PH-bionic trigger type multi-layer intelligent seed coating agent for improving salt tolerance of crops as well as preparation method and application of pH-bionic trigger type multi-layer intelligent seed coating agent

Abstract

The invention belongs to the technical field of salt-resistant planting of crops in coastal saline-alkali soil, and provides a pH-responsive bionic triggering three-layer intelligent seed coating agent as well as preparation and application thereof in order to solve the problem that seedlings of crops such as corns in saline-alkali soil are difficult to emerge. According to the invention, a three-layer synergistic coating structure is constructed on the surface of the seed by adopting the idea of taking the seed as the place and taking the soil acid-base condition as a response signal, and the release process is regulated and controlled by the outer layer, so that the salt tolerance of the seed is improved. The coating sequentially comprises a melatonin-nano-selenium active core layer, a nano-selenium active core layer, a nano-selenium active core layer and a nano-selenium active core layer from inside to outside, the pectin-sodium carboxymethyl cellulose-monosaccharide slow-release adhesive layer forms a water-retaining buffer layer, so that salt diffusion is delayed, and active components are continuously released; the silk fibroin and chitosan or carboxymethyl chitosan form a pH response regulation and control layer, permeability is regulated along with acid-base change, and on-demand release is achieved. The three layers of coatings maintain Na < + > / K < + > balance through salt resistance, water retention and pH response effects, and promote stable germination and growth of crops in the saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of coastal saline-alkali soil planting technology, specifically relating to a pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance, its preparation method and application. Background Technology

[0002] With global population growth and increasingly scarce arable land resources, the development and utilization of marginal lands such as saline-alkali land is of significant strategic importance for ensuring national food security. my country has a wide distribution of saline-alkali soils. For example, coastal areas (such as the Yellow River Delta) are affected by seawater intrusion and climate factors. High concentrations of salt (mainly Na⁺ and Cl⁻) in the soil, often accompanied by extreme pH environments, can cause severe osmotic stress, ion toxicity, and oxidative stress. This leads to impaired seed water absorption, disordered cell metabolism, and inhibited radicle elongation, ultimately severely reducing germination rates and crop yields. "Planting suitable crops for suitable land," which involves enhancing the resilience of seeds to adapt to saline-alkali environments, is a key path for sustainable agricultural development.

[0003] Seed coating technology, as an important pre-sowing treatment method, aims to improve the seed germination microenvironment and enhance early stress resistance by constructing functional membrane or gel layers on the seed surface. However, existing seed coating technologies still have significant limitations in addressing saline-alkali stress: First, their functions are relatively singular and their environmental adaptability is insufficient. Traditional chemical pesticide-type coatings are mainly used for pest and disease control, offering limited improvement to abiotic stresses such as salt and drought, and posing a risk of environmental residues. While nutrient-based coatings can supplement micronutrients, they often suffer from unstable active ingredient forms, excessively rapid release, or asynchrony with seedling needs. Second, the film adhesion and mechanical stability are insufficient. Some coatings are prone to powdering and detachment during storage, transportation, and mechanized sowing, leading to functional failure and potential dust pollution. Third, they lack intelligent response and controllable slow-release mechanisms. The release of active components in most biopolymer coatings relies mainly on simple diffusion, making it difficult to achieve timed and on-demand supply based on soil pH changes or seedling growth stages. Furthermore, the large pH fluctuations in saline-alkali soils highlight the necessity of environmental response regulation.

[0004] It is worth noting that some mucilaginous seeds in nature adapt to arid or water-scarce environments. Their seed coats, upon absorbing water, release polysaccharide mucus and rapidly swell, forming a gel-like outer layer that envelops the seed, thus creating a "seed microenvironment" conducive to germination. This microenvironment possesses functions such as water retention, buffering of external solute changes, and regulation of nutrient migration, providing biomimetic inspiration for the design of seed coating materials under adverse conditions: by constructing a polysaccharide hydrogel layer with water-absorbing and swelling capabilities on the seed surface, the moisture and solute conditions around the seed can be improved in low water potential and high salinity environments, enhancing the stability of germination and seedling establishment. Existing research shows that hydrophilic polysaccharides such as pectin and sodium carboxymethyl cellulose can form a three-dimensional network structure under ionic cross-linking conditions, exhibiting gelling, water-retention, and adhesive properties, making them suitable for constructing coating layers with both slow-release and adhesion functions. However, the regulation of active ingredient release in such polysaccharide layers largely depends on diffusion processes and lacks a response to external environmental signals, making responsive release difficult.

[0005] On the other hand, chitosan and carboxymethyl chitosan, as natural amino polysaccharide materials, possess excellent film-forming properties, adhesion, and certain bioactivity, making them widely used in agricultural coatings. Furthermore, the protonation and deprotonation behavior of the amino and carboxyl groups in their molecular chains allows them to exhibit differences in swelling degree, permeability, and network density under different pH conditions, providing a material basis for constructing pH-responsive control layers. However, existing formulations mostly employ single-layer coatings or simple compounding of chitosan-based materials, and a control system that uses soil pH changes as a trigger signal to regulate the release behavior of salt-resistant active components in the inner layer has not yet been established. This makes it difficult to meet the "on-demand supply" requirements of active substances for seed germination under saline-alkali soil conditions.

[0006] Melatonin, as a small-molecule regulator related to plant stress response, has the potential to scavenge reactive oxygen species, maintain ion homeostasis, and regulate osmotic balance. Selenium (especially nano-selenium) has high bioactivity and can participate in antioxidant systems and enhance plant stress resistance. Although there are research reports on the application of melatonin, selenium, or pH-responsive materials in agriculture, there is still a lack of technical solutions that integrate the synergistic utilization of salt-resistant active components, the construction of a biomimetic hydrogel layer microenvironment, and the pH-responsive regulation of chitosan or carboxymethyl chitosan through a layered structure, using soil acidity / alkalinity conditions as a trigger signal to achieve controlled release of active substances and synergistic salt-resistant effects. Therefore, developing a smart seed coating technology that can respond to soil acidity / alkalinity conditions and achieve on-demand release of salt-resistant active substances through a multi-layered synergistic structure has important theoretical significance and application value for improving the emergence quality and salt tolerance of crops in saline-alkali soils. Summary of the Invention

[0007] To address the challenges of severe salt stress and large pH fluctuations during seed germination in saline-alkali soils, and the difficulty of achieving environmental response regulation with existing seed coatings, this invention, based on the design concept of "planting in suitable locations," uses soil acidity and alkalinity as a response trigger signal. It constructs a three-layer intelligent coating structure on the seed surface with clearly defined functions and synergistic effects from the inside out. The outer pH response regulation layer controls and regulates the release process of the salt-resistant active components in the inner layer, thereby enabling the on-demand release of active substances in saline-alkali environments and improving seed salt tolerance and seedling emergence stability.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A pH-bionic triggering multilayer smart seed coating agent for improving crop salt tolerance. The seed coating agent covers the seed surface and includes, from the inside out, a first active core layer, a second slow-release attachment layer, and a third pH-responsive regulation layer.

[0010] Furthermore, the first active core layer is a salt-resistant active substance loading layer, which is composed of melatonin-nano selenium composite material. Melatonin is loaded onto the surface of nano selenium through adsorption, coordination or coating to form a stable nanocomposite system.

