Cold-resistant growth-promoting biological agent and application thereof in staple food grain crops

Abstract

The invention relates to the field of agriculture, and discloses a cold-resistant growth-promoting biological agent and application thereof in staple food grain crops. The biological preparation is prepared from the following raw materials in parts by weight: 0.01 to 5 parts of plant hormone raw materials, 10 to 50 parts of organic osmotic adjusting substances, 5 to 30 parts of microbial metabolites, 0.5 to 5 parts of anti-freezing protective agents, 0.1 to 2 parts of surfactants, 0.3 to 8 parts of carriers, 0.5 to 10 parts of inorganic salts, 0.1 to 5 parts of trace elements, 1 to 15 parts of oligosaccharide substances and 20 to 80 parts of deionized water. The plant hormone raw material is brassinolide; the organic osmotic adjusting substance is betaine. Through cooperation of multiple components including brassinolide, betaine, glycerol and chitosan oligosaccharide, preventive treatment is performed on staple food grain crops before low-temperature stress, a plant stress resistance mechanism is activated, antifreeze protein synthesis is induced, cell osmotic pressure balance is maintained, and the survival ability of the crops in a low-temperature environment is remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the field of agricultural technology, in particular to a cold-resistant growth-promoting biological preparation and its application in staple crops. Background Art

[0002] Staple crops such as rice, wheat, and corn occupy a central position in global agricultural production, but their growth cycles are often threatened by environmental factors such as seasonal low temperatures, late spring frosts, and low temperatures in cold regions. Low temperature stress significantly inhibits enzyme activity within plant cells and disrupts the fluidity of cell membrane lipids, leading to reduced root water and nutrient absorption capacity, impeded photosynthesis, cell dehydration and shrinkage, and even apoptosis. This, in turn, can lead to low seedling survival rates, stagnant growth, and insufficient grain filling, severely impacting crop yield stability and quality.

[0003] Traditional cold-resistant methods such as covering for insulation and spraying of chemical agents have limitations such as high operating costs, short cold-resistant period, and easy to cause environmental pollution or pesticide residues. It is difficult to fundamentally improve the crop's own cold-resistant mechanism and growth compensation ability. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a cold-resistant growth-promoting biological agent and its application in staple food crops, which solves the limitations of the existing technology such as high operating costs, short cold resistance period, easy to cause environmental pollution or pesticide residues, and difficulty in fundamentally improving the crop's own cold resistance mechanism and growth compensation ability.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: The cold-resistant growth-promoting biological preparation comprises the following raw materials in parts by weight: 0.01-5 parts of plant hormone raw materials, 10-50 parts of organic osmotic regulating substances, 5-30 parts of microbial metabolites, 0.5-5 parts of antifreeze protectants, 0.1-2 parts of surfactants, 0.3-8 parts of carriers, 0.5-10 parts of inorganic salts, 0.1-5 parts of trace elements, 1-15 parts of oligosaccharides, and 20-80 parts of deionized water.

[0006] Preferably, the plant hormone raw material is brassinolide; The organic osmotic regulating substance is betaine; The microbial metabolite is yeast extract; The antifreeze protectant is glycerol; The surfactant is sodium lauryl sulfonate; The carrier is kaolin; The inorganic salt is potassium dihydrogen phosphate; The trace element is calcium ion; The oligosaccharide substance is chitosan oligosaccharide.

[0007] Preferably, the chitosan oligosaccharide has a deacetylation degree of ≥90% and is prepared by the following steps: dissolving chitosan in an acetic acid solution, adding a complex enzyme for hydrolysis, wherein the complex enzyme is composed of cellulase and protease in a mass ratio of 1:2, controlling the hydrolysis temperature to 45-55°C, and the reaction time to 3-6 hours. The final product is purified by ultrafiltration to obtain chitosan oligosaccharide with a molecular weight of ≤5000Da.

[0008] Preferably, the particle size distribution of the carrier is such that 80% of the particles are ≤50 μm and the specific surface area is ≥20 m² / g.

[0009] Preferably, the method for preparing the cold-resistant growth-promoting biological agent comprises the following steps: S1, dissolving the plant hormone raw materials, organic osmotic regulating substances, antifreeze protective agents, and surfactants in deionized water, and stirring in a liquid mixing tank to form a uniform solution; S2, adding the carrier, inorganic salt, and trace elements into a powder mixer for premixing, and then transferring to a high-speed shear disperser for mixing with the solution obtained in S1; S3, pre-cooling the oligosaccharide substance in a freezing treatment device and then putting it into a constant temperature reactor, then adding the microbial metabolites and keeping it warm; S4, the mixed material is homogenized and then packaged.

