction is complete, cool naturally to room temperature to obtain hyaluronic acid-poly(N-isopropylacrylamide) composite gel for later use.

[0046] D: Add 0.6 kg of hyaluronic acid-methacrylic anhydride complex to 1.5 kg of hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35℃ and keep constant temperature, add 0.001 kg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and crosslink under ultraviolet light with a wavelength of 365 nm and an intensity of 10-20 mW / cm² for 20 min to obtain a double composite gel, and keep warm at 35℃ for later use;

[0047] E: Add 1kg of proline, 1kg of inositol and 1.5kg of antifreeze protein to 3.5kg of water and stir to disperse. Then add 20kg of gelatin gelling agent and 2kg of heat-insulating double composite gel to obtain the cold-resistant agent.

[0048] Example 3: Preparation of Cold Resistance Agent

[0049] A: Add 3 kg of gelatin to 10 times its weight of water and stir to disperse. Add 0.2 kg of mannuronic acid, mix well, and adjust the pH to 7. Stir and mix at 200 r / min for 30 min, then heat to 60℃ and keep warm for 10 min. Then add 2 kg of glycerin, stir and mix well, and let cool naturally to room temperature to obtain gelatin gel.

[0050] B: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Add 0.036kg of methacrylic anhydride to 1.2kg of hyaluronic acid solution, mix well, adjust the pH to 8, stir at room temperature for 8 hours, and allow to cool naturally to room temperature after the reaction is complete to obtain a hyaluronic acid-methacrylic anhydride complex for later use.

[0051] C: Dissolve hyaluronic acid in water to prepare 3 kg of 1 wt% hyaluronic acid solution. Adjust the pH of the hyaluronic acid solution to 8, then add 0.03 kg of N-isopropylacrylamide and mix well. Then add 0.003 kg of ammonium persulfate and mix well. Heat to 70℃ and stir for 6 h. After the reaction is complete, cool naturally to room temperature to obtain hyaluronic acid-poly(N-isopropylacrylamide) composite gel, which is ready for use.

[0052] D: Add 1.2 kg of hyaluronic acid-methacrylic anhydride complex to 3 kg of hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35℃ and keep constant temperature, add 0.002 kg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and crosslink under ultraviolet light with a wavelength of 365 nm and an intensity of 20 mW / cm² for 30 min to obtain a double composite gel, and keep warm at 35℃ for later use;

[0053] E: Add 2kg of proline, 2kg of inositol and 3kg of antifreeze protein to 7kg of water and stir to disperse. Then add 30kg of gelatin gelling agent and 4kg of heat-insulating double composite gel to obtain the cold-resistant agent.

[0054] Comparative Example 1: Preparation of Cold Resistance Agent

[0055] In contrast to Example 1, the only difference is that in the preparation of the cold-resistant agent in Comparative Example 1, mannulic acid was not added to treat the gelatin in step A, as shown below:

[0056] A: Add 2.4 kg of gelatin to 10 times its weight of water and stir to disperse. Heat to 55°C and keep warm for 15 minutes. Then add 1.5 kg of glycerin and stir to mix evenly. Allow to cool naturally to room temperature to obtain gelatin gel.

[0057] B~E: Same as in Example 1.

[0058] Comparative Example 2: Preparation of Cold Resistance Agent

[0059] Compared with Example 1, the only difference is that glycerol is not added in step A of the preparation of the antifreeze in Comparative Example 2, while the other steps are the same as in Example 1.

[0060] Comparative Example 3: Preparation of Cold Resistance Agent

[0061] Compared with Example 1, the only difference is that in the preparation of the cold-resistant agent in Comparative Example 3, the pH was adjusted to 6 in step A. All other steps were the same as in Example 1.

[0062] Comparative Example 4: Preparation of Cold Resistance Agent

[0063] Compared with Example 1, the only difference is that in the preparation of the cold-resistant agent in Comparative Example 4, the pH was adjusted to 8 in step A. All other steps were the same as in Example 1.