[0011] Furthermore, the second slow-release adhesion layer is a composite solution composed of pectin, sodium carboxymethyl cellulose, and monosaccharides. A slow-release network structure is constructed through ionic cross-linking to achieve the slow release of active ingredients and enhance adhesion to the seed surface.

[0012] Furthermore, the third pH-responsive control layer is composed of chitosan or carboxymethyl chitosan as pH-responsive functional components, and silk fibroin as a structural support material to form a composite network with the chitosan or carboxymethyl chitosan. This network is used to adjust the swelling and permeability of the coating layer according to changes in soil acidity and alkalinity, thereby achieving on-demand control of the release of the inner active components.

[0013] Furthermore, the first active core layer also includes functional nanoparticles or carbon-based nanoparticles.

[0014] The functional nanoparticles are magnesium oxide nanoparticles (MgO NPs).

[0015] Preparation method of pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance

[0016] (1) Preparation of the first active core layer:

[0017] 1) Take sodium selenite aqueous solution as selenium precursor, add melatonin solution to sodium selenite aqueous solution, the melatonin solution is prepared by dissolving in a small amount of ethanol and then diluting;

[0018] 2) Slowly add ascorbic acid solution as a reducing agent under stirring conditions to generate nano-selenium particles and simultaneously form a melatonin-nano-selenium composite system. Continue stirring for 30-60 min, adjust the pH of the system to 6-7, and obtain a stable dispersion of melatonin-nano-selenium composite nanomaterials.

[0019] 3) Immerse the seeds in a melatonin-nano-selenium composite nanomaterial dispersion, ensuring that the seeds are completely immersed in the solution. After 5-30 minutes, remove the seeds and place them in a ventilated and dry place to air dry for 2-12 hours to obtain the first layer of seed coating.

[0020] (2) Preparation of the second sustained-release adhesion layer:

[0021] 1) A complex polysaccharide solution was prepared using pectin and sodium carboxymethyl cellulose as raw materials;

[0022] 2) Add the mixed monosaccharide solution to the complex polysaccharide solution and stir thoroughly until completely dissolved;

[0023] 3) Add calcium chloride (CaCl2) solution to adjust the mechanical strength of the hydrogel and let it stand for 12 h; add 2 M NaOH solution with 70% of the pectin molar amount, and after 24~48 h, immerse the first layer of seed coating obtained in step (1) into the above system to form a hydrogel structure on the seed surface; after the hydrogel is formed, soak for 5 min, take it out, and air dry for 24~48 h to obtain the second layer of hydrogel seed coating with slow-release adhesion function.

[0024] (3) Preparation of the third pH-responsive control layer:

[0025] 1) Prepare a silk fibroin-polysaccharide composite solution according to the required ratio. Dissolve the polysaccharide in an aqueous acetic acid solution with a volume fraction of 0.5% to 1.5% as a solvent, and adjust the pH of the solution to 5.0 to 5.5. Then mix the silk fibroin solution and the polysaccharide solution and stir thoroughly to obtain a homogeneous silk fibroin-polysaccharide composite solution.

[0026] 2) Immerse the seeds with the second coating obtained in step (2) into the silk fibroin-polysaccharide composite solution to deposit and form a third outer coating structure on the seed surface. During or after the coating process, add sodium tripolyphosphate to the silk fibroin-polysaccharide composite solution to form an ionic cross-linking or covalent cross-linking network between the silk fibroin and chitosan or carboxymethyl chitosan molecules, thereby constructing a silk fibroin-polysaccharide composite regulatory layer structure with pH response characteristics.

[0027] Furthermore,

[0028] The sodium selenite aqueous solution in (1) has a concentration of 0.5–5 mM and a melatonin concentration of 0.15 mg / mL.-1 The molar ratio of ascorbic acid to sodium selenite is 4:1;

[0029] Alternatively, functional nanomaterials or carbon-based nanomaterials may be added to the melatonin-selenium nanocomposite nanomaterial dispersion of (1); the concentration of the functional nanomaterials in the melatonin-selenium nanocomposite nanomaterial dispersion is 10–100 mg / L. -1 .

[0030] Furthermore,

[0031] In (2), the mass ratio of pectin to sodium carboxymethyl cellulose is 1:1, and the total mass fraction of the complex polysaccharide system is 5-10%.

[0032] Alternatively, in (2), the mixed monosaccharides include D-(+)-xylose, L-(+)-arabinose, DL-arabinose, and L-rhamnose, with a molar ratio of 30:9:9:14, and the total mass fraction of the monosaccharides is 2-5%;

[0033] Or, in (2), the final concentration of CaCl2 is 5 mM.

[0034] Furthermore,

[0035] In (3), the polysaccharide is chitosan or carboxymethyl chitosan, and the mass ratio of silk fibroin to polysaccharide is 1:3, 3:1 or 1:1.

[0036] Alternatively, in (3), the final concentration of sodium tripolyphosphate is 5 mM.

[0037] Application of pH-bionic triggering multilayer intelligent seed coating agent for improving crop salt tolerance. The coating agent can alleviate the inhibitory effect of salt stress on crops, improve seed germination rate, promote root development, increase seedling biomass, and improve crop nutrition and photosynthetic physiological characteristics.

[0038] The beneficial technical effects of this invention are as follows:

[0039] By constructing a multi-layered coating structure on the seed surface, consisting of a salt-resistant active core layer, a biomimetic water-retaining and slow-release attachment layer, and a pH-responsive control layer, the functional layers work synergistically. Specifically, the salt-resistant active core layer provides antioxidant and ion homeostasis regulation support for seed germination and early seedling growth; the biomimetic water-retaining and slow-release attachment layer enhances the adhesion stability of the coating on the seed surface and creates a water-retaining and buffering microenvironment around the seed; and the pH-responsive control layer utilizes the environmental responsiveness of chitosan or carboxymethyl chitosan to adjust the permeability of the coating layer according to changes in soil pH, thereby regulating the release process of the active substances in the inner layer.

[0040] Through the synergistic effect of the above-mentioned multi-layered structure, the seed coating of this invention can maintain good water absorption and swelling performance and structural stability in saline-alkali soil environments, achieving a continuous supply of active substances and effectively reducing Na+ under salt stress conditions. + Excessive entry into the plant maintains Na + / K + It balances ions and enhances the antioxidant defense capabilities of plants, thereby promoting stable germination and early growth of crops in saline-alkali soils, showing good application prospects and promotion value. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the structure of the multilayer intelligent seed coating agent of the present invention;

[0042] Figure 2 The effects of different proportions of silk fibroin-carboxymethyl chitosan seed coating on maize growth are shown in the following figures: (a) maize growth at 7 days, (b) germination rate under non-salt stress, (c) germination rate under salt stress, (d) fresh weight, (e) shoot length, (f) root length, and (g) seed vigor.

[0043] Figure 3 The effects of different proportions of silk fibroin-carboxymethyl chitosan seed coating on maize root growth are shown in the figures: (a) total root length, (b) root surface area, (c) root volume, and (d) number of root tips.

[0044] Figure 4 The effects of different proportions of silk fibroin-chitosan seed coating on maize growth are shown in the following figures: (a) maize growth at 7 days, (b) germination rate under non-salt stress, (c) germination rate under salt stress, (d) fresh weight, (e) shoot length, (f) root length, and (g) seed vigor.