[0010] Preferably, the powder mixing in S2 is carried out using a planetary power mixer, and the cavity temperature is controlled to be ≤40°C during the mixing process.

[0011] Preferably, the homogenization treatment of S4 adopts a high-pressure homogenizer, and the homogenization pressure is not less than 50 MPa.

[0012] Preferably, the heat preservation treatment in S3 is carried out in a vacuum drying oven, and the moisture content of the material after treatment is ≤5%.

[0013] Preferably, the pre-cooling temperature of the oligosaccharide substance in S3 is 0-5°C.

[0014] Preferably, the cold-resistant growth-promoting biological agent is used in staple crops, and the cold-resistant growth-promoting biological agent is used for preventive treatment of crops before low temperature stress occurs.

[0015] The present invention provides a cold-resistant growth-promoting biological agent and its application in staple crops. It has the following beneficial effects: 1. The present invention uses the synergistic combination of brassinolide, betaine, glycerol, and chitosan oligosaccharide to preventively treat staple crops before low-temperature stress, thereby activating plant stress resistance mechanisms, inducing antifreeze protein synthesis, and maintaining cell osmotic pressure balance, significantly enhancing the survival ability of crops in low-temperature environments.

[0016] 2. The present invention stabilizes the cell membrane structure through betaine and glycerol, and chitosan oligosaccharides increase the membrane charge density to reduce the damage of ice crystals to the cell membrane, effectively alleviating the damage of low temperature stress to the crop cell membrane, maintaining the integrity of the cell structure to ensure the stability of plant physiological functions.

[0017] 3. The yeast extract and calcium ions contained in the present invention promote the root vitality of crops, enhance the efficiency of photosynthesis and regulate energy metabolism after low temperature stress, and cooperate with cold-resistant ingredients to achieve growth compensation under adverse conditions, thereby promoting plant height growth and biomass accumulation.

[0018] 4. The preparation process of the present invention controls the cavity temperature to ≤40°C with a planetary power mixer, the homogenization pressure of the high-pressure homogenizer to ≥50MPa, and the material moisture content to ≤5% in a vacuum drying oven. Combined with the adsorption and sustained-release properties of the kaolin carrier, it ensures that the active ingredients remain stable and evenly dispersed during the preparation process, ensuring that the preparation continues to effectively exert its cold-resistant and growth-promoting effects under low temperature stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a flow chart of the preparation method of the cold-resistant growth-promoting biological preparation. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Please see the attached Figure 1 The embodiment of the present invention provides a cold-resistant growth-promoting biological preparation, which includes the following raw materials in parts by weight: 0.01-5 parts of plant hormone raw materials, 10-50 parts of organic osmotic regulating substances, 5-30 parts of microbial metabolites, 0.5-5 parts of antifreeze protectants, 0.1-2 parts of surfactants, 0.3-8 parts of carriers, 0.5-10 parts of inorganic salts, 0.1-5 parts of trace elements, 1-15 parts of oligosaccharides, and 20-80 parts of deionized water.

[0022] The raw material of plant hormone is brassinolide; The organic osmotic regulating substance is betaine; The microbial metabolite is yeast extract; The antifreeze protectant is glycerol; The surfactant is sodium lauryl sulfate; The carrier is kaolin; The inorganic salt is potassium dihydrogen phosphate; The trace element is calcium ion; The oligosaccharide substance is chitosan oligosaccharide.

[0023] Specifically, plant hormone raw materials (brassinolide): Brassinolide is a highly effective plant growth regulator that can activate stress resistance signal pathways to enhance plants' tolerance to low temperatures, promote cell division and elongation, induce antifreeze protein synthesis and maintain cell membrane structure stability, regulate stomatal closure to reduce water loss, and coordinate plant cold resistance and growth promotion processes.

[0024] Organic osmotic regulating substance (betaine): Betaine, as a small molecule compatible solute, maintains osmotic pressure balance by accumulating in cells, preventing cell dehydration and shrinkage under low temperature stress. Its polar groups can stabilize membrane protein structure, reduce mechanical damage to cell membranes caused by ice crystals, and improve cell stability of plants in low temperature environments.

[0025] Microbial metabolites (yeast extract): Yeast extract is rich in amino acids, nucleotides, vitamins and trace elements, which can provide plants with nutritional supplements under adverse conditions, enhance photosynthesis and energy metabolism. The ingredients such as trehalose it contains can synergistically play an osmotic protection role, activate root vitality and improve nutrient absorption efficiency.

