Method for improving seedling stage waterlogging tolerance of sesame by using melatonin and application thereof

Abstract

This invention discloses a method for enhancing the waterlogging tolerance of sesame seedlings using melatonin and its application. The method utilizes alginate oligosaccharides, boric acid, melatonin, calcium chloride, polyalkylene oxide-modified heptamethyltrisiloxane, and deionized water as raw materials. Through a stepped temperature control and multi-channel synchronous assembly process, a cross-linked network is constructed to physically encapsulate melatonin, effectively shielding its photosensitive area to slow down UV photolysis. This method features low surface tension, allowing easy penetration into microcracks and stomata on plant leaves. After a small amount of water evaporates, an in-situ sol-gel phase transition is triggered, forming a hydrogel microfilm that adheres to the leaf tissue, resisting natural rainfall washout and achieving slow-release of melatonin. This invention effectively solves the problems of easy loss and photolysis of conventional exogenous agents, and the components synergistically activate basic plant immune and antioxidant pathways, systematically enhancing the waterlogging tolerance of sesame seedlings.

Description

Technical Field

[0001] This invention relates to the fields of agricultural chemicals and crop cultivation technology, specifically to a method and its application for improving the waterlogging resistance of sesame seedlings using melatonin. Background Technology

[0002] Sesame is an important oilseed crop in my country. It is highly nutritious, with seeds rich in fat, protein, total sugar, and trace elements. It is believed to have benefits such as strengthening the kidneys and lowering cholesterol. As an important oilseed crop, sesame's root system is highly sensitive to changes in soil moisture. During the seedling stage, sesame seedlings are often subjected to waterlogging stress caused by continuous rainfall or poor drainage. Waterlogging can lead to localized soil hypoxia, interfering with normal respiration and metabolism, causing the accumulation of reactive oxygen species and membrane lipid peroxidation damage, thus inhibiting plant growth.

[0003] To alleviate crop waterlogging stress, the application of exogenous plant growth regulators or stress-resistance inducers is a common intervention in agricultural production. Melatonin, as an antioxidant and biosignaling molecule, plays a certain role in regulating plant physiology and resisting abiotic stress. Conventional single-aqueous formulations have high surface tension, making it difficult for the sprayed solution to penetrate the hydrophobic waxy layer on the surface of sesame leaves, and most of it remains on the leaf surface. Under rainfall conditions, the solution adhering to the leaf surface is easily washed away by rainwater, resulting in a low actual leaf retention rate. In addition, melatonin molecules are prone to photolysis when exposed to natural light and ultraviolet light, leading to the inactivation of the active ingredient. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method and its application for improving the waterlogging resistance of sesame seedlings using melatonin, thereby solving the problem that melatonin molecules are easily deactivated by ultraviolet light when exposed to the environment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin is prepared from the following raw materials in parts by weight: 0.8 to 1.5 parts alginate oligosaccharide; 3.0 to 8.0 parts boric acid; 80 to 120 parts melatonin; 3.0 to 8.0 parts calcium chloride; 0.3 to 0.8 parts polyalkylene oxide modified heptamethyltrisiloxane; 10 to 15 parts anhydrous ethanol for pre-dissolving; and the balance being deionized water and an appropriate amount of pH adjuster.

[0006] By adopting the above technical solution, synergistic effects are achieved at both the microscopic and macroscopic physicochemical levels through the use of specific types and proportions of polysaccharide molecules, inorganic salts, plant active ingredients, and surfactants. The specific reaction and structural assembly process is as follows: Borate ions undergo a reversible dynamic condensation reaction with adjacent free diol structures on the alginate oligosaccharide chain to construct a weakly cross-linked primary network backbone. Free calcium ions coordinate with active sites such as free carboxyl groups on the alginate oligosaccharide chain, inducing the polysaccharide chain to spontaneously generate an egg-box structure contraction. Within the same spatiotemporal window of polysaccharide chain contraction and cross-linking, free melatonin monomers are physically embedded in the micro-cavities enclosed by a dynamic cross-linking network by utilizing multiple hydrogen bonds and the steric hindrance of the polymer electron cloud. Polyalkylene oxide-modified heptamethyltrisiloxane is oriented at the fluid interface, significantly reducing the macroscopic static surface tension of the fluid. Therefore, the following beneficial effects are achieved: the physicochemical exposure state of free melatonin is completely changed; the dense ternary supramolecular topology provides effective spatial masking for its indole ring conjugated system, significantly prolonging the photolysis half-life of the active substance; and the extremely low surface tension provides the necessary kinetic conditions for the fluid to cross the hydrophobic waxy layer of plant leaves and penetrate into leaf microcracks and stomatal cavities.

[0007] Preferably, the surface tension of the compound aqueous solution is 20 to 22 mN / m, and the final pH value of the compound aqueous solution is stable between 6.0 and 6.5.

[0008] By adopting the above technical solution, it can be ensured that the fluid has the ultimate wetting power to penetrate into the plant stomata, and the slightly acidic environment can maintain the long-term stability of the reversible covalent bond of boric acid and the coordination bond of calcium ions, preventing the formulation from prematurely gelling or the degradation and failure of active ingredients during storage.

[0009] Preferably, the weight-average molecular weight of alginate oligosaccharides ranges from 1500 Da to 3000 Da.

[0010] By adopting the above technical solution, the macromolecular framework is guaranteed to have both excellent water solubility and permeability and film-forming mechanical strength, avoiding the problem that the molecular weight is too large, resulting in high fluid viscosity that prevents atomization and penetration into the pores, or that the molecular weight is too small, resulting in a loose cross-linking network and a lack of drug encapsulation capacity.