[0045] Figure 5 The effects of different proportions of silk fibroin-carboxymethyl chitosan seed coating on maize root growth are shown in the figures: (a) total root length, (b) root surface area, (c) root volume, and (d) number of root tips.

[0046] Figure 6 The following are radar charts showing the comprehensive evaluation of the salt resistance performance of the silk fibroin-polysaccharide seed coating on maize seedlings: (a) radar chart of the salt resistance performance index of the silk fibroin-carboxymethyl chitosan seed coating on maize seedlings; (b) radar chart area of ​​the salt resistance performance index of the silk fibroin-carboxymethyl chitosan seed coating on maize seedlings; (c) radar chart of the salt resistance performance index of the silk fibroin-chitosan seed coating on maize seedlings; and (d) radar chart area of ​​the salt resistance performance index of the silk fibroin-chitosan seed coating on maize seedlings.

[0047] Figure 7The swelling kinetics curves of the third layer silk fibroin-chitosan composite membrane of the present invention in different pH buffer solutions are shown.

[0048] Figure 8 This is the cumulative release curve of selenium, the active ingredient in seed coating, under different pH conditions according to the present invention;

[0049] Figure 9 The compressive stress-strain curves of seed coatings with different numbers of layers according to the present invention are shown.

[0050] Figure 10 The effect of the multi-layer intelligent seed coating agent of the present invention on maize growth under salt stress is shown in the figures: (a) plant height, (b) plant stem.

[0051] Figure 11 The effects of the multilayer intelligent seed coating agent of the present invention on the photosynthetic performance and related indicators of maize under salt stress are as follows: (a) chlorophyll, (b) nitrogen content, (c) net photosynthetic rate (Pn), (d) stomatal conductance (Gs), (e) intercellular CO2 concentration Ci, (f) transpiration rate (Tr), (g) water use efficiency (WUE), and (h) stomatal extreme value (Ls).

[0052] Figure 12 The effects of the multilayer intelligent seed coating agent of the present invention on the antioxidant system and membrane lipid peroxidation level of maize under salt stress, (a) SOD, (b) MDA;

[0053] Figure 13 To illustrate the effect of the multilayer intelligent seed coating agent of this invention on the ionic homeostasis of maize under salt stress, (a) Na + (b)K + . Detailed Implementation

[0054] The specific implementation method will be further described below with reference to the accompanying drawings.

[0055] Example 1:

[0056] Preparation method of pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance Figure 1 Here is a schematic diagram of the structure of the seed coating agent of this application:

[0057] 1. Preparation of the first layer of melatonin-nano selenium composite active core layer (C1):

[0058] (1) Preparation of composite precursor solution

[0059] First, prepare the selenium precursor solution. Weigh 86.5 mg of sodium selenite (Na₂SeO₃, anhydrous), dissolve it in deionized water, and bring the volume to 100 mL to obtain a 5.0 mM sodium selenite aqueous solution. Store it in the dark. Simultaneously, prepare the melatonin solution: Accurately weigh 15 mg of melatonin powder, dissolve it in a small amount of anhydrous ethanol, and bring the volume to 0.5 mL. Under magnetic stirring, slowly add the melatonin solution to the sodium selenite solution, mixing thoroughly to form a selenium-melatonin mixed precursor system. The final concentration of melatonin is 0.15 mg / mL. -1 .

[0060] (2) In-situ reduction and synthesis of nanocomposite materials

[0061] In the above selenium-melatonin mixed precursor system, under magnetic stirring (800-1000 rpm) at room temperature and in the dark, ascorbic acid aqueous solution was added dropwise as a reducing agent. Specifically, 10.0 mL of 0.20 M ascorbic acid solution was added at a rate of 0.5-1.0 mL / min. -1 The ascorbic acid was added dropwise to the reaction system at a rate of approximately 4:1 to achieve a molar ratio of ascorbic acid to sodium selenite. After the addition was complete, the reaction was stirred for 30–60 min.

[0062] During the reaction, the solution color gradually changes from light to orange-red and then tends to stabilize, indicating... Selenium is reduced to zero valence and forms stable, dispersed nano-selenium particles. Melatonin binds to the surface of the nano-selenium particles during the reaction through physical adsorption or hydrogen bonding, resulting in a melatonin-modified nano-selenium composite dispersion. After the reaction, the pH of the system is adjusted to 6.0–7.0 using dilute hydrochloric acid or sodium hydroxide solution to improve dispersion stability.

[0063] (3) Functional modification

[0064] To further enhance the mechanical strength or specific biological functions of the composite material, functional nanoparticles (such as SiO2 NPs, ZnO NPs, Fe2O3 NPs, TiO2 NPs, and MgO NPs) were introduced in some parallel experimental groups in this embodiment. Specifically, the target nanoparticles were added to the above selenium-melatonin composite dispersion, with the concentration controlled at 60 mg / L. -1 The mixture is then subjected to ultrasonic dispersion for 5-15 minutes to prepare a multi-component composite active core layer slurry.

[0065] (4) Preparation of the first layer of seed coating

[0066] Immerse the seeds in the above solution, ensuring that the seeds are completely submerged. After 5 to 30 minutes, remove them and place them in a well-ventilated and dry place to air dry for 2 to 12 hours to obtain the first layer of seed coating.

[0067] 2. Preparation of the second sustained-release adhesion layer (C2):

[0068] The second layer is a slow-release adhesion layer, which uses a composite polysaccharide system composed of pectin and sodium carboxymethyl cellulose as the matrix material. Various monosaccharide components can be introduced into the composite polysaccharide system, and a hydrogel structure with a certain mechanical strength can be formed under the regulation of calcium ions and alkaline conditions. This structure is used to coat the first active core layer and realize the slow-release of active ingredients.

[0069] In one embodiment, the method for preparing the sustained-release adhesion layer includes the following steps:

[0070] (1) Preparation of matrix solution: Weigh pectin and sodium carboxymethyl cellulose and prepare a composite polysaccharide matrix solution at a mass ratio of 1:1, and control the total mass fraction of the composite polysaccharide solution to be 5%.

[0071] (2) Functional component doping: A mixed monosaccharide component is introduced into the composite polysaccharide matrix solution. The mixed monosaccharide component consists of D-(+)-xylose, L-(+)-arabinose, DL-arabinose, and L-rhamnose, with a molar ratio of 30:9:9:14. The total amount of the mixed monosaccharides added is controlled to be 2.35% of the mass of the composite polysaccharide solution. High-speed stirring is used to ensure that all components are completely dissolved to form a homogeneous system.

[0072] (3) Ionic pre-crosslinking: CaCl2 solution was added to the above system for pre-crosslinking treatment. The final concentration of CaCl2 was adjusted to 5 mM and the system was allowed to stand for 12 h to induce the initial interaction between polysaccharide molecular chains.

[0073] (4) Gelation and coating: A 2 M NaOH solution was added to the system, with the addition amount being 70% of the molar amount of pectin, to adjust the pH value of the system and initiate the gelation reaction. After 24-48 h, the first layer of seed coating prepared in step 1 of this example was immersed into the reaction system by the impregnation method. When in contact with the second layer of solution, cross-linking and gelation occurred at the interface, thereby further fixing and constructing a stable polysaccharide network structure.

[0074] (5) Curing and treatment: After soaking for 5 minutes, take it out and place it in a ventilated environment for air drying for 24 hours to obtain the second layer of hydrogel seed coating with slow-release adhesion function.