[0026] Cryoprotectant (glycerol): Glycerol, as a small molecule alcohol antifreeze, inhibits ice crystal growth by lowering the freezing point and increasing the viscosity of the solution. At the same time, it reduces the free water content in the cell by combining with water molecules through hydroxyl groups, reduces the risk of freezing, and maintains the structural integrity of the cell in a low-temperature environment.

[0027] Surfactant (sodium dodecyl sulfate): Sodium dodecyl sulfate is an anionic surfactant that can reduce the surface tension of the solution, promote uniform mixing of fat-soluble and water-soluble components, improve the stability of the preparation, and form micelles to encapsulate active substances, avoiding efficacy attenuation due to phase separation and ensuring the synergistic effect of all components.

[0028] Carrier (kaolin): Kaolin acts as a solid carrier, using its porous structure to adsorb liquid active ingredients to form a stable solid-liquid dispersion system. Its layered structure can slowly release active ingredients, prolong their retention time on the plant surface, and provide trace elements. Its particle size and specific surface area parameters directly affect the adsorption efficiency and efficacy of the preparation.

[0029] Inorganic salts (potassium dihydrogen phosphate): Potassium dihydrogen phosphate is a phosphorus-potassium compound fertilizer that can supplement phosphorus and potassium ions. Phosphorus participates in sugar metabolism to enhance frost resistance, and potassium stabilizes protein structure to prevent enzyme inactivation. The synergistic effect of the two can enhance the strength of plant root cell walls, reduce low temperature damage to the roots, and maintain metabolic balance under adverse conditions.

[0030] Trace elements (calcium ions): Calcium ions act as second messengers, transmitting stress resistance signals by activating calmodulin, inducing the expression of cold-responsive genes, and at the same time combining with cell wall pectin to enhance cell structural stability, reduce cell rupture caused by low temperatures, and improve plant cold resistance and growth capabilities by regulating the enzyme activity environment.

[0031] Oligosaccharides (chitosan oligosaccharides, degree of deacetylation ≥ 90%): Chitosan oligosaccharides are produced by hydrolysis of chitosan with complex enzymes. They are rich in amino groups and can increase the membrane charge density and reduce ice crystal attachment damage by binding to the negative charge on the cell membrane surface, thereby inducing plants to produce phytoalexins and pathogenesis-related proteins. Their degradation products can participate in cell wall repair and enhance plant antifreeze and immunity.

[0032] Deionized water: Deionized water is used as a solvent to dissolve hydrophilic ingredients and adjust the concentration of the preparation to ensure uniform dispersion of the components. Its high purity prevents metal ions from interfering with the stability of the active ingredients. The water phase ratio can be adjusted according to different spraying requirements to prepare preparations suitable for different scenarios.

[0033] The chitosan oligosaccharide has a deacetylation degree of ≥90% and is prepared by the following steps: dissolving chitosan in an acetic acid solution, adding a complex enzyme for hydrolysis, wherein the complex enzyme is composed of cellulase and protease in a mass ratio of 1:2, controlling the hydrolysis temperature at 45-55°C, and the reaction time at 3-6 hours; and finally purifying the final product by ultrafiltration to obtain chitosan oligosaccharide with a molecular weight of ≤5000Da.

[0034] Specifically, chitosan oligosaccharide is prepared by dissolving chitosan in acetic acid solution, hydrolyzing it at 45-55°C for 3-6 hours using a complex enzyme consisting of cellulase and protease in a mass ratio of 1:2, and then purified by ultrafiltration to obtain a product with a deacetylation degree ≥ 90% and a molecular weight ≤ 5000Da. The high deacetylation degree makes it rich in amino groups, which can effectively bind to the negative charge on the cell membrane surface to increase the membrane charge density and reduce ice crystal attachment damage. At the same time, it acts as an elicitor to induce plants to produce phytoalexins and pathogenesis-related proteins, and participates in cell wall repair through the degradation product glucosamine, significantly enhancing the plant's antifreeze ability and cell repair efficiency.

[0035] The particle size distribution of the carrier is 80% of the particles ≤ 50μm, and the specific surface area is ≥ 20m² / g.

[0036] Specifically, the carrier is kaolin with a particle size distribution of 80% of the particles ≤ 50μm and a specific surface area ≥ 20m² / g. Its small particle size and high specific surface area characteristics give it a strong adsorption capacity, which can fully adsorb the active ingredients to form a stable dispersion system. At the same time, it slowly releases effective substances through the porous structure, prolongs the action time on the plant surface, ensures that antifreeze protectants, plant hormones and other ingredients are evenly released and absorbed, and thereby enhances the cold-resistant and growth-promoting effect of the preparation under low temperature stress.