[0011] Preferably, the alginate oligosaccharide is prepared by the following specific enzymatic hydrolysis process: sodium alginate powder is dissolved in deionized water at a mass concentration of 1.5% to 2.0%, and the pH is adjusted to 7.0 to 7.5 to prepare a substrate solution; alginate lyase is added at a ratio of 50 to 100 U / g substrate, and the solution is hydrolyzed at a constant temperature of 40°C to 45°C and a rotation speed of 120 to 150 rpm for 4 to 6 hours; after the hydrolysis is completed, the temperature is raised to 95°C and held for 15 minutes to inactivate the enzyme, and the supernatant is collected by cooling and centrifugation; 3 to 4 times the volume of anhydrous ethanol is slowly added for precipitation, and the precipitate is washed with anhydrous ethanol, pre-frozen, and vacuum freeze-dried to obtain the final product.

[0012] By adopting the above technical solution, the targeted cleavage of specific glycosidic bonds by alginate lyase can be used to accurately and controllably obtain target polysaccharide fragments with extremely narrow molecular weight distribution, ensuring the consistency and reproducibility of the node density in subsequent supramolecular network assembly.

[0013] A method for improving the waterlogging resistance of sesame seedlings using melatonin includes the following steps: S1. Add deionized water to the reactor, then add alginate oligosaccharide powder and boric acid in sequence, and stir evenly. Use a pH adjuster to adjust the initial pH of the substrate solution to a neutral-acidic or alkaline range, turn on the heating to raise the temperature, and stir at a constant temperature to form a low-viscosity homogeneous fluid. S2. Cool the reaction by introducing cold water into the jacket of the reactor; dissolve melatonin in anhydrous ethanol and calcium chloride in deionized water in advance, and use a constant flow pump to slowly add the melatonin ethanol solution and calcium chloride aqueous solution to the reactor simultaneously. After the addition is completed, maintain the temperature and continue stirring to promote the completion of topological network contraction and hydrogen bonding with melatonin. S3. Use a pH adjuster to adjust the pH of the substrate solution back to slightly acidic, add polyalkylene oxide modified heptamethyltrisiloxane, add deionized water to the target volume, increase the stirring speed to perform high-shear homogenization, and filter to obtain melatonin compound aqueous solution.

[0014] By adopting the above technical solution, the thermodynamic obstacles in the assembly process were overcome by using stepped temperature control and multi-channel synchronous assembly technology. The high-temperature pre-crosslinking stage breaks the activation energy barrier of the dynamic condensation of boric acid and diol structures, promoting the full expansion of the network skeleton; the subsequent forced cooling gives the macromolecular chains the physical kinetic energy for contraction and folding. At this time, free calcium ions and melatonin molecules are introduced simultaneously through dual channels, achieving a precise match between ion coordination contraction and the physical embedding of drug molecules on the time axis, avoiding macroscopic flocculation and precipitation, and constructing a stable fluid that is microscopically dense and macroscopically homogeneous.

[0015] Preferably, in S1, the initial pH value is adjusted to 6.8 to 7.2, the temperature is raised to 55°C to 65°C, the constant temperature stirring time is 40 to 60 minutes, and the stirring speed is 150 to 250 rpm.

[0016] By adopting the above technical solution, a suitable weakly neutral environment and abundant heat energy input are maintained, which maximizes the dissociation of borate ions and their pre-bonding with the cis-diol structure of sugar rings.

[0017] Preferably, in S2, the cooling rate is controlled at 2°C to 3°C / min, and the target cooling temperature is 30°C to 35°C; the melatonin ethanol solution and the calcium chloride aqueous solution are added simultaneously using a dual-channel constant flow pump, with a constant drop rate of 8 mL / min, and the constant temperature stirring time after the drop is completed is 30 to 45 minutes.

[0018] By adopting the above technical solution, the contraction rate of the macromolecular chain under the temperature gradient is precisely set, so that the closure speed of the spatial cavity is highly matched with the diffusion rate of melatonin molecules, ensuring high drug loading and strong binding.

[0019] Preferably, in S3, the pH value is lowered and stabilized at 6.0 to 6.5, the stirring speed for high-shear homogenization is 300 to 400 rpm, and the homogenization time is 15 to 20 minutes.

[0020] By adopting the above technical solution, high mechanical shear force is used to forcibly disperse and orient surfactant molecules and adsorb them at the interface, thereby blocking non-specific aggregation between macromolecules and stabilizing the mesoscopic physical phase of the final fluid.

[0021] A method for enhancing the waterlogging resistance of sesame seedlings using melatonin involves spraying the above-mentioned compound aqueous solution evenly onto both sides of the sesame seedling leaves using a spraying device when the sesame seedlings have grown to the 4 true leaf stage and are facing continuous rainfall or the initial stage of waterlogging. The amount of the agent applied should be such that the leaves are fully moistened without obvious dripping.

[0022] By adopting the above technical solution, the application of pesticides is targeted at the leaf age window during which sesame is most sensitive to external environmental stress. High contact surface area is achieved by spraying, which stimulates the plant's endogenous immune awakening and multi-pathway stress resistance physiological response.

[0023] Preferably, after the compound aqueous solution penetrates into the microcracks in the cuticle and the lower cavity of the stomatal cavities of sesame leaves, accompanied by a small amount of water evaporation, the concentration of medium and large molecules crosses the critical gel concentration, thereby triggering an in-situ sol-gel phase transition. This results in the spontaneous formation of a viscoelastic hydrogel microfilm within the microtopological structure of the leaf surface, which resists the erosion of natural rainfall and achieves a long-term sustained release of melatonin into the mesophyll cells.

[0024] By adopting the above technical solution, an intelligent environmental response is achieved, where the aqueous solution maintains extremely low viscosity in non-target areas to facilitate spraying and spreading, while spontaneously solidifying upon entering the target physiological space. This hydrogel microfilm can solidify the internal structure of leaves in situ, completely solving the problem of pesticide loss and shedding under extreme heavy rainfall conditions. It ensures a constant low concentration of active substances slowly released and penetrating into the tissue over time, continuously amplifying the transcription level of downstream defense genes and systematically improving the crop's flood resistance limit.