[0075] 3. Preparation of the third pH-responsive control layer (C3)

[0076] This embodiment aims to construct a third pH-responsive network layer composed of silk fibroin and chitosan (or carboxymethyl chitosan). By screening the mass ratio of the two, the optimal outer layer formulation with stress-promoting germination performance is determined. Furthermore, through independent membrane swelling tests and release experiments of the complete coating system, the response mechanism of this layer structure to environmental pH and its controlled release effect on the inner active substances are verified.

[0077] In one embodiment, the method for preparing the pH-responsive layer includes the following steps:

[0078] This embodiment is based on the design concept of "constructing a pH-responsive network with chitosan or carboxymethyl chitosan to regulate the release behavior of the inner active components according to the soil acidity and alkalinity environment". It provides a method for preparing a third pH-responsive regulatory layer (C3) and coating it on the seed surface. The method is based on the composite film formation of silk fibroin and chitosan or carboxymethyl chitosan. Through ionic crosslinking or covalent crosslinking, a stable three-dimensional responsive network is formed in the outer layer. This makes the outer membrane exhibit adjustable swelling and permeability under different pH environments, and further has a controllable influence on the release kinetics of the active substances loaded in the first two layers. Finally, a multi-layer coated seed with a complete outer regulatory structure is obtained.

[0079] 1) Preparation of silk fibroin-polysaccharide complex solution

[0080] First, prepare a chitosan or carboxymethyl chitosan solution according to the target formulation. Chitosan is preferably dissolved in an aqueous acetic acid solution with a volume fraction of 0.5% to 1.5%. The pH of the solution is adjusted to 5.0 to 5.5 under continuous stirring to ensure that the polysaccharide system has good solubility and subsequent cross-linking activity. Then, the silk fibroin solution is mixed with the polysaccharide solution at a set mass ratio (e.g., silk fibroin:chitosan or silk fibroin:carboxymethyl chitosan is 1:3, 3:1 or 1:1) and stirred thoroughly to obtain a homogeneous silk fibroin-polysaccharide composite solution.

[0081] 2) Preparation of the third seed coating

[0082] Seeds with the second coating layer completed are used as the third coating layer substrate. They are immersed in the above-mentioned silk fibroin-polysaccharide composite precursor solution for coating, so that the composite material is adsorbed and deposited on the seed surface and gradually forms a continuous outer wet film. In order to ensure the uniformity of the film layer and the integrity of the coverage, it is preferable to keep a gentle stirring or roller coating during the immersion process, and control the immersion time within the range of 5 to 30 minutes according to the seed type and the target film thickness. This allows the composite components to be fully wetted and evenly spread on the seed coat surface, while avoiding excessive deposition that would result in an excessively thick film layer and affect the water and air permeability.

[0083] 3) Cross-linking network formation and pH-responsive structure construction

[0084] After the outer wet film is formed, a crosslinking agent is added to solidify the third layer, thereby inducing the formation of an ionic or covalent crosslinking network between silk fibroin and chitosan or carboxymethyl chitosan. The crosslinking agent can be a polyphosphate crosslinking agent (e.g., sodium tripolyphosphate) to achieve ionic crosslinking between polysaccharide segments, or a natural crosslinking agent to achieve covalent or composite crosslinking between silk fibroin and polysaccharides. Under the crosslinking action, the outer layer transforms from the initial composite wet film into a three-dimensional network structure with a certain gel content and structural strength, thereby exhibiting differentiated swelling and permeability characteristics in different pH environments and providing a controllable channel for the diffusion and migration of active components in the inner layer. In another embodiment, the crosslinking agent can also be added during the impregnation and coating process to achieve simultaneous deposition and solidification, thereby further improving the adhesion stability of the film layer.

[0085] 4) Drying, curing, and preparation of the complete outer control layer

[0086] After the cross-linking reaction is complete, the seeds are removed and air-dried under ventilated conditions until the surface film is stable and non-adhesive, resulting in multi-layered coated seeds. The resulting outer layer is a silk fibroin-chitosan or silk fibroin-carboxymethyl chitosan composite film, in which chitosan or carboxymethyl chitosan provides pH-responsive properties, and silk fibroin is used to improve the structural integrity and mechanical stability of the film, thereby helping to regulate the release behavior of the inner active components by the outer layer.

[0087] Example 2:

[0088] Screening for the optimal ratio of the third pH-responsive control layer:

[0089] To determine the optimal composite ratio of the third layer material, coated seeds with different silk fibroin to chitosan mass ratios (1:3, 1:1, 3:1) and different silk fibroin to carboxymethyl chitosan mass ratios (1:3, 1:1, 3:1) were subjected to germination experiments under non-salt stress and salt stress (NaCl solution simulating salt stress) conditions. Based on the optimal ratio obtained through screening, its swelling response and ability to regulate the release behavior of active ingredients under different pH conditions were further verified.

[0090] 1. Plate germination experiment

[0091] The experiment included a silk fibroin-chitosan coating group (silk fibroin:chitosan ratio of S:CH 1:3, 3:1, 1:1), a silk fibroin-carboxymethyl chitosan coating group (silk fibroin:carboxymethyl chitosan ratio of S:CAR 1:3, 3:1, 1:1), and a blank control group (CK). Each treatment was replicated in six cases (n=6). Seedling growth images, germination rate, fresh weight, shoot length, root length, and seed vigor index were continuously monitored and recorded for 7 days. Root morphology indicators such as total root length, root surface area, root volume, and root tip number were quantitatively analyzed using a root scanning system. The optimal silk fibroin-chitosan and silk fibroin-carboxymethyl chitosan ratios were screened through plate germination experiments to determine the best polysaccharide material for subsequent experiments.

[0092] 2. pH response characteristics

[0093] A third pH-responsive control layer (a silk fibroin-chitosan composite membrane, abbreviated as C3) was prepared using the optimal formulation. The dried membrane was cut to the same size (e.g., 10 mm × 10 mm), and the initial dry weight W0 was recorded. The membrane samples were placed in buffer solutions at pH 5.0, 7.0, and 9.0, respectively, and swelling experiments were conducted at room temperature (25℃). Samples were taken and weighed at preset time points (0, 1, 2, 4, 6, 8, 12, 18, 24, 30, 36, 42, 48, 54, 60, and 68 h; after removing surface liquid, the wet weight W was measured). t The formula for calculating the swelling ratio (SR) is: SR (%) = (W / W) t -W0) / W0×100% Parallel samples (n=6) were set up at each time point, and swelling curves were plotted to compare the swelling behavior under different pH conditions.

[0094] 3. Determination of sustained-release behavior of active ingredients

[0095] To evaluate the sustained-release behavior of active ingredients in three-layer coated seeds under different environmental conditions, selenium was used as a tracer. Equal numbers of coated seeds were placed in Erlenmeyer flasks, and 50 mL of pre-prepared and temperature-equilibrated release media (pH 5.0, 7.0, and 9.0 buffer solutions) were added. Three replicates were set for each group to ensure complete seed immersion. The flasks were incubated at 25°C.

[0096] Samples of 1 mL were taken at 6h, 12h, 24h, 36h, 48h, 60h, and 72h for testing, and an equal volume of fresh buffer solution was added at the same temperature. The supernatant was centrifuged and filtered, and the Se concentration was determined by ICP-MS. The cumulative release at each time point was calculated, and a cumulative release curve from 0 to 72h was plotted.