[0037] The present invention also provides a method for preparing a cold-resistant growth-promoting biological agent, comprising the following steps: S1, dissolving the plant hormone raw materials, organic osmotic regulating substances, antifreeze protective agents, and surfactants in deionized water, and stirring in a liquid mixing tank to form a uniform solution; S2, adding the carrier, inorganic salt, and trace elements into a powder mixer for premixing, and then transferring to a high-speed shear disperser for mixing with the solution obtained in S1; S3, pre-cooling the oligosaccharide substance in a freezing treatment device and then putting it into a constant temperature reactor, then adding the microbial metabolites and keeping it warm; S4, the mixed material is homogenized and then packaged.

[0038] The powders in S2 are mixed using a planetary power mixer, and the chamber temperature is controlled to be ≤40°C during the mixing process.

[0039] The homogenization treatment of S4 adopts a high-pressure homogenizer with a homogenization pressure of not less than 50MPa.

[0040] The insulation treatment of S3 is carried out in a vacuum drying oven, and the moisture content of the material after treatment is ≤5%.

[0041] The pre-cooling temperature of the oligosaccharide substances in S3 is 0-5°C.

[0042] Specifically, S1 dissolves plant hormone raw materials (such as brassinolide), organic osmotic regulating substances (such as betaine), antifreeze protective agents (such as glycerol) and surfactants in deionized water, and stirs them in a liquid mixing tank to form a uniform solution to achieve sufficient dissolution and dispersion of hydrophilic components; S2 premixes the carrier (such as kaolin), inorganic salts (such as potassium dihydrogen phosphate), and trace elements (such as calcium ions) in a powder mixer, and then transfers them to a high-speed shear disperser to mix with the S1 solution. The planetary power mixer controls the cavity temperature to ≤40°C to avoid high temperature destruction of the active ingredients. The high-speed shear ensures that the solid and liquid phases are evenly dispersed and form a stable Fixed suspension system; S3 pre-cools oligosaccharides (such as chitosan oligosaccharides) at 0-5°C and then places them into a constant temperature reactor. Microbial metabolites (such as yeast extract) are added and kept in a vacuum drying oven until the moisture content is ≤5%. Low-temperature pre-cooling prevents chitosan oligosaccharides from denaturing due to temperature fluctuations, while the vacuum environment accelerates water evaporation and avoids oxidation reactions, ensuring sufficient interaction between oligosaccharides and metabolites; S4 homogenizes the mixture using a high-pressure homogenizer (pressure ≥ 50MPa), using high-pressure shear force to break up particle agglomerates and uniformize the particle size distribution. Finally, the mixture is packaged to obtain a preparation with high stability and balanced release of active ingredients. This process maximizes the retention of the activity of each ingredient by precisely controlling temperature, pressure, and mixing sequence, ensuring the cold-resistant and growth-promoting efficacy of the preparation under low-temperature stress.

[0043] The present invention also provides the use of a cold-resistant growth-promoting biological agent in staple crops. The cold-resistant growth-promoting biological agent is used for preventive treatment of crops before low temperature stress occurs.

[0044] Specifically, when the cold-resistant growth-promoting biological agent of the present invention is applied to staple crops (such as rice, wheat, and corn), preventive spraying before low-temperature stress occurs can activate the plant's stress resistance mechanism in advance: brassinolide induces antifreeze gene expression, betaine and glycerol maintain cellular water balance through osmotic regulation, chitosan oligosaccharides stimulate phytoalexin synthesis, yeast extract supplements energy metabolism substrates, and the kaolin slow-release system ensures the continued action of the active ingredients. Preventive treatment allows crops to form a complete cold-resistant protective barrier before the onset of low temperatures, significantly reducing cell membrane damage (a decrease in relative leaf conductivity), improving low-temperature survival rates, and promoting growth in plant height and fresh weight after growth resumes, thereby ensuring stable production and increased income for staple crops in cold regions or under adverse conditions such as late spring frosts, providing effective technical support for safe food production.

[0045] The following is an introduction with reference to specific embodiments: Example

[0046] Raw material composition (parts by weight) The plant hormone raw material is 0.01 parts of brassinolide, the organic osmotic regulating substance is 10 parts of betaine, the microbial metabolite is 5 parts of yeast extract, the antifreeze protectant is 0.5 parts of glycerol, the surfactant is 0.1 parts of sodium dodecyl sulfate, the carrier is 0.3 parts of kaolin (particle size distribution 80% particles ≤ 50μm, specific surface area ≥ 20m² / g), the inorganic salt is 0.5 parts of potassium dihydrogen phosphate, the trace element is 0.1 parts of calcium ions, the oligosaccharide substance is 1 part of chitosan oligosaccharide (deacetylation degree ≥ 90%, molecular weight ≤ 5000Da, by dissolving chitosan in acetic acid solution, adding a complex enzyme consisting of cellulase and protease in a mass ratio of 1:2, hydrolyzing at 45°C for 3 hours, and ultrafiltration purification), and 80 parts of deionized water.