[0025] This invention provides a method for improving the waterlogging resistance of sesame seedlings using melatonin and its application. It has the following beneficial effects: 1. This invention constructs a dense cross-linked network structure internally by combining alginate oligosaccharides, boric acid, and calcium ions with a step-temperature controlled preparation process. Utilizing multiple hydrogen bonds and the steric hindrance of polymer segments, free melatonin molecules are physically embedded within the cavities of the network micro-regions. This shields the photosensitive conjugated region of melatonin, reduces the photodegradation rate caused by ultraviolet radiation, and prolongs the half-life and field persistence of the core active substance.

[0026] 2. This invention reduces the surface tension by adding polyalkylene oxide-modified heptamethyltrisiloxane, enabling it to penetrate into the microcracks and substomatal cavities of plant leaves. After entering the stomatal microenvironment, the concentration of macromolecules increases and exceeds the critical gel concentration along with water evaporation, triggering an in-situ sol-gel phase transition. The formed hydrogel microfilm can effectively adhere to the interior of the leaf structure, resisting mechanical erosion and dissolution loss caused by natural rainfall, and achieving the slow release of active substances into the mesophyll tissue.

[0027] 3. The alginate oligosaccharide of this invention, as an endogenous oligosaccharide analog in plants, triggers a basic immune response. The released calcium ions act as second messengers in cell signal transduction, and the two interact with the melatonin-mediated antioxidant pathway. The combined intervention of the components increases the activity of antioxidant enzymes such as superoxide dismutase, reduces the accumulation of reactive oxygen species and malondialdehyde in cells, maintains the metabolic homeostasis and osmotic balance of plant cells under waterlogging stress, and systematically improves the waterlogging tolerance of sesame seedlings. Attached Figure Description

[0028] Figure 1 This is a step diagram of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0031] Melatonin, chemically known as N-acetyl-5-methoxytryptamine, has a purity of ≥99.0%. Sodium alginate, whose polymer backbone is a linear block copolymer composed of repeating units β-D-mannuronic acid and α-L-guluronic acid linked by 1,4-glycosidic bonds, has a purity ≥90.0%; Alginate lyase, with an enzyme activity ≥10000U / g, is used for the directed catalytic cleavage of glycosidic bonds in sodium alginate molecules. Anhydrous calcium chloride, industrial grade, purity ≥96.0%; Boric acid, industrial grade, purity ≥99.5%; The cloud point of a 0.1% aqueous solution of polyalkylene oxide modified heptamethyltrisiloxane is limited to 40°C to 50°C, and the surface tension at 25°C is limited to 20 mN / m to 22 mN / m. Alginate oligosaccharides are low molecular weight products prepared by enzymatic hydrolysis of sodium alginate, wherein the repeating units and their arrangement are maintained as linear block copolymers of β-D-mannuronic acid and α-L-guluronic acid linked by 1,4-glycosidic bonds.

[0032] Preparation Example 1: This preparation example provides a method for preparing alginate oligosaccharides with a median weight-average molecular weight, comprising the following steps: Accurately weigh 18.0 g of sodium alginate powder and dissolve it in 1000 mL of deionized water to prepare a substrate solution with a mass concentration of approximately 1.8%. Adjust the pH of the substrate solution to 7.2 using a 0.1 mol / L sodium hydroxide aqueous solution. Add alginate lyase at a ratio of 75 U / g substrate. Place the reaction in a constant temperature water bath shaker at 42°C and maintain the enzymatic hydrolysis at 135 rpm for 5 hours. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 95°C and maintain it for 15 minutes. The alginate lyase protein was denatured and inactivated by centrifugation at 9000 rpm for 15 minutes after natural cooling to room temperature. The supernatant was collected. Anhydrous ethanol was slowly added dropwise to the supernatant for alcohol precipitation. After standing overnight, the precipitate was collected by filtration. The precipitate was washed twice with anhydrous ethanol and then pre-frozen in a -80°C freezer. Finally, it was freeze-dried under vacuum for 48 hours to obtain alginate oligosaccharide powder with a weight average molecular weight of 2200 Da.

[0033] Preparation Example 2: This preparation example provides a method for preparing alginate oligosaccharides with a lower weight-average molecular weight, including the following steps: Accurately weigh 15.0 g of sodium alginate powder and dissolve it in 1000 mL of deionized water to prepare a substrate solution with a mass concentration of approximately 1.5%. Adjust the pH of the substrate solution to 7.5 using a 0.1 mol / L sodium hydroxide aqueous solution. Add alginate lyase at a ratio of 100 U / g substrate. Place the reaction in a constant temperature water bath shaker at 45°C and maintain the enzymatic hydrolysis at 150 rpm for 6 hours. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 95°C and maintain it at a constant temperature for 15 hours. Centrifuge for 15 minutes to denature and inactivate the alginate lyase protein; after naturally cooling to room temperature, centrifuge at 10,000 rpm for 15 minutes and collect the supernatant; slowly add 4 times the volume of anhydrous ethanol to the supernatant for alcohol precipitation, let stand overnight, and collect the precipitate by vacuum filtration; wash the precipitate 3 times with anhydrous ethanol, then pre-freeze it in a -80℃ freezer, and finally freeze-dry it under vacuum for 48 hours to obtain alginate oligosaccharide powder with a weight average molecular weight of 1500 Da.

[0034] Preparation Example 3 provides a method for preparing alginate oligosaccharides with the upper limit of weight-average molecular weight, including the following steps: Accurately weigh 20.0 g of sodium alginate powder and dissolve it in 1000 mL of deionized water to prepare a substrate solution with a mass concentration of approximately 2.0%. Adjust the pH of the substrate solution to 7.0 using 0.1 mol / L dilute hydrochloric acid or sodium hydroxide aqueous solution. Add alginate lyase at a ratio of 50 U / g substrate. Place the reaction in a constant temperature water bath shaker at 40°C and maintain the enzymatic hydrolysis at 120 rpm for 4 hours. After the enzymatic hydrolysis is completed, rapidly raise the temperature to 95°C and maintain it at a constant temperature. The alginate lyase protein was denatured and inactivated for 15 minutes. After cooling to room temperature, the mixture was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. Three times the volume of anhydrous ethanol was slowly added to the supernatant for alcohol precipitation. After standing overnight, the precipitate was collected by filtration. The precipitate was washed twice with anhydrous ethanol, then pre-frozen in a -80°C freezer, and finally freeze-dried under vacuum for 48 hours to obtain alginate oligosaccharide powder with a weight average molecular weight of 3000 Da.