[0097] 4. Mechanical property testing

[0098] To evaluate the mechanical properties of the coating material, compression tests were used to characterize gel samples from different treatment groups. The following treatment groups were established: group C1C2, which introduced a second polysaccharide layer on top of the core layer; and group L123, which constructed a third silk fibroin-chitosan composite structure on top of the core and second polysaccharide layers but without nanoparticles. The mechanical properties of the multilayer coating were investigated. Non-shrinkage compression tests were performed on circular gel samples (n = 3, diameter 38 mm, thickness 16 mm) using an electronic universal testing machine. The entire experiment was filmed up to 30% strain compression. Compression stress-strain curves were output.

[0099] Experimental results:

[0100] (1) Effects of different proportions of silk fibroin-carboxymethyl chitosan (S:CAR) seed coating on maize germination

[0101] The effects of different proportions of silk fibroin-carboxymethyl chitosan (S:CAR) seed coating on maize germination are shown in the following results. Figure 2 As shown, different ratios of silk fibroin-carboxymethyl chitosan (S:CAR) composite coating significantly regulate maize germination and early growth. Under non-salt conditions, S:CAR (1:1) showed the best overall effect, significantly increasing maize fresh weight, shoot length, root length, and seed vigor by 36.03%, 24.73%, 25.18%, and 35.81%, respectively. The overall ratio showed a pattern of 1:1 > 3:1 > 1:3. Under salt stress (100 mM NaCl), S:CAR (1:1) still exhibited the strongest mitigation effect, significantly increasing fresh weight, shoot length, and root length by 29.32%, 36.44%, and 31.15% compared to the control (CK), and improving seed vigor by 35.37%; while other ratios had limited growth-promoting effects. These results indicate that S:CAR (1:1) maintains a stable advantage under both salt stress and non-salt conditions and is the preferred ratio for silk fibroin-carboxymethyl chitosan seed coating.

[0102] (2) Effects of different proportions of silk fibroin-carboxymethyl chitosan (S:CAR) seed coating on maize root development

[0103] The effects of different proportions of silk fibroin-carboxymethyl chitosan (S:CAR) composite hydrogel seed coating on maize roots under 100 mM NaCl salt stress are as follows: Figure 3As shown in the figure. Under non-salt conditions, compared with the CK group, the S:CAR (1:1) treatment group significantly promoted root development, with a total root length increase of approximately 29.94%, root surface area increase of approximately 63.09%, root volume increase of approximately 39.36%, and root tip number increase of approximately 155.05%. Under 100 mM NaCl salt stress, compared with the CK group, S:CAR (1:1) coating significantly increased total root length by approximately 22.45%, root surface area by approximately 9.08%, root volume by approximately 37.46%, and root tip number by approximately 80.54%. The results confirm that S:CAR (1:1) coating has a dual effect of promoting normal growth and alleviating salt stress, which is attributed to the synergistic mechanism of material properties and root architecture remodeling. On the one hand, the high water-retention capacity of carboxymethyl chitosan and the stable network structure of silk fibroin form a physical buffer layer in the rhizosphere, effectively delaying high salt infiltration and reducing water loss, thereby alleviating physiological drought caused by salt stress. On the other hand, and more importantly, this coating greatly enhances root tip differentiation capacity and increases root surface area. This well-developed root system configuration enhances the plant's ability to capture water and nutrients in low-water-potential saline environments, improving maize's salt tolerance from a morphological perspective.

[0104] (3) Effects of different ratios of silk fibroin-chitosan (S:CH) seed coating on maize germination

[0105] The effects of different ratios of silk fibroin-chitosan seed coating on maize germination are as follows: Figure 4 As shown, under both saline and non-salt conditions, all six treatment ratios significantly promoted maize fresh weight, shoot length, root length, and seed vigor. Specifically, under non-salt conditions, compared with the control (CK), each treatment increased maize fresh weight by 35.29%–53.23%, shoot length by 26.25%–47.60%, root length by 14.96%–52.01%, and seed vigor by 16.19%–53.63%; under saline conditions, each treatment increased maize fresh weight by 37.19%–48.30%, shoot length by 35.27%–40.42%, root length by 23.40%–37.30%, and seed vigor by 49.20%–62.30%. Under both saline and non-salt conditions, the S:CH (3:1) treatment showed the best results for all indicators. Its fresh weight, shoot length, root length, and seed vigor increased by 53.23%, 47.60%, 52.01%, and 53.63% respectively compared to the control under non-salt conditions, and by 48.30%, 40.42%, 37.30%, and 62.30% respectively under saline conditions. S:CH (3:1) is the best treatment for promoting maize growth and improving maize salt tolerance.

[0106] (4) Effects of different ratios of silk fibroin-chitosan (S:CH) seed coating on maize root development

[0107] The effects of different ratios of silk fibroin-chitosan seed coating on maize root development are as follows: Figure 5 As shown, overall, S:CH(3:1) performed best. Under non-salt conditions, it increased total root length by approximately 59.05%, root surface area by approximately 36.08%, root volume by approximately 49.17%, and root tip number by approximately 58.84%. Under salt conditions, compared with the CK group, S:CH(3:1) increased total root length by approximately 45.26%, root surface area by approximately 38.28%, root volume by approximately 106.67%, and root tip number by approximately 56.25%. These results indicate that S:CH(3:1) composite hydrogel coating not only promotes root growth under normal conditions but also significantly alleviates the damage to root architecture caused by salt stress and improves salt tolerance. Its mechanism of action may come from the following synergistic effects: First, the composite hydrogel forms a water-retaining coating layer around the seed. The film-forming and water-retaining properties of chitosan and the network stability of a high proportion of silk fibroin work together to delay salt entry and reduce water loss in the early stage of stress, thereby alleviating "physiological drought"; Second, chitosan has the biological activity of promoting growth and inducing stress resistance. The amino acids produced by the degradation of silk fibroin may also provide nutrition for the early growth of seedlings; Third, more importantly, the S:CH (3:1) ratio may form a more suitable pore and mechanical structure, which facilitates root penetration and material exchange, and significantly increases the number of root tips, root surface area and root volume, morphologically reshaping a more "developed" root system, improving the plant's ability to obtain water and mineral nutrients in low water potential and high salt environment, thereby enhancing salt stress adaptability.

[0108] The combined effects of silk fibroin-polysaccharide composite seed coating on salt tolerance-related indicators of maize under salt stress were evaluated using radar charts to screen for the optimal ratio. The results are as follows: Figure 6 As shown in the radar chart, the overall response of each treatment to multiple indicators such as shoot length, root length, and root morphology is clearly reflected. A larger coverage area and a more even distribution indicate a better overall salt tolerance effect. Based on this analysis, Figure 6 (a)~(b) show that the combination of silk fibroin and carboxymethyl chitosan (S:CAR) can promote seedling growth to varying degrees, with S:CAR (1:1) showing the best overall performance and a greater improvement in most traits. In contrast, Figure 6(c)~(d) show that the silk fibroin:chitosan complex (S:CH) has a more significant promoting effect. Among them, S:CH (3:1) is significantly better than other ratios in terms of average indicators and overall expansion in radar charts, and has a more comprehensive and stable improvement on shoot length, root length, and root-related traits. Combining the antibacterial and resistance-inducing effects of chitosan and the good film-forming and water-retention properties of silk fibroin, it is speculated that the S:CH (3:1) ratio is more conducive to forming a reasonable balance between membrane permeability, water absorption, and structural strength. However, an excessively high proportion of chitosan may lead to an overly dense membrane, affecting seed imbibition and oxygen exchange. In conclusion, it is recommended that S:CH (3:1) be given priority as the optimal ratio for further verification and application research in subsequent seed coating experiments.