[0047] Preparation method S1: Dissolve 0.01 parts of brassinolide, 10 parts of betaine, 0.5 parts of glycerol, and 0.1 parts of sodium lauryl sulfate in 80 parts of deionized water and stir in a liquid mixing tank to form a uniform solution; S2: 0.3 parts of kaolin, 0.5 parts of potassium dihydrogen phosphate, and 0.1 parts of calcium ions were pre-mixed in a powder mixer, and then transferred to a high-speed shear disperser to mix with the solution obtained in S1; S3: 1 part of chitosan oligosaccharide was pre-cooled to 0°C in a freezing treatment device and then placed in a constant temperature reactor. 5 parts of yeast extract were added and the mixture was kept warm in a vacuum drying oven until the moisture content of the material was ≤5%; S4: The mixed material is homogenized by a high-pressure homogenizer (homogenizing pressure 50 MPa) and then packaged. Example

[0048] Raw material composition (parts by weight) The plant hormone raw materials are 5 parts of brassinolide, the organic osmotic regulating substance is 50 parts of betaine, the microbial metabolite is 30 parts of yeast extract, the antifreeze protectant is 5 parts of glycerol, the surfactant is 2 parts of sodium dodecyl sulfate, the carrier is 8 parts of kaolin (particle size distribution 80% particles ≤ 50μm, specific surface area ≥ 20m² / g), the inorganic salt is 10 parts of potassium dihydrogen phosphate, the trace element is 5 parts of calcium ion, the oligosaccharide substance is 15 parts of chitosan oligosaccharide (deacetylation degree ≥ 90%, molecular weight ≤ 5000Da, by dissolving chitosan in acetic acid solution, adding a complex enzyme consisting of cellulase and protease in a mass ratio of 1:2, hydrolyzing at 55°C for 6 hours, and ultrafiltration purification), and 20 parts of deionized water.

[0049] Preparation method S1: Dissolve 5 parts of brassinolide, 50 parts of betaine, 5 parts of glycerol, and 2 parts of sodium lauryl sulfate in 20 parts of deionized water and stir in a liquid mixing tank to form a uniform solution; S2: 8 parts of kaolin, 10 parts of potassium dihydrogen phosphate, and 5 parts of calcium ions were pre-mixed in a powder mixer, and then transferred to a high-speed shear disperser to mix with the solution obtained in S1; S3: 15 parts of chitosan oligosaccharide were pre-cooled to 5°C in a freezing treatment device and then placed in a constant temperature reactor. 30 parts of yeast extract were added and the mixture was kept warm in a vacuum drying oven until the moisture content of the material was ≤5%; S4: The mixed material is homogenized by a high-pressure homogenizer (homogenizing pressure 60MPa) and then packaged. Example

[0050] Raw material composition (parts by weight) The plant hormone raw materials are 2 parts of brassinolide, the organic osmotic regulating substance is 30 parts of betaine, the microbial metabolite is 15 parts of yeast extract, the antifreeze protectant is 2 parts of glycerol, the surfactant is 1 part of sodium dodecyl sulfate, the carrier is 4 parts of kaolin (particle size distribution 80% particles ≤ 50μm, specific surface area ≥ 20m² / g), the inorganic salt is 5 parts of potassium dihydrogen phosphate, the trace element is 2 parts of calcium ion, the oligosaccharide substance is 8 parts of chitosan oligosaccharide (deacetylation degree ≥ 90%, molecular weight ≤ 5000Da, by dissolving chitosan in acetic acid solution, adding a complex enzyme consisting of cellulase and protease in a mass ratio of 1:2, hydrolyzing at 50°C for 4.5 hours, and ultrafiltration purification), and 50 parts of deionized water.

[0051] Preparation method S1: Dissolve 2 parts of brassinolide, 30 parts of betaine, 2 parts of glycerol, and 1 part of sodium lauryl sulfate in 50 parts of deionized water and stir in a liquid mixing tank to form a uniform solution; S2: Add 4 parts of kaolin, 5 parts of potassium dihydrogen phosphate, and 2 parts of calcium ions into a planetary power mixer for premixing (control the chamber temperature ≤ 40°C during the mixing process), and then transfer to a high-speed shear disperser to mix with the solution obtained in S1; S3: 8 parts of chitosan oligosaccharide were pre-cooled to 2°C in a freezing treatment device and then placed in a constant temperature reactor. 15 parts of yeast extract were added and the mixture was kept warm in a vacuum drying oven until the moisture content of the material was ≤5%; S4: The mixed material is homogenized by a high-pressure homogenizer (homogenizing pressure 55MPa) and then packaged.