[0064] Comparative Example 5: Preparation of Cold Resistance Agent

[0065] Compared with Example 1, the only difference is that steps B, C, and D were omitted in the preparation of the cold-resistant agent in Comparative Example 5, i.e., the double composite gel was not added to prepare the cold-resistant agent, as shown below:

[0066] A: Same as Example 1;

[0067] B: Add 1.5 kg of proline, 1.5 kg of inositol and 2 kg of antifreeze protein to 5 kg of water and stir to disperse. Then add to 24 kg of gelatin gelling agent, heat to 25°C and stir to mix evenly to obtain a sol-state antifreeze agent.

[0068] Comparative Example 6: Preparation of Cold Resistance Agent

[0069] In contrast to Example 1, the only difference is that in Comparative Example 6, ultraviolet light crosslinking was not performed in step D during the preparation of the antifreeze agent; instead, the two-component composite gel was directly mixed, as shown below:

[0070] A-C: Same as in Example 1;

[0071] D: Add 0.9 kg of hyaluronic acid-methacrylic anhydride complex to 2.2 kg of hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35°C and keep at a constant temperature to obtain a double composite gel, and keep at 35°C for later use;

[0072] E: Same as in Example 1.

[0073] Comparative Example 7: Preparation of Cold Resistance Agent

[0074] Compared with Example 1, the only difference is that proline was not added in step E of the preparation of the anti-cold agent in Comparative Example 7, while the other steps were the same as in Example 1.

[0075] Comparative Example 8: Preparation of Cold Resistance Agent

[0076] Compared with Example 1, the only difference is that in the preparation of the antifreeze agent in Comparative Example 8, inositol was not added in step E. All other steps were the same as in Example 1.

[0077] Comparative Example 9: Preparation of Cold Resistance Agent

[0078] Compared with Example 1, the only difference is that antifreeze protein was not added in step E of the preparation of the antifreeze agent in Comparative Example 9, while the other steps are the same as in Example 1.

[0079] Comparative Example 10: Preparation of Cold Resistance Agent

[0080] In contrast to Example 1, the difference lies in the preparation of the anti-cold agent in Comparative Example 10. Instead of preparing a gelled formulation, it was prepared as a solution. Gelatin was not used to load proline or similar substances; instead, it was directly added to water to prepare a mixed solution, as detailed below:

[0081] Add 1.5 kg of glycerin to 24 kg of water, then add 1.5 kg of proline, 1.5 kg of inositol and 2 kg of antifreeze protein to 5 kg of water, stir to disperse, and then mix evenly to obtain the antifreeze agent.

[0082] Example 4: Methods to improve the cold resistance of Michelia champaca

[0083] (1) According to the weather forecast, on the evening of the day before the arrival of the cold snap, the cold-resistant agent was prepared according to the method of Example 1. The sol-state cold-resistant agent was evenly sprayed onto the surface of the Michelia figo plant at a rate of 0.8 kg / plant.

[0084] (2) Three days after the cold snap in spring, weigh out urea, potassium dihydrogen phosphate and carbendazim according to the mass ratio of urea: potassium dihydrogen phosphate: carbendazim = 50:20:1. Then mix urea and potassium dihydrogen phosphate and add 250 times the mass of water and stir to dissolve. Then add carbendazim and stir to mix evenly to obtain mixed liquid fertilizer. Spray the mixed liquid fertilizer evenly on the surface of the Michelia figo plant at a rate of 0.6 kg / plant. After that, watering, pest and disease control and other maintenance can be carried out in accordance with the conventional methods.

[0085] Experiment 1: Determination of the melting temperature of the antifreeze agent

[0086] The melting temperature (gel-sol transition temperature) of the cold-resistant agents prepared in Example 1 and Comparative Examples 1 to 9 was tested. Specifically, after the cold-resistant agents in each group were prepared, samples were taken from each group and the melting temperature was tested. The results are shown in Table 1.