[0109] (5) Results of pH responsiveness experiment

[0110] The third pH-responsive regulating layer (C3) is composed of silk fibroin and chitosan. The swelling kinetics curves in different pH buffer solutions are shown below. Figure 7 As shown, in the swelling kinetics experiment from 0 to 68 h, the C3 film exhibited a significant pH-dependent swelling behavior: the swelling rate was fastest at pH 5.0, followed by pH 7.0, while the swelling degree was lowest at pH 9.0. This difference indicates that the silk fibroin-chitosan composite network has typical pH-triggered volume response characteristics, providing a structural basis for the pH difference in subsequent release behavior. The mechanism may be due to the charge state and electrostatic interaction between silk fibroin and chitosan under different pH conditions. Under slightly acidic conditions, the protonation degree of chitosan increases, and the network structure is relatively larger, which is more conducive to the penetration of water molecules. As the pH increases, silk fibroin becomes negatively charged, forming an electrostatic attraction with the positively charged chitosan, enhancing the intersegmental interaction and increasing the network density, thereby limiting the entry of water molecules and inhibiting swelling. In the acid-base alternating cycle experiment, the cross-linked C3 film still maintained good swelling-shrinkage reversibility and structural stability; in contrast, the non-cross-linked film is prone to softening or local dissolution under high water content conditions, leading to a decrease in response repeatability. The above results indicate that the cross-linked network structure helps to improve the stability and durability of the membrane in an aqueous environment.

[0111] (6) Results of sustained-release performance of active ingredients

[0112] For details on the cumulative release curves of selenium, the active ingredient in seed coatings under different pH conditions, please refer to [link / reference needed]. Figure 8 Studies on the release behavior of active ingredients in coated seeds under different pH conditions have revealed that the complete three-layer coating system exhibits significant environmentally responsive sustained-release characteristics.

[0113] The cumulative release of selenium also exhibited a typical controlled-release curve: rapid release in the early stages, followed by a decrease and smoothing of the release rate. The release rate showed a significant pH dependence: pH 5.0 > pH 7.0 > pH 9.0. At 72 h, the cumulative release was highest at pH 5.0 (87.4 ± 3.24%), moderate at pH 7.0 (67.73 ± 5.21%), and lowest at pH 9.0 (47 ± 3.21%).

[0114] Higher release rates under acidic conditions are consistent with a stronger swelling tendency: at pH 5.0, membrane swelling leads to increased diffusion channels, thus promoting the release of active ingredients. A more porous membrane with higher water content and more abundant diffusion channels facilitates the migration of selenium from the coating matrix, resulting in higher cumulative release. Under alkaline conditions, the amino groups of chitosan are in a deprotonated state, causing membrane shrinkage and increased diffusion resistance, inhibiting the release process. Furthermore, the rhizosphere is a highly active microenvironment directly influenced by the root system, exhibiting significant pH fluctuations. To improve the efficiency of obtaining insoluble nutrients such as phosphorus and iron, crops often induce localized acidity near the root surface through H⁺ efflux and the secretion of organic acids such as citric acid, malic acid, and oxalic acid. Chitosan coating can utilize this acidic signal to achieve responsive controlled release, enhancing membrane hydrophilicity and significantly swelling, thereby accelerating the release of active selenium (such as nano-selenium) driven by the concentration gradient. Under neutral or alkaline conditions, deprotonation leads to a denser structure and restricted diffusion, thus achieving delayed release and "on-demand supply." The above results indicate that the three-layer coating structure can achieve controlled release of active ingredients through a pH-responsive mechanism, thereby better matching the dynamic needs for nutrients and regulatory factors during seed germination and early growth.

[0115] (7) Mechanical properties

[0116] For details of the compressive stress-strain curves of seed coatings with different numbers of layers, please refer to [link / reference]. Figure 9 Both the C1C2 and C1C2C3 groups exhibited typical nonlinear stress-strain responses of hydrogel materials within the 0–30% strain range, meaning that the compressive stress continuously increased with increasing compressive strain. Compared to the bilayer structure containing only C1C2, the introduction of the third C3 hydrogel layer shifted the stress-strain curve of the C1C2C3 group upwards overall, especially in the medium-to-high strain region, indicating that the introduction of the third layer further enhanced the compressive load-bearing capacity of the overall coating system. From a mechanical behavior perspective, the C3 layer participates in load-bearing during compression and provides constraint to the inner structure, making the coating less prone to local collapse under pressure, thus enabling it to withstand higher compressive stress at the same strain level. The compressive stress of the C1C2C3 group was higher than that of the C1C2 group, indicating that the introduction of the third hydrogel layer effectively enhanced the coating structure's resistance to compression damage. This characteristic has important mechanical significance for maintaining the integrity of the seed coating structure under actual working conditions such as sowing, transportation, and soil compression.

[0117] Example 3

[0118] The effects of multi-layer seed coating on maize growth under salt stress:

[0119] In this embodiment, the preparation method of the multi-layer intelligent seed coating agent is generally the same as in Example 1, except that: in the first active core layer, MgO NPs are further introduced into the melatonin-nano selenium composite system, and the concentration of MgO NPs is 60 mg / L. -1 The composition and preparation method of the second sustained-release coating layer are the same as in Example 2; the third outer coating layer is constructed using the optimal ratio of silk fibroin and chitosan (mass ratio of 3:1) obtained from the aforementioned screening. Except for the above differences, the remaining preparation steps and conditions are consistent with the aforementioned examples.

[0120] Experimental verification:

[0121] 1. Test soil

[0122] Soil samples were collected from the coastal saline soil area of ​​Dongying City, Shandong Province (118°37'E, 37°18'N). The topsoil from 0 to 20 cm was obtained using the five-point sampling method. After natural air drying, the soil was sieved through a 2 mm sieve to remove stones and plant debris.

[0123] Table 1 Basic physical and chemical properties of soil

[0124] pH <![CDATA[EC(μS cm -1 )]]> <![CDATA[Total salt content (g kg -1 ).]]> 8.33±0.06 224.9±3.25 3.8±0.12

[0125] 2. Test plants

[0126] Maize was selected as the test plant.

[0127] 3. Potted plant experiment

[0128] To investigate the effect of multi-layer intelligent seed coating structure on salt tolerance of maize, a 30-day pot experiment was conducted. Saline-alkali soil was used as the test soil, and different treatment systems were constructed using a multi-layer seed coating method. The following treatment groups were set up: naked seed control group (CK); treatment group with only a carrier layer applied (Car); treatment group with melatonin-nano selenium as an active core layer (C1); treatment group with a second polysaccharide structure further introduced on top of the core layer (C1C2); treatment group with a third silk fibroin-chitosan composite structure constructed on top of the core layer and the second polysaccharide layer but without nanoparticles (L123); treatment group with MgO NPs introduced into the complete three-layer structure (L123+NP); and treatment group with the pH-responsive outer layer removed from the L123+NP system, i.e., only silk fibroin was added to the third layer without chitosan (L123+NP-npH). Each treatment was set up with 6 replicates.