[0035] Example 1: This example provides a compound aqueous solution that utilizes melatonin to enhance the waterlogging resistance of sesame seedlings, and its preparation and application method, including the following steps: In a reactor equipped with a temperature detection probe, a heating and cooling jacket, and a variable frequency mechanical stirrer, 800 mL of deionized water was added, followed by 1.0 g of alginate oligosaccharide powder prepared in Preparation Example 1 and 5.0 parts by weight of boric acid. The mechanical stirrer was turned on and the speed was set to 200 rpm. A 0.1 mol / L sodium hydroxide aqueous solution was added dropwise to precisely adjust the initial pH of the substrate solution to 7.0. The jacket heating was turned on and the temperature was raised to 60°C. The mixture was stirred at this temperature for 45 minutes to form a low-viscosity homogeneous fluid. Close the heating jacket and circulate cold water into the cooling jacket to rapidly cool the reaction to 35°C at a cooling rate of 2 to 3°C / min. Dissolve 100 parts by weight of melatonin powder in 10 mL of anhydrous ethanol beforehand. Simultaneously, dissolve 5.0 parts by weight of anhydrous calcium chloride in 150 mL of deionized water. Slowly and synchronously add the ethanol solution containing melatonin and the calcium chloride aqueous solution to the reaction vessel using a dual-channel constant flow pump at a flow rate of 8 mL / min. After the addition is complete, maintain the temperature at 35°C and continue stirring for 30 minutes to promote the completion of topological network contraction and hydrogen bonding. The final pH value was slowly adjusted down and stabilized at 6.2 using 0.1 mol / L dilute hydrochloric acid; then, approximately 0.05% by mass of polyalkylene oxide-modified heptamethyltrisiloxane was added, and deionized water was added to bring the total volume to 1000 mL; the stirring speed was increased to 350 rpm, and high-shear homogenization was performed for 15 minutes; after mixing, the mixture was filtered through a 200-mesh filter to obtain the melatonin compound aqueous solution; One day before the sesame seedlings reach the stage of having 4 true leaves and are facing continuous rainfall or waterlogging stress, use a conventional backpack sprayer to evenly spray the above-mentioned compound water-based agent on the foliage, ensuring that the droplets fully cover both sides of the sesame leaves, and that the leaves are moist but not dripping.

[0036] Example 2: This example provides a compound aqueous solution that utilizes melatonin to enhance the waterlogging resistance of sesame seedlings, and its preparation and application method, including the following steps: Add 800 mL of deionized water to the reactor, then add 0.8 g of alginate oligosaccharide powder prepared in Preparation Example 2 and 3.0 parts by mass of boric acid; turn on the mechanical stirrer and set the speed to 150 rpm; add 0.1 mol / L sodium hydroxide aqueous solution dropwise to adjust the initial pH of the substrate solution to 6.8; turn on the jacket heating to raise the temperature to 55°C and stir at this temperature for 40 minutes. The reaction was rapidly cooled to 30°C by introducing cold water; 80 parts by weight of melatonin powder were pre-dissolved in 10 mL of anhydrous ethanol; at the same time, 3.0 parts by weight of anhydrous calcium chloride were dissolved in 150 mL of deionized water; the two solutions were simultaneously and slowly added dropwise to the reaction vessel; after the addition was completed, the temperature was maintained at 30°C and stirring was continued for 30 minutes. The pH value was adjusted and stabilized at 6.0 using 0.1 mol / L dilute hydrochloric acid; 0.3 g of polyalkylene oxide modified heptamethyltrisiloxane was added, and deionized water was added to bring the total volume to 1000 mL; the speed was increased to 300 rpm, and high-shear homogenization was performed for 15 minutes, followed by filtration to obtain the final product. In the early stages of waterlogging, agricultural plant protection drones were used to evenly spray the above-mentioned compound water-based agent onto the leaves of sesame seedlings.

[0037] Example 3: This example provides a compound aqueous solution that utilizes melatonin to enhance the waterlogging resistance of sesame seedlings, and its preparation and application method, including the following steps: Add 800 mL of deionized water to the reactor, then add 1.5 g of alginate oligosaccharide powder prepared in Preparation Example 3 and 8.0 parts by weight of boric acid; turn on the mechanical stirrer and set the speed to 250 rpm; add 0.1 mol / L sodium hydroxide aqueous solution dropwise to adjust the initial pH of the substrate solution to 7.2; turn on the jacket heating to raise the temperature to 65°C and stir at this temperature for 60 minutes. The reaction was rapidly cooled to 35°C by introducing cold water; 120 parts by weight of melatonin powder were pre-dissolved in 15 mL of anhydrous ethanol; at the same time, 8.0 parts by weight of anhydrous calcium chloride were dissolved in 150 mL of deionized water; the two solutions were simultaneously and slowly added dropwise to the reaction vessel; after the addition was completed, the temperature was maintained at 35°C and stirring was continued for 45 minutes. The pH value was adjusted and stabilized at 6.5 using 0.1 mol / L dilute hydrochloric acid; 0.8 g of polyalkylene oxide modified heptamethyltrisiloxane was added, and deionized water was added to bring the total volume to 1000 mL; the speed was increased to 400 rpm, and high-shear homogenization was performed for 20 minutes, followed by filtration. One day before the sesame seedlings reach the stage of having 4 true leaves and are facing continuous rainfall, use a backpack sprayer to evenly spray the leaves on both sides.