[0087] Table 1

[0088] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Melting temperature (°C) 23.2 28.9 25.2 24.6 22.5 24.8 23.6 23.5 23.4 23.7

[0089] Based on the data analysis in Table 1, we can conclude that:

[0090] (1) The melting temperature of the cold-resistant agent in Example 1 of the present invention is 23.2℃, which means that it can be transformed into a sol state when heated to 23.2℃. This indicates that the phase transition temperature of the cold-resistant agent prepared according to the method of the present invention is low. It can be transformed into a sol at a low temperature, which is suitable for uniform spraying onto the plant surface and will not have an adverse effect on the plant.

[0091] (2) In Comparative Example 1, no mannouronic acid was added to treat the gelatin during the preparation of the antifreeze agent. The interaction between the gelatin molecular chains is strong, and more heat is needed to break the interaction between the molecular chains and transform it into a sol, which requires a higher temperature. In Comparative Example 2, no glycerol was added during the preparation of the antifreeze agent. The addition of glycerol changed the interaction between the internal water and the gelatin, and the combined effect reduced the melting temperature of the gelatin. In Comparative Example 3, the pH was adjusted to 6 in step A during the preparation of the antifreeze agent. The decrease in pH reduced the charge density generated by mannouronic acid, which had a poor effect on reducing the interaction between the gelatin molecular chains, and the melting temperature increased to a certain extent. In Comparative Example 4, the pH was adjusted to 8 in step A during the preparation of the antifreeze agent. The increase in pH further increased the charge density, which further reduced the interaction between the gelatin molecular chains, and the melting temperature further decreased. However, as the main matrix material, the gelatin gelling agent is prone to damage to the plant if sprayed on the plant surface at too high a pH. The temperature of 23.2°C in Example 1 of this invention is already within the suitable temperature range for spraying plants.

[0092] (3) No double composite gel was added during the preparation of the anti-cold agent in Comparative Example 5. The hyaluronic acid-poly(N-isopropylacrylamide) gel in the double composite gel exists in a sol state, which is beneficial to synergistically reduce the phase transition temperature of the anti-cold agent. Without the double composite gel, the melting temperature increases to a certain extent.

[0093] Experiment 2: Michelia champaca Cold Resistance Experiment

[0094] The cold-resistant agents prepared in Example 1 and Comparative Examples 1-10 were subjected to a cold-resistant experiment at Xi'an Botanical Garden. The experiment was divided into 12 groups: experimental group 1, control group 1-10, and blank control group. The cold-resistant agents and methods used in each group are as follows:

[0095] Experimental Group 1: The cold-resistant agent of Example 1 and the cold-resistant method of Example 4 were used;

[0096] Control groups 1-10: The cold-resistant agents used in comparative examples 1-10 and the cold-resistant method used in Example 4 were used respectively;

[0097] Blank control group: No cold-resistant agent was used; instead, an equal amount of water was sprayed directly at the same time as the cold-resistant agent was sprayed.

[0098] Specifically, each group used 1-year-old potted Michelia figo with consistent growth as experimental plants, with 10 plants in each group. Since 1-year-old seedlings were used for the experiment, the amount of cold-resistant agent sprayed was 0.1 kg / plant. Because the melting temperature of the cold-resistant agents in Comparative Examples 1 and 2 was relatively high, the cold-resistant agent in Control Group 1 was heated to 29℃ to melt before spraying; the cold-resistant agent in Control Group 2 was heated to 26℃ to melt before spraying.

[0099] After each plant was sprayed with a cold-resistant agent, it was placed in an artificial climate chamber with a daytime temperature of 5℃, a nighttime temperature of -3℃, and a humidity of 65% to simulate a late spring frost. The treatment lasted for 4 days. After the 4-day treatment, the cold resistance of each group of plants (including malondialdehyde (MDA) content and chlorophyll content) was measured. Then, the plants were treated for 3 days with a daytime temperature of 15℃ and a nighttime temperature of 10℃. After that, they were sprayed with 0.1 kg / plant of mixed liquid fertilizer and then transferred to the outdoor natural environment for growth. After 6 months, the plant height growth of each group was measured. The experiment was repeated 3 times, and the data are shown in Table 2.