[0129] The tested maize seeds (Zea mays L.) were first disinfected with 10% H2O2 for 30 min, and then repeatedly rinsed with sterile deionized water to remove residual disinfectant. Seeds coated with different treatment systems were sown in flowerpots filled with saline-alkali soil and cultured under the same conditions for subsequent determination of plant growth, physiological and biochemical indicators.

[0130] 4. Experimental Results

[0131] (1) Results of the effect of multi-layer intelligent seed coating agent on maize growth under salt stress

[0132] The effects of multi-layer intelligent seed coating agent on maize growth under salt stress are as follows: Figure 10 As shown in the examples, the results of the examples indicate that, compared with the control group, the multi-layer coating system significantly improved the overall plant growth indicators. Regarding plant height, compared with CK, the carrier layer treatment alone increased height by 26.07%, indicating a relatively limited promoting effect, suggesting that the carrier itself is not the main source of function. After introducing the melatonin-nano selenium active core layer, plant height increased by 54.93% compared to CK, indicating that the active core plays a key role in promoting vegetative growth. Based on this, further introducing a second polysaccharide structure and a third silk fibroin-chitosan composite structure increased plant height by 83.89% and 105.06% respectively compared to CK. Plant height continued to increase with the number of coating layers, indicating that the multi-layer structure can better protect the active substances, stabilize the system, and achieve more effective release regulation. The introduction of MgO NPs in the complete three-layer coating showed the best effect, increasing plant height by 114.44% compared to CK. Removing the pH-responsive outer layer reduced the increase to 107%, significantly weakening the advantage, indicating that the pH-responsive outer layer makes an important contribution to maintaining its growth-promoting advantage in the three-layer system containing MgO NPs.

[0133] The plant growth indicators also showed a consistent trend: the carrier layer increased by 24.68% compared to the control (CK), the active core layer by 31.19%, the addition of the second layer by 33.53%, and the addition of the third layer by 34.81%. The treatment with the introduction of MgONPs in the complete three-layer coating showed the most significant improvement, increasing by 46.88% compared to the control (CK); the improvement decreased to 34.61% after removing the pH-responsive outer layer. In general, the more complete the coating layers and the more rational the structure, the better the growth-promoting effect on maize.

[0134] (2) Results of the effect of multi-layer intelligent seed coating agent on chlorophyll and nitrogen content of maize seedlings under salt stress

[0135] The effects of multi-layer intelligent seed coating on chlorophyll and nitrogen content in maize leaves and seedlings under salt stress, such as Figure 11As shown in a~b. Overall, coating treatments can increase chlorophyll and nitrogen content, with more complete structures resulting in more significant increases. Regarding chlorophyll content, compared to the control (CK), the Car carrier layer (Car) only increased by about 17.19%, a relatively limited increase; the addition of the active core layer (C1) increased chlorophyll by 19.38%. With increasing coating layers, C1C2 and L123 continued to increase by about 21.88% and 25.31%, respectively, indicating that multi-layer structures can more stably maintain leaf metabolism. The L123+NP treatment group, with its complete three layers and the introduction of MgO NPs, showed the most significant increase, resulting in a significant chlorophyll increase of about 38.13%; after removing the pH-responsive outer layer, the increase dropped to about 31.35%, indicating that the pH-responsive outer layer plays a crucial role in maintaining chlorophyll content. Regarding nitrogen content, Car and C1 increased by 19.01% and 21.81%, respectively, with C1 showing a more significant increase, indicating that the active core layer significantly enhances nitrogen absorption and assimilation. C1, C2, and L123 further increased by 26.60% and 29.39%, respectively, demonstrating that the multilayer structure has a sustained promoting effect on nitrogen accumulation. The L123+NP treatment group had the highest content, with a significant increase of 31.79% in nitrogen content; after removing the pH-responsive outer layer, the nitrogen content increased by 29.79%. Considering both indicators, the active core layer can significantly increase the chlorophyll and nitrogen content of maize seedlings, with the increase further enhanced with the increase in the number of coating layers. Furthermore, the effect was best after adding MgO NPs to the three-layer structure.

[0136] (3) Results of the effect of multilayer intelligent seed coating agent on photosynthetic parameters of maize seedlings under salt stress

[0137] The effects of multi-layer intelligent seed coating agent on photosynthetic parameters of maize seedlings under salt stress are as follows: Figure 11 As shown in c~h. Overall, compared with CK, all coating treatments improved leaf gas exchange and CO2 assimilation processes, and the improvement increased with the improvement of the structure from single-layer to multi-layer. The carrier layer Car had a relatively limited effect on photosynthetic enhancement, but it still increased Pn, Gs, and Tr by 74.25%, 50.07%, and 45.00%, respectively, indicating that the carrier mainly played a certain role in buffering and protection. After adding the active core layer (C1), the three indicators were further significantly improved to 93.94%, 60.55%, and 82.50%, indicating that the active component was the key source for promoting photosynthetic capacity enhancement. With the advancement of coating structure, the promoting effects of C1C2 and L123 on Pn, Gs and Tr are further enhanced: compared with CK, C1C2 increases by 118.18%, 78.98% and 105.00% respectively, and L123 increases by 142.43%, 94.87% and 127.50% respectively, indicating that the multilayer structure is more conducive to stable function and maintaining a high gas exchange level under salt stress.

[0138] Ci decreased under all coating treatments, with a greater decrease in the more complete the structure (Car, C1, C1C2, and L123 decreased by 44.26%, 45.90%, 49.18%, and 52.40%, respectively). Combined with the continuous increase in Pn, it can be inferred that the enhanced photosynthesis is not only due to improved stomatal conductance but also closely related to improved mesophyll assimilation efficiency. Among all treatments, L123+NP with complete three layers and the introduction of MgO NPs showed the best effect. Compared with the control (CK), Pn, Gs, and Tr increased by 157.58%, 107.72%, and 132.50%, respectively, while Ci decreased by 57.38%, indicating that it enhanced both gas exchange and significantly improved CO2 assimilation and utilization. Water-related indicators also improved simultaneously, with WUE increasing by 10.13% and Ls by 193.62%, indicating that this system was more effective in regulating stomatal limitation and water use under salt stress. In contrast, while L123+NP–npH, lacking a pH-responsive outer layer, still significantly improved Pn, Gs, and Tr (by 145.46%, 98.77%, and 130.00%, respectively, while Ci decreased by 53.44%, WUE increased by 6.58%, and Ls increased by 180.26%), it was weaker overall than L123+NP. This indicates that the pH-responsive outer layer can further stabilize the coordination between stomatal regulation and CO2 assimilation, thereby achieving a more sustained and greater boost in photosynthesis.

[0139] (4) Effects of multilayer smart seed coating agent on antioxidant system and membrane lipid peroxidation level of maize seedlings under salt stress

[0140] Malondialdehyde (MDA) is one of the typical end products of membrane lipid peroxidation and can indicate the degree of oxidative damage to cell membranes; superoxide dismutase (SOD) is a scavenger of superoxide anion free radicals (…). The activity of key enzymes in the membrane lipid peroxidation group reflects the plant's antioxidant defense capacity. Under salt stress, the control group showed higher levels of membrane lipid peroxidation and relatively insufficient antioxidant defense. Compared with the control group, all coating treatments showed a synchronous change of "increased SOD and decreased MDA", indicating that coating can alleviate salt stress damage by enhancing the antioxidant system and reducing membrane lipid peroxidation.