[0038] Example 4 provides a compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin, along with its preparation and application method. This example, based on Example 1, fine-tunes the internal crosslinking and coordination ratios, and includes the following steps: Add 800 mL of deionized water to the reactor, then add 1.0 g of alginate oligosaccharide powder prepared in Preparation Example 1 and 8.0 parts by weight of boric acid; turn on the mechanical stirrer and set the speed to 200 rpm to adjust the initial pH of the substrate solution to 7.0; heat to 60 °C and stir for 45 minutes. Rapidly cool to 35°C; dissolve 100 parts by weight of melatonin powder in 10 mL of anhydrous ethanol; simultaneously dissolve 3.0 parts by weight of anhydrous calcium chloride in 150 mL of deionized water; add these solutions dropwise to the reaction vessel and maintain the temperature at 35°C while stirring for 30 minutes. The initial pH of the substrate solution was lowered and stabilized at 6.2; 0.5 g of polyalkylene oxide modified heptamethyltrisiloxane was added, and deionized water was added to 1000 mL; the rotation speed was increased to 350 rpm, and the mixture was homogenized for 15 minutes, then filtered to obtain the final product. One day before the sesame seedlings reach the stage of having 4 true leaves and are facing continuous rainfall, use a backpack sprayer to evenly spray the leaves.

[0039] Comparative Example 1 differs from Example 1 in that it does not contain alginate oligosaccharide, boric acid, anhydrous calcium chloride, or polyalkylene oxide-modified heptamethyltrisiloxane; it is simply a pure aqueous solution containing 100 μmol / L melatonin. All other aspects are the same.

[0040] Comparative Example 2 differs from Example 1 in that it does not contain alginate oligosaccharide powder, but is otherwise identical.

[0041] Comparative Example 3 differs from Example 1 in that boric acid was not added, but all other aspects are the same.

[0042] Comparative Example 4 differs from Example 1 in that anhydrous calcium chloride was not added, but all other aspects are the same.

[0043] Comparative Example 5 differs from Example 1 in that it does not contain polyalkylene oxide-modified heptamethyltrisiloxane, but is otherwise identical.

[0044] Comparative Example 6 differs from Example 1 in that the preparation process was changed. A conventional one-pot mixing method at room temperature was adopted, in which alginate oligosaccharide, boric acid, melatonin ethanol solution, calcium chloride aqueous solution and polyalkylene oxide modified heptamethyltrisiloxane were all added to water at room temperature and stirred directly until homogeneous. The proportions of the remaining components were the same.

[0045] Comparative Example 7 differs from Example 1 in that the alginate oligosaccharide is replaced in equal amounts with sodium polyacrylate, which has a similar molecular weight but does not contain adjacent diol structures; all other aspects are the same.

[0046] Test Example 1: The test was conducted using a fully automatic surface tension meter under the conditions of an ambient temperature of 25℃ and a relative humidity of 60%. The sample liquid to be tested was injected into the injection pump, the droplet extrusion rate was controlled and the droplet was suspended at the needle tip. The system automatically acquired the droplet profile image and fitted the Yang-Laplace equation. After the reading stabilized, it was read continuously for 5 times and the average value was taken to determine the static surface tension of each group of aqueous solutions. A rotational rheometer equipped with a temperature control system and a solvent trap was used. A parallel plate fixture with a diameter of 40 mm and a gap of 1.0 mm was selected. The test sample was placed on the test platform and the test temperature was set to 25℃. In oscillation mode, the strain was fixed at 1% and the test frequency was 1Hz. A constant temperature stage was used to assist the slow evaporation of water to simulate the natural micro-evaporation process of leaves in the field. The changes in storage modulus and loss modulus over time were continuously recorded. The mass fraction of solid content at the intersection of G' and G'' curves was recorded as the critical gel concentration, and the time required to reach the phase transition point was also recorded.

[0047] Table 1. Surface tension and rheological phase change parameters of the compound aqueous agents of each embodiment and comparative example.

[0048] The surface tension of Examples 1 to 3 consistently decreased to the range of 20 mN / m to 22 mN / m, significantly lower than that of deionized water and Comparative Example 5, which lacked a siloxane surfactant. This extremely low surface tension is the physical prerequisite for the liquid to overcome the hydrophobic repulsion of the waxy layer on the plant leaf surface, demonstrating that the fluid possesses the kinetic conditions to penetrate into microcracks and the substomatal cavity of the leaf surface. In the rheological phase transition test, in Examples 1 to 3, when the solid content increased to 2.3 wt% to 3.0 wt% due to slight evaporation of water, the storage modulus of Examples 1 to 3 rapidly increased and exceeded the loss modulus, indicating that the fluid spontaneously transformed from a viscosity-dominated sol state to an elasticity-dominated hydrogel state. Comparative Example 3 lacked boric acid, exhibiting only a unidirectional interaction between calcium ions and free carboxyl groups, lacking a dynamic covalent condensation network between borate ions and adjacent diols, resulting in no dynamic covalent condensation network throughout the test. The method crosses the gel point; Comparative Example 7 uses sodium polyacrylate without adjacent diols to replace alginate oligosaccharides, which also loses spatial crosslinking sites and always maintains liquid characteristics. Its final storage modulus is only 12.3 Pa, and it cannot form a gel film with mechanical strength; Comparative Example 5 has gelling ability, but due to the lack of interfacial tension regulation, the droplets present as tall spherical shapes with large contact angles on the test platform. The small specific surface area leads to an extremely slow water evaporation rate, and the phase transition time is greatly extended to 46.2 minutes. In actual field conditions, the droplets are very likely to roll off due to gravity or wind before completing the phase transition; The above physical quantitative indicators directly confirm the necessity of boric acid crosslinking and calcium bridge coordination network design, and verify the real existence of the mechanism of in-situ sol-gel phase transition locking active substances when a small amount of water is lost in the stomatal microenvironment.