[0100] Table 2

[0101] MDA content (μmol / g) Chlorophyll content (mg / g) Plant height growth (cm) Experimental group 1 1.22 1.92 23.8 Control group 1 1.41 1.87 22.4 Control group 2 1.57 1.70 20.5 Control group 3 1.31 1.90 23.6 Control group 4 1.62 1.66 19.4 Control group 5 1.64 1.63 19.0 Control group 6 1.45 1.84 22.0 Control group 7 1.79 1.54 18.0 control group 8 1.72 1.59 18.2 Control group 9 1.67 1.62 18.8 control group 10 1.88 1.50 17.4 Blank control 2.03 1.34 16.5

[0102] Based on the data analysis in Table 2, we can conclude that:

[0103] (1) After low-temperature treatment, the malondialdehyde content of Michelia figo in experimental group 1 was low and the chlorophyll content was high, indicating that the Michelia figo in experimental group 1 could still maintain a good physiological state under low-temperature treatment, with a low degree of membrane peroxidation. Moreover, after 6 months of application of mixed liquid fertilizer, the plant height increased significantly, indicating that the cold resistance agent prepared in this invention can effectively improve the cold resistance of Michelia figo in low-temperature environment, thus safely passing through the late spring cold. Combined with the later spraying of mixed liquid fertilizer, the good growth condition of Michelia figo can be guaranteed. In the blank control group, the malondialdehyde content of Michelia figo seedlings increased significantly and the chlorophyll content decreased significantly after low-temperature treatment, indicating that low-temperature treatment caused great cold damage to the seedlings, and the subsequent growth condition was poor. The plant height increase was 6.9 cm lower than that of Michelia figo seedlings in experimental group 1.

[0104] (2) In control group 2, no glycerin was added during the preparation of the anti-cold agent. In an environment of -3°C, the water is easy to freeze and form ice crystals, which cause physical damage to Michelia figo. In experimental group 1, glycerin was added and the freezing point of the water in the anti-cold agent was lowered, thus avoiding damage caused by the formation of ice crystals.

[0105] (3) In the preparation of the cold-resistant agent in control group 4, the pH was adjusted to 8 in step A. The pH of the cold-resistant agent was too alkaline, which caused alkaline stress to Michelia champaca and the malondialdehyde content increased significantly. In the preparation of the cold-resistant agent in control group 5, no double composite gel was added. The stomata of Michelia champaca were blocked, which affected normal physiological metabolism. The malondialdehyde content increased and the chlorophyll content decreased. In the preparation of the cold-resistant agent in control group 6, no ultraviolet light crosslinking was performed. Instead, the double composite gel was directly mixed. A stable network structure was not formed in the double composite gel, which affected the formation of the porous channel skeleton structure after the cold-resistant agent was sprayed, and affected normal physiological metabolism.

[0106] (4) No proline was added during the preparation of the cold-resistant agent in control group 7, no inositol was added during the preparation of the cold-resistant agent in control group 8, and no antifreeze protein was added during the preparation of the cold-resistant agent in control group 9. The enzyme activity and cell membrane stability of the plants in control groups 7, 8, and 9 were reduced, and the water in the cells was prone to crystallization, causing physical damage and poor cold resistance. This indicates that the combined effect of proline and inositol and antifreeze protein can better improve the cold resistance of Michelia and ensure the normal growth of Michelia in low temperature environment.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for improving the cold resistance of Michelia champaca, characterized in that, The method is as follows: (1) According to the weather forecast, on the evening of the day before the arrival of the cold snap, spray the sol-state anti-cold agent evenly onto the surface of the Michelia figo plant at a rate of 0.1-2 kg / plant. (2) Three days after the cold snap in spring, spray the mixed liquid fertilizer evenly on the surface of the Michelia figo plant at a rate of 0.1-1 kg / plant. Then, follow the conventional management method.