[0052] Comparative Example Comparative Example 1 (without plant hormone raw materials) The difference from Example 3 is that 2 parts of brassinolide are removed from the raw material composition, and the remaining raw materials, parts, and preparation method are the same as those in Example 3.

[0053] Comparative Example 2 (no oligosaccharides) The difference from Example 3 is that 8 parts of chitosan oligosaccharide are removed from the raw material composition, and the remaining raw materials, parts, and preparation methods are consistent with those in Example 3.

[0054] Comparative Example 3 (without antifreeze protectant) The difference from Example 3 is that 2 parts of glycerol are removed from the raw material composition, and the remaining raw materials, parts, and preparation method are the same as those in Example 3.

[0055] Comparative Example 4 (Carrier Particle Size Does Not Meet Standards) The difference from Example 3 is that the carrier is changed to kaolin with a particle size distribution of 80% particles > 50 μm and a specific surface area < 20 m² / g. The remaining raw materials, parts, and preparation method are the same as those in Example 3.

[0056] Comparative Example 5 (Insufficient Deacetylation Degree of Chitosan Oligosaccharide) The difference from Example 3 is that the oligosaccharide substance is replaced with chitosan oligosaccharide with a deacetylation degree of 80% (the other preparation conditions are the same), and the other raw materials, parts, and preparation method are consistent with Example 3.

[0057] Table 1: Comparative effect table of embodiments and comparative examples. Comparison project explanation: Survival rate (%): This represents the percentage of seedlings that survived three days of growth after low-temperature stress (72 hours at 4°C). It is a core indicator of a plant's cold tolerance. A higher survival rate indicates a more robust cryoprotective effect on plant cells and tissues, effectively mitigating low-temperature-induced apoptosis and physiological dysfunction.

[0058] Detection method: After the low temperature treatment, the number of seedlings in each group with upright stems, no severe wilting of leaves and active heart leaves was counted. The percentage of surviving plants to the total number of plants in each group was the survival rate (30 plants in each group, the average value was taken).

[0059] ≥80%: The preparation has a complete cold protection mechanism, which can significantly enhance the plant's tolerance to low temperatures and is suitable for use on crops in cold regions.

[0060] 60%-80%: Moderate cold resistance, can provide protection in mild low temperature environments, but the effect is limited in extreme low temperatures.

[0061] <60%: Insufficient cold protection and severe damage to plant cells, indicating that key ingredients of the preparation are missing or the ratio is unreasonable.

[0062] Leaf relative conductivity (mS / cm): This metric measures the conductivity of leaf cell exudate after low-temperature stress to quantify the extent of cell membrane damage. Low temperatures disrupt cell membrane integrity, leading to electrolyte leakage. Higher conductivity indicates greater membrane permeability and more severe damage. This indicator is negatively correlated with plant cold tolerance.

[0063] Detection method: Take the leaves after low-temperature treatment, wash the surface impurities with deionized water, cut into 1 cm² small pieces, add 20 ml of deionized water, vacuum filter for 5 minutes, and let it stand for 30 minutes. Use a conductivity meter to measure the conductivity of the extract (μS / cm). At the same time, measure the saturated conductivity after boiling for 5 minutes. Relative conductivity = (initial conductivity / saturated conductivity) × 100%.

[0064] <10mS / cm: The cell membrane is slightly damaged and the preparation effectively maintains the stability of the membrane structure.

[0065] 10-15mS / cm: Membrane damage is moderate, the plant's cold resistance is limited, and it needs to rely on exogenous protective ingredients.

[0066] >15mS / cm: The membrane system is severely damaged, cell function is disordered, and the preparation lacks core antifreeze protection components.

[0067] Plant Height Growth Rate (%): This refers to the percentage increase in plant height three days after recovery from low-temperature stress compared to pre-treatment, reflecting the activity of the plant's apical meristem and stem elongation capacity. A higher growth rate indicates a more significant effect of the agent in alleviating low-temperature growth inhibition and promoting vegetative organ development.

[0068] Detection method: Measure the vertical height from the base of the seedling to the tip of the leaf (initial plant height) before low-temperature treatment, and measure again after growth recovery. Plant height growth rate = (final plant height - initial plant height) / initial plant height × 100% (measure 10 plants in each group and take the average value).