[0049] Test Example 2: Accurately transfer 20 mL of each solution from each example and the relevant comparative example into a quartz petri dish with an inner diameter of 60 mm, keeping the liquid level consistent to eliminate the interference of optical path difference on the test results; Place the quartz petri dish containing the sample in a UV aging test chamber; turn on the UV LED light source with a main wavelength of 365nm, and adjust the radiation intensity to stabilize at 45W / m². 2 The test environment temperature was set to be constant at 25℃; At light exposure times of 0, 4, 8, 12 and 24 hours, 0.5 mL samples were taken from each culture dish and immediately transferred to brown light-protected centrifuge tubes to terminate the light reaction. The sample solution was diluted with ultrapure water at a volume ratio of 1:10, filtered through a 0.22 μm polytetrafluoroethylene microporous membrane, and the concentration of melatonin was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were reversed-phase C24. 18 The chromatographic column was used with a methanol-water mixture as the mobile phase, the detection wavelength was set to 278 nm, and the flow rate was set to 1.0 mL / min. The peak areas at each time point were recorded, the melatonin residual rate was calculated, and the degradation half-life was calculated by fitting the first-order kinetic equation.

[0050] Table 2. Melatonin Residual Rate and Degradation Half-Life of Each Test Sample under Continuous UV Irradiation Table 2.

[0051] The compound aqueous solutions prepared in Examples 1 to 4 exhibited a clear physicochemical protective effect on melatonin, with degradation half-lives ranging from 41.8 to 53.1 hours. Compared to the pure water baseline in Comparative Example 1, the retention time of melatonin was extended by 6 to 8 times. Comparative Example 2, lacking the addition of an alginate oligosaccharide backbone, showed a degradation curve that essentially overlapped with the pure water group, demonstrating that the absence of a macromolecular carrier prevented the formation of a mesoscopic-dimensional physical encapsulation region, resulting in the complete exposure of melatonin molecules to the ultraviolet radiation field. Comparative Example 6 had the same composition as Example 1, but employed a single-stage mixing process at room temperature, resulting in a degradation half-life of only 14.8 hours. This difference directly verifies the necessity of the stepped temperature control process in this invention. Room temperature mixing cannot overcome the activation energy barrier of the dynamic condensation between boric acid and adjacent diols, and... The lack of physical kinetic energy for macromolecular chain contraction during cooling resulted in a macroscopically homogeneous but mesoscopically unavailable ternary supramolecular topology, failing to provide steric hindrance to mask the melatonin conjugated resonance region. In Comparative Example 7, replacing alginate oligosaccharides with sodium polyacrylate in equal amounts reduced the half-life to 18.1 hours. Although sodium polyacrylate can undergo some cross-linking under the action of calcium ions, its main chain lacks adjacent diol structures, preventing the formation of a reversible covalent backbone with borate ions. The constructed network is loose and lacks specific binding sites for melatonin hydrogen bonding. The above experimental data confirm that the specific polysaccharide backbone, metal ion coordination, and step-temperature controlled assembly process are all indispensable, collectively constituting the microenvironment that restricts the electronic transition of the melatonin indole ring, thus confirming the objective validity of the steric hindrance anti-photolysis mechanism.

[0052] Test Example 3: Potted sesame seedlings with uniform growth and at the 4-true-leaf stage were selected; a set volume of the test solution from each example and comparative example was evenly spread on both sides of the third and fourth true leaves of each seedling using a micropipette; 5 biological replicates were set up for each treatment. The treated plants were placed in an artificial climate chamber with a temperature of 28°C, relative humidity of 55%, and a constant surface wind speed of 0.5 m / s, and allowed to air dry naturally for 2 hours to simulate the actual water evaporation process in the field. Transfer the plants to an artificial rainfall simulation platform; set the rainfall intensity to 35 mm / h, and control the average kinetic energy of raindrops within the standard range for conventional rainstorms; continuously rinse for 45 minutes; after rinsing, let the plants stand for 30 minutes to drain the surface free water; The marked test leaves were cut and weighed fresh. First, the leaf surface was gently rinsed three times with 15 mL of methanol solution. The eluent was collected and diluted to 50 mL for the determination of surface retention. Then, the rinsed leaves were cut into small pieces, ground into powder with liquid nitrogen, and extracted with an appropriate amount of methanol using ultrasound for 30 minutes. The supernatant was collected by centrifugation and diluted to 50 mL for the determination of the amount of osmotic fluid inside the mesophyll tissue. The melatonin concentration in the two extracts was determined by high performance liquid chromatography, and the absolute retention amount and total retention rate of melatonin per unit fresh weight of leaves were calculated.

[0053] Table 3. Melatonin target retention of each test solution after simulated rainfall.

[0054] The compound aqueous solutions prepared in Examples 1 to 4 exhibited extremely strong physical adsorption and anti-loss capabilities after being subjected to high-intensity simulated rainfall, with the overall total retention rate remaining stable between 77.4% and 81.3%. The permeate penetration was significantly higher than the surface retention, proving that the main component had penetrated the cuticle and entered the plant tissue. The pure aqueous solution in Comparative Example 1 had a total retention rate of only 6.8% after rinsing, demonstrating that conventional water-soluble agents are essentially ineffective under extreme weather conditions. Comparative Example 5 lacked polyalkylene oxide-modified heptamethyltrisiloxane, resulting in high surface tension. The solution could not spread on the hydrophobic waxy layer and could not penetrate into the stomatal cavity by capillary force. Data showed that its internal permeation decreased significantly to 3.56 μg / gFW, and the droplets remaining on the surface easily rolled off under the impact of rainwater kinetic energy, leading to a total retention rate of only 21.0%. This confirms that ultra-low surface tension is a key factor in overcoming hydrophobic barriers. The necessary conditions for barrier function; Comparative Example 3 lacks the boric acid crosslinking structure. Although it has a certain permeability, after penetrating into the stomatal microenvironment or adhering to the leaf surface, it cannot undergo in-situ sol-gel phase transition during water evaporation. The fluid fails to transform into a viscoelastic hydrogel film and is ultimately diluted and carried away by rainwater, with a total retention rate of only 35.1%. This verifies that the physical shear resistance provided by dynamic covalent bonds at the critical gel concentration is the key to locking in active substances. Comparative Example 6 uses a room temperature mixing process, and its overall retention rate is much lower than that of the examples. This indicates that without a stepped temperature control process, the macromolecular chains fail to effectively shrink and entangle in three-dimensional space. The resulting gel network has a loose microstructure, insufficient water retention and mechanical strength, and cannot effectively resist the continuous mechanical shearing and dissolution effects of raindrops. The above data fully confirm the effectiveness of the synergistic anti-erosion mechanism of interfacial penetration and in-situ phase transformation film.