2. The method for improving the cold resistance of Michelia champaca according to claim 1, characterized in that, The cold-resistant agent in step (1) includes the following raw materials: Gelatin, mannulic acid, glycerin, hyaluronic acid, methacrylic anhydride, N-isopropylacrylamide, ammonium persulfate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, antifreeze protein, proline, inositol.

3. The method for improving the cold resistance of Michelia champaca according to claim 2, characterized in that, The method for preparing the cold-resistant agent is as follows: A: Add gelatin to 10 times its weight of water and stir to disperse. Add mannouric acid, mix well and adjust the pH to 7. Stir and mix at 100-200 r / min for 30 min, then heat to 50-60℃ and keep warm for 10-20 min. Then add glycerin, stir and mix well, and let cool naturally to room temperature to obtain gelatin gel. B: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Add methacrylic anhydride to the hyaluronic acid solution, mix well, and adjust the pH to 8. Stir the reaction at room temperature for 6-8 hours. After the reaction is complete, cool naturally to room temperature to obtain the hyaluronic acid-methacrylic anhydride complex, which is ready for use. C: Dissolve hyaluronic acid in water to prepare a 1wt% hyaluronic acid solution. Adjust the pH of the hyaluronic acid solution to 8, add N-isopropylacrylamide and mix well. Then add ammonium persulfate and mix well. Heat to 60-70℃ and stir for 4-6 hours. After the reaction is complete, cool naturally to room temperature to obtain hyaluronic acid-poly(N-isopropylacrylamide) composite gel, which is ready for use. D: Add the hyaluronic acid-methacrylic anhydride complex to the hyaluronic acid-poly(N-isopropylacrylamide) composite gel, heat to 35℃ and keep constant temperature, add 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and crosslink under ultraviolet light with a wavelength of 365nm and an intensity of 10-20mW / cm² for 20-30 minutes to obtain a double composite gel, and keep it at 35℃ for later use; E: Add proline, inositol, and antifreeze protein to water and stir to disperse. Then add the gelatin gelling agent, and then add the heat-insulating double composite gel and stir to mix evenly to obtain the antifreeze agent. Heat to 25°C to obtain the sol-state antifreeze agent, which can then be sprayed on Michelia figo.

4. The method for improving the cold resistance of Michelia champaca according to claim 3, characterized in that, In step A, the mass ratio of gelatin, mannuronic acid, and glycerin is (2-3):(0.1-0.2):(1-2).

5. A method for improving the cold resistance of Michelia champaca according to claim 4, characterized in that, In step B, the mass ratio of hyaluronic acid solution to methacrylic anhydride is (0.6-1.2):(0.018-0.036).

6. The method for improving the cold resistance of Michelia champaca according to claim 5, characterized in that, In step C, the mass ratio of hyaluronic acid solution, N-isopropylacrylamide, and ammonium persulfate is (1.5–3): (0.015–0.03): (0.0002–0.003).

7. A method for improving the cold resistance of Michelia champaca according to claim 6, characterized in that, In step D, the mass ratio of hyaluronic acid-methacrylic anhydride complex, hyaluronic acid-poly(N-isopropylacrylamide) composite gel, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is (0.6-1.2):(1.5-3):(0.001-0.002).

8. A method for improving the cold resistance of Michelia champaca according to claim 7, characterized in that, In step E, the mass ratio of gelatin gelling agent, proline, inositol, antifreeze protein, and dual composite gel is (20-30): (1-2): (1-2): (1.5-3): (2-4).

9. A method for improving the cold resistance of Michelia champaca according to claim 8, characterized in that, The method for preparing the mixed liquid fertilizer in step (2) is as follows: Mix urea and potassium dihydrogen phosphate, then add 200-300 times their weight of water and stir to dissolve. Then add carbendazim and stir to mix evenly to obtain a mixed liquid fertilizer.

10. A method for improving the cold resistance of Michelia champaca according to claim 9, characterized in that, The mass ratio of urea, potassium dihydrogen phosphate, and carbendazim in the mixed fertilizer solution is 50:20:1.

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