[0069] ≥15%: The preparation synergistically promotes cell division and elongation, effectively offsetting the growth inhibition caused by low temperature.

[0070] 10%-15%: The growth promotion effect is limited and can only partially restore low temperature damage.

[0071] <10%: Growth is basically stagnant, lack of growth-promoting active ingredients or imbalance between cold resistance and growth-promoting functions.

[0072] Fresh Weight Increase (%): This measure assesses biomass accumulation efficiency by measuring the change in overall seedling fresh weight. It comprehensively reflects the root system's ability to absorb water and nutrients, photosynthetic intensity, and cellular metabolic activity. A higher fresh weight increase indicates that the formulation is effective in maintaining normal plant metabolism while enhancing stress resistance.

[0073] Detection method: Weigh the fresh weight of the whole seedling (including the root system) before low-temperature treatment (initial fresh weight) and after recovery of growth. Fresh weight increase rate = (final fresh weight - initial fresh weight) / initial fresh weight × 100% (5 plants were randomly selected from each group, washed and dried, and then weighed to take the average value).

[0074] Grading standards and significance: ≥20%: The preparation significantly improves the carbon and nitrogen metabolism level of plants and promotes the accumulation of photosynthetic products.

[0075] 15%-20%: Metabolic activity is partially restored and growth potential is moderate.

[0076] <15%: Substance synthesis is blocked, energy metabolism is disordered, and low temperature causes irreversible damage.

[0077] Example and comparative example effect discussion: Cold protection effect: Examples 1-3 all employed a full-ingredient synergistic formulation (brassinolide + betaine + glycerol + chitosan oligosaccharide, etc.), achieving survival rates of 78%-88%. Example 2 (the upper limit of raw materials) achieved the highest survival rate (88%) due to the synergistic effect of high concentrations of brassinolide (5 parts) and chitosan oligosaccharide (15 parts), demonstrating that highly active ingredients can enhance plant stress resistance gene expression and osmotic regulation. Example 3 (intermediate value) achieved a survival rate of 85%, balancing cost and effectiveness, verifying the rationality of the formulation concentration range.

[0078] Comparative Examples 1-3, lacking core ingredients (brassinolide, chitosan oligosaccharide, and glycerol), had survival rates of only 58%-70%. Comparative Example 3 (no glycerol) had the lowest survival rate (58%), demonstrating the irreplaceable role of cryoprotectants in maintaining cellular osmotic pressure and preventing ice crystal damage. Comparative Example 1 (no brassinolide) had a survival rate of 62%, confirming the critical role of plant hormones in activating cold-resistance signaling pathways.

[0079] Cell membrane damage control (leaf relative conductivity): The conductivity of Examples 1-3 was 7.8-9.5 mS / cm, significantly lower than that of the comparative example (10.3-16.8 mS / cm), demonstrating that the full-ingredient formula effectively maintains cell membrane integrity. Example 2 (conductivity 7.8 mS / cm) minimized membrane damage due to the dual permeability protection provided by betaine (50 parts) and glycerol (5 parts). The conductivities of Comparative Examples 1 and 3 exceeded 15 mS / cm, reflecting the severe damage to the membrane system caused by the absence of core ingredients, which positively correlated with the survival rate data.

[0080] The conductivity of comparative examples 4-5 (the carrier particle size does not meet the standard and the deacetylation degree of chitosan oligosaccharide is insufficient) is 10.3-11.4 mS / cm, indicating that the carrier adsorption efficiency and chitosan oligosaccharide activity (deacetylation degree ≥ 90%) affect the release of active ingredients, indirectly aggravating membrane damage.

[0081] Growth promotion effect (plant height and fresh weight growth rate): Examples 1-3 achieved plant height increases of 16.2%-19.8% and fresh weight increases of 20.1%-24.5%, significantly outperforming the comparative examples (9.8%-17.3%). Example 2 (upper limit) achieved optimal growth indicators due to the high concentration of yeast extract (30 parts) and calcium ions (5 parts), which promoted root development and photosynthate accumulation. Comparative Examples 1-3, lacking growth-promoting and cold-resistant synergistic ingredients, saw plant height and fresh weight increases of less than 15%, demonstrating that cold-resistant or growth-promoting properties alone are insufficient to compensate for growth under adverse conditions.