[0141] The effects of multilayer smart seed coating agents on the antioxidant system and membrane lipid peroxidation level of maize seedlings under salt stress, such as Figure 12As shown, in terms of SOD, Car, C1, and C1C2 increased by 6.67%, 10.67%, and 13.33% respectively compared to CK, indicating that basic coating can activate the antioxidant system to a certain extent. Further multi-layer coating resulted in even more significant improvements: L123 increased by 20.00%, L123+NP increased by 32.00%, and L123+NP–npH increased by 23.33%. This demonstrates that with the layering of coating and functional components, the seedlings' ability to scavenge ROS induced by salt stress is more fully enhanced, and the antioxidant defense line is more easily and quickly established. The L123+NP treatment group significantly increased the SOD level of maize seedlings. Regarding MDA, all treatments decreased compared to CK: Car decreased by 6.67%, C1 decreased by 8.00%, and C1C2 decreased by 20.67%. Multi-layer coating further amplified the decrease: L123 decreased by 26.67%, L123+NP decreased by 33.33%, and L123+NP–npH decreased by 25.00%. The continuous decrease in MDA and the gradual increase in SOD corroborate each other, indicating that the coating treatment not only enhanced the activity of antioxidant enzymes in maize but also reduced the accumulation of membrane lipid peroxidation end products, thus demonstrating a protective effect on cell membrane stability. In summary, the multilayer intelligent seed coating agent alleviates oxidative damage under salt stress by enhancing ROS scavenging ability and reducing the level of membrane lipid peroxidation reflected by MDA, with the effect increasing with increasing structural complexity. L123+NP showed the best overall performance, indicating that it has a more significant application potential in improving the salt tolerance of maize seedlings under the conditions of this experiment.

[0142] (5) Results of the effect of multilayer smart seed coating agent on ionic homeostasis of maize under salt stress

[0143] Under salt stress, the Na+ content in the aboveground parts of plants... + With K + The content and its equilibrium relationship are important indicators reflecting the ionic homeostasis. Na + Excessive accumulation in the aboveground parts can cause ion toxicity and interfere with cell metabolism and membrane system stability; K + These are key ions for maintaining osmotic regulation, stomatal movement, and various enzymatic reactions. Therefore, by reducing the Na+ content in the aboveground parts... + Accumulate and increase K in the aboveground parts + Maintaining a horizontal level can be used to characterize the ability of the coating system to regulate ion homeostasis under salt stress. The results of the effect of multilayer smart seed coating agents on maize ion homeostasis under salt stress are as follows: Figure 13 As shown, compared with the control (CK), all coating treatments reduced the Na+ content in the aboveground parts. + The content of Na+ in the aboveground parts decreases, and the degree of decrease gradually increases with the coating structure from single-layer to multi-layer. Car and Cl respectively reduce the Na+ content in the aboveground parts. +The levels decreased by 5.45% and 9.09%; when the coating structure progressed to two layers (C1C2) and three layers (L123), the aboveground Na... + The percentages decreased to 21.82% and 25.45% respectively, indicating that the multilayer structure is more conducive to suppressing Na+. + Accumulation in the aboveground parts. Furthermore, after introducing MgO NPs into the three-layered structure, L123+NPs cause Na+ accumulation in the aboveground parts. + Reduced by 49.09%; removal of the pH-responsive outer layer of L123+NP–npH reduced the Na+ content in the aboveground parts. + A 35.15% reduction indicates that retaining the pH-responsive outer layer effectively inhibits Na+. + The facilitating effect of accumulation is more effective. In K + In terms of efficacy, compared with CK, all coating treatments improved the K content of the aboveground parts. + The content of K in the aboveground parts increases with structural progression. Car and C1 respectively reduce the content of K in the aboveground parts. + Increased by 4.80% and 5.60%; C1C2 and L123 increased by 14.40% and 15.60% respectively. After introducing MgO NPs into the three-layer structure, L123+NP increased the Ka of the aboveground part. + Increased by 28.00%; L123+NP–npH reduced K in the aboveground parts + The increase of 23.60% indicates that the complete system promotes K + It is superior in terms of maintenance.

[0144] Comprehensive Na + With K + The changes indicate that the multi-layer intelligent seed coating system can reduce the accumulation of Na+ in the aboveground parts of corn under salt stress. + Multiple K retention + This effectively regulates ion homeostasis, which is more conducive to normal plant growth. Among them, the coating system with a three-layer structure, introducing nanoparticles and retaining the pH-responsive outer layer, shows a more significant improvement in this ion balance.

[0145] To address the problems of uncontrollable release of active ingredients, difficulty in responding to soil pH fluctuations, and limited effectiveness in regulating early crop salt tolerance under saline-alkali soil conditions, existing seed coating technologies employ a "seed-based, site-appropriate" design concept. Using soil pH as a trigger signal, a three-layer intelligent coating structure with clearly defined functions and synergistic effects is constructed on the seed surface. The outer pH-responsive control layer gates and regulates the release process of the inner salt-resistant active components, enabling on-demand release of active substances in saline-alkali environments. This invention provides a pH-biomimetic triggered multilayer intelligent seed coating agent and its preparation method for improving crop salt tolerance. The coating agent comprises, from the inside out, a salt-resistant active core layer, a biomimetic slow-release attachment layer, and a pH-responsive control layer, achieving stable loading, slow-release supply, and responsive regulation of active components in response to changes in soil pH. Experimental results demonstrate that this multilayer coating system significantly improves seedling growth and photosynthetic performance under salt stress conditions, enhancing antioxidant defense capabilities, reducing membrane lipid peroxidation levels, and regulating Na+. + / K + Ion balance effectively alleviates salt stress damage, with a coating system exhibiting superior overall salt tolerance due to its complete three-layer structure, the introduction of functional nanoparticles, and the retention of a pH-responsive outer layer. In summary, this invention, by constructing a multi-layer coating system on the seed surface—comprising salt-resistant active components, a biomimetic water-retaining and slow-release structure, and a pH-responsive regulating layer—forms an intelligent seed coating technology adaptable to changes in saline-alkali soil environments. This technology demonstrates good application feasibility and promising prospects, and can be used for pre-sowing treatment of crops in saline-alkali land to improve seedling emergence quality and seedling salt tolerance.

[0146] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance, characterized in that: The seed coating agent covers the seed surface and consists of a first active core layer, a second slow-release attachment layer, and a third pH-responsive regulation layer from the inside out. The pH-responsive regulation layer uses soil acidity and alkalinity as a response trigger signal and regulates the release process of the salt-resistant active components in the inner layer by adjusting its own permeability.

2. The pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 1, characterized in that: The first active core layer is composed of melatonin-nano selenium composite material. Melatonin is loaded onto the surface of nano selenium through adsorption, coordination or coating to form a stable nanocomposite system.

3. The pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 1, characterized in that: The second slow-release adhesion layer is a composite solution composed of pectin, sodium carboxymethyl cellulose and monosaccharides. A slow-release network structure is constructed through ionic cross-linking to achieve the slow release of active ingredients and enhance adhesion to the seed surface.

4. The pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 1, characterized in that: The third pH-responsive control layer is composed of chitosan or carboxymethyl chitosan as pH-responsive functional components, and silk fibroin as a structural support material to form a composite network with the chitosan or carboxymethyl chitosan. This network is used to adjust the