[0055] Test Example 4: Select plump and robust sesame seeds and sow them evenly in flowerpots filled with standardized substrate; after the sesame seedlings emerge, thin them out, leaving 3 seedlings of uniform growth in each pot; the experiment adopted a randomized block design with a total of 3 biological replicates. The experiment included seven treatment groups: a normal water supply group; a waterlogging treatment group; and five treatment groups with different melatonin concentrations. Groups M1 to M5 were prepared using the same step temperature control process, polysaccharide backbone, and adjuvant ratios as in Example 1. The only variable was the final concentration of melatonin added to the system, which were 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L, respectively. Once the sesame seedlings have grown to the stage of having 4 true leaves, the application of pesticides will begin. Starting one day before the date of the waterlogging stress, the leaves of the seedlings in each treatment group will be evenly sprayed with a conventional handheld sprayer every day. The amount of pesticide applied will be such that the leaves are fully moistened without any liquid pooling or dripping. The spraying will continue for 3 consecutive days. The M0 group will be sprayed with an equal amount of deionized water at the same time. After the pesticide application process was completed, all pots except the CK group were subjected to waterlogging treatment; water was poured into the pots and the water level was kept constant at 2cm above the soil surface for 6 days; the CK group was not flooded and water was managed according to the conventional field capacity. After the 6-day waterlogging stress period, open the drainage hole at the bottom of the pot to drain the water; perform destructive sampling of the plant, separate the sesame seedling roots from the substrate, rinse them clean with running water, and use a root scanning analyzer to measure the total root length, root surface area, root diameter and root volume morphological indicators. Functional leaves of the plant were collected simultaneously, quickly frozen in liquid nitrogen, and then stored in an ultra-low temperature freezer at -80℃. The supernatant of the frozen leaf tissue was extracted and homogenized. The contents of malondialdehyde and hydrogen peroxide were determined, and the activities of superoxide dismutase, peroxidase and catalase were detected. The contents of soluble protein and free proline were also determined.

[0056] Table 4. Effects of different concentrations of exogenous melatonin on root morphology and antioxidant physiological indicators of waterlogged sesame seedlings.

[0057] According to the data in Table 4, six consecutive days of waterlogging stress severely inhibited the physiological functions of sesame seedlings. Compared with the control group (CK) with normal water supply, the total root length, root surface area, and root volume of the M0 group decreased by an average of 49.98%, 45.15%, and 12.99%, respectively, and the activities of soluble protein and POD decreased significantly. Simultaneously, the hypoxic environment induced membrane lipid peroxidation, leading to a significant increase in leaf MDA and H2O2 contents by an average of 68.32% and 129.27%, respectively. Although the plants instinctively increased SOD, CAT activity, and proline content as a self-defense mechanism, this was insufficient to offset the damage caused by waterlogging.

[0058] After applying the compound aqueous solution, exogenous melatonin effectively alleviated the inhibitory effect caused by waterlogging, which was directly reflected in the overall recovery of total root length, root surface area, root diameter, and root volume of seedlings. Among them, the M2 treatment with a concentration of 100 μmol / L showed the best alleviating effect on waterlogged sesame. Compared with the waterlogged control group M0, the leaf MDA content of the M1 and M2 treatment groups decreased by 13.02%~24.17%, and the H2O2 content decreased by 13.94%~25.81%, significantly reducing the degree of oxidative damage to cell membranes. In terms of antioxidant enzyme systems, the activities of SOD, POD, and CAT in the M1 and M2 groups were further amplified, increasing by 1. The concentrations of soluble protein and proline, which play a role in osmotic regulation, increased by 6.59%~22.19%, 36.47%~74.84%, and 3.26%~13.76%, respectively. Meanwhile, the contents of soluble protein and proline, which regulate osmosis, also increased by 47.68%~48.67% and 22.46%~33.25%, respectively. Principal component analysis and membership function comprehensive evaluation of multidimensional indicators confirmed that the waterlogging tolerance of each treatment group was M2>M1>M3>M4>M5>M0. The foliar targeted slow-release melatonin solution effectively cleared excess toxic free radicals in the plant, maintained the stability of cell membrane structure and osmotic balance, and substantially improved the overall stress resistance threshold of sesame seedlings.

[0059] Test Example 5: The waterlogging-resistant sesame variety PYH and the waterlogging-sensitive sesame variety JHM were selected as test materials.

[0060] The experimental design was the same as the previous test example, using the optimal concentration selected in the early screening, that is, using a compound aqueous solution containing 100 μmol / L melatonin for foliar spraying.

[0061] Destructive sampling was performed on the sesame roots of each treatment group at 0, 3, and 6 days after the start of waterlogging during the seedling stage, as well as during the subsequent budding and flowering stages.

[0062] The collected root samples were flash-frozen in liquid nitrogen and then used for subsequent determination of osmotic regulators, antioxidant enzyme activity, endogenous hormones, proteomics, and expression analysis of key genes.

[0063] Table 5. Regulation of physiological responses of optimal concentrations of exogenous melatonin to different waterlogging-resistant sesame varieties.