[0082] In Comparative Example 4 (the carrier particle size does not meet the standard), the growth index is close to that of Example 1 (lower limit) due to the decreased sustained-release efficiency of the active ingredient, but is still lower than that of Example 3, which confirms the influence of the carrier performance on the stability of the formulation; in Comparative Example 5 (the deacetylation degree of chitosan oligosaccharide is 80%), the growth-promoting effect is weaker than that of Example 3 due to the reduction of active groups, indicating that the deacetylation degree of chitosan oligosaccharide is positively correlated with its stress resistance induction ability.

[0083] Carrier and ingredients work synergistically: Examples 1-3 used kaolin with a particle size of ≤50 μm and a specific surface area of ​​≥20 m² / g. Their high adsorption capacity ensured uniform dispersion of the active ingredients, resulting in superior survival rates, conductivity, and growth indicators compared to Comparative Example 4 (standard kaolin), demonstrating the critical role of carrier physical parameters in formulation efficacy. Furthermore, Example 3 employed a planetary power mixer (chamber temperature ≤40°C) and a high-pressure homogenizer (55 MPa) to prevent high-temperature degradation of the active ingredients and maintain the bioactivity of the chitosan oligosaccharide and yeast extract. However, the Comparative Example lacked strict control over process parameters, indirectly compromising efficacy.

[0084] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A cold-resistant growth-promoting biological agent, characterized in that: The invention comprises the following raw materials in parts by weight: 0.01-5 parts of plant hormone raw materials, 10-50 parts of organic osmotic regulating substances, 5-30 parts of microbial metabolites, 0.5-5 parts of antifreeze protectants, 0.1-2 parts of surfactants, 0.3-8 parts of carriers, 0.5-10 parts of inorganic salts, 0.1-5 parts of trace elements, 1-15 parts of oligosaccharides, and 20-80 parts of deionized water.

2. The cold-resistant growth-promoting biological preparation according to claim 1, characterized in that The plant hormone raw material is brassinolide; The organic osmotic regulating substance is betaine; The microbial metabolite is yeast extract; The antifreeze protectant is glycerol; The surfactant is sodium lauryl sulfonate; The carrier is kaolin; The inorganic salt is potassium dihydrogen phosphate; The trace element is calcium ion; The oligosaccharide substance is chitosan oligosaccharide.

3. The cold-resistant growth-promoting biological preparation according to claim 2, characterized in that The chitosan oligosaccharide has a deacetylation degree of ≥90% and is prepared by the following steps: dissolving chitosan in an acetic acid solution, adding a complex enzyme for hydrolysis, wherein the complex enzyme is composed of cellulase and protease in a mass ratio of 1:2, controlling the hydrolysis temperature at 45-55°C, and the reaction time at 3-6 hours; and finally purifying the final product by ultrafiltration to obtain chitosan oligosaccharide with a molecular weight of ≤5000Da.

4. The cold-resistant growth-promoting biological preparation according to claim 1, characterized in that The particle size distribution of the carrier is such that 80% of the particles are ≤50 μm and the specific surface area is ≥20 m² / g.

5. A method for preparing a cold-resistant growth-promoting biological agent, for use in the cold-resistant growth-promoting biological agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, dissolving the plant hormone raw materials, organic osmotic regulating substances, antifreeze protective agents, and surfactants in deionized water, and stirring in a liquid mixing tank to form a uniform solution; S2, adding the carrier, inorganic salt, and trace elements into a powder mixer for premixing, and then transferring to a high-speed shear disperser for mixing with the solution obtained in S1; S3, pre-cooling the oligosaccharide substance in a freezing treatment device and then putting it into a constant temperature reactor, then adding the microbial metabolites and keeping it warm; S4, the mixed material is homogenized and then packaged.

6. The method for preparing the cold-resistant growth-promoting biological agent according to claim 5, characterized in that: The powders in S2 are mixed using a planetary power mixer, and the cavity temperature is controlled to be ≤40°C during the mixing process.

7. The method for preparing the cold-resistant growth-promoting biological agent according to claim 5, characterized in that: The homogenization treatment of S4 adopts a high-pressure homogenizer, and the homogenization pressure is not less than 50MPa.

8. The method for preparing the cold-resistant growth-promoting biological agent according to claim 5, characterized in that: The heat preservation treatment of S3 is carried out in a vacuum drying oven, and the moisture content of the material after treatment is ≤5%.

9. The method for preparing the cold-resistant growth-promoting biological agent according to claim 5, characterized in that: The pre-cooling temperature of the oligosaccharide substance in S3 is 0-5°C.

10. Application of a cold-resistant growth-promoting biological agent in staple crops, the cold-resistant growth-promoting biological agent according to any one of claims 1 to 4 being characterized in that: The cold-resistant growth-promoting biological preparation is used for preventive treatment of crops before low-temperature stress occurs.

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