[0064] Waterlogging significantly inhibited the normal growth of sesame seedlings and damaged the root structure. According to the data in Table 5, there were significant differences in the response of different genotypes to waterlogging. Among them, sensitive varieties were more severely affected: under waterlogging conditions alone, their plant height and root biomass decreased by 23.61% and 29.25% respectively compared with the CK group; while the decrease in waterlogging tolerant varieties was relatively smaller, with plant height and root biomass decreasing by 22.87% and 23.64% respectively. At the same time, waterlogging induced severe oxidative stress. The accumulation of MDA and H2O2 in the JHM group increased significantly by 88.51% and 203.87% respectively, showing severe membrane lipid peroxidation damage. Exogenous application of 100 μmol / L melatonin significantly alleviated the aforementioned adverse effects. In terms of morphological development, melatonin reduced the inhibitory effect of waterlogged sesame growth, resulting in a significant increase in plant height and root biomass for both JHM and PYH compared to waterlogging alone. At the physiological and metabolic level, compared to waterlogging alone, waterlogged plants treated with melatonin showed reduced soluble protein and proline content, thereby reducing the excessive accumulation of osmotic regulators and conserving energy for growth. Simultaneously, melatonin treatment significantly reduced the accumulation of MDA and H2O2, mitigating oxidative damage. Since the waterlogging-tolerant PYH itself has good antioxidant capacity and endogenous hormone stability, and its damage is relatively minor, the phenotypic rescue and index reversal effects of exogenous melatonin on waterlogging-sensitive JHM are particularly prominent. At the proteomics and molecular level, further analysis of the samples identified 1846 differentially expressed proteins. Enrichment analysis showed that these differentially expressed proteins were mainly concentrated in core metabolic pathways, phenylpropane biosynthesis, glutathione metabolism, and reactive oxygen species (ROS) metabolism. Targeted uptake of melatonin triggered the upregulation of genes such as superoxide dismutase (SOD) while downregulating the expression of hydrogen peroxide-induced proteins, which was highly consistent with the results of the aforementioned macroscopic physiological indicators. Protein-protein interaction analysis further highlighted key proteins such as RPL6 and RPL4, which play an important role as potential hub nodes in the melatonin-mediated ROS metabolism network. This invention systematically elucidates the multiple mechanisms by which melatonin confers waterlogging tolerance in plants from both phenotypic and underlying molecular levels, laying a theoretical foundation for its application in waterlogged areas.

[0065] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound aqueous solution that utilizes melatonin to enhance the waterlogging resistance of sesame seedlings, characterized in that, It is composed of the following raw materials in parts by weight: 0.8-1.5 parts of alginate oligosaccharide; 3.0-8.0 parts of boric acid; 80-120 parts of melatonin; 3.0-8.0 parts of calcium chloride; 0.3-0.8 parts of polyalkylene oxide modified heptamethyltrisiloxane; 10-15 parts of anhydrous ethanol for presolubilization; and the balance being deionized water and an appropriate amount of pH adjuster.

2. The compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 1, characterized in that, The surface tension of the compound aqueous solution is 20~22 mN / m, and the final pH value of the compound aqueous solution is stable between 6.0 and 6.

5.

3. The compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 1, characterized in that, The weight-average molecular weight of the alginate oligosaccharide ranges from 1500 Da to 3000 Da.

4. The compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 1, characterized in that, The alginate oligosaccharide was prepared by the following specific enzymatic hydrolysis process: sodium alginate powder was dissolved in deionized water at a mass concentration of 1.5% to 2.0%, and the pH was adjusted to 7.0 to 7.5 to prepare a substrate solution; alginate lyase was added at a ratio of 50 to 100 U / g substrate, and the solution was hydrolyzed at a constant temperature of 40℃ to 45℃ and 120 to 150 rpm for 4 to 6 hours; after the hydrolysis was completed, the temperature was raised to 95℃ and held for 15 minutes to inactivate the enzyme, and the supernatant was collected by cooling and centrifugation; 3 to 4 times the volume of anhydrous ethanol was slowly added for precipitation, and the precipitate was washed with anhydrous ethanol, pre-frozen, and vacuum freeze-dried to obtain the final product.

5. A method for preparing a compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin, as described in any one of claims 1-4, characterized in that... Includes the following steps: S1. Add deionized water to the reactor, then add alginate oligosaccharide powder and boric acid in sequence, and stir until homogeneous. Use a pH adjuster to adjust the initial pH of the substrate solution to a neutral to slightly acidic or alkaline range, turn on the heating to raise the temperature, and stir at a constant temperature to form a low-viscosity homogeneous fluid. S2. Cool the reaction by introducing cold water into the jacket of the reactor; dissolve melatonin in anhydrous ethanol and calcium chloride in deionized water in advance, and use a constant flow pump to slowly add the melatonin ethanol solution and calcium chloride aqueous solution to the reactor simultaneously. After the addition is completed, maintain the temperature and continue stirring to promote the completion of topological network contraction and hydrogen bonding with melatonin. S3. Use a pH adjuster to adjust the pH of the substrate solution back to slightly acidic, add polyalkylene oxide modified heptamethyltrisiloxane, add deionized water to the target volume, increase the stirring speed to perform high-shear homogenization, and filter to obtain melatonin compound aqueous solution.

6. The method for preparing a compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 5, characterized in that, In S1, the initial pH value is adjusted to 6.8~7.2, the temperature is raised to 55℃~65℃, the constant temperature stirring time is 40~60 minutes, and the stirring speed is 150~250 rpm.

7. The method for preparing a compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 5, characterized in that, In S2, the cooling rate is controlled at 2℃~3℃ / min, and the target cooling temperature is 30℃~35℃; the melatonin ethanol solution and calcium chloride aqueous solution are added simultaneously by a dual-channel constant flow pump, the addition flow rate is constant at 8mL / min, and the constant temperature stirring time after the addition is completed is 30~45 minutes.

8. The method for preparing a compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 5, characterized in that, In S3, the pH value is lowered and stabilized at 6.0~6.5, the stirring speed for high shear homogenization is 300~400 rpm, and the homogenization time is 15~20 minutes.

9. A method for applying a compound aqueous solution that utilizes melatonin to enhance the waterlogging resistance of sesame seedlings, as described in any one of claims 1-4, characterized in that: When sesame seedlings have grown to the stage of 4 true leaves and are facing continuous rainfall or the initial stage of waterlogging, the compound aqueous solution is evenly sprayed onto both sides of the sesame seedling leaves using a spraying device. The amount of the solution should be such that the leaves are fully moistened without obvious dripping.

10. The application method of the compound aqueous solution for enhancing the waterlogging resistance of sesame seedlings using melatonin according to claim 9, characterized in that: The compound aqueous solution, along with a small amount of water evaporation, forms a viscoelastic hydrogel microfilm on the leaf surface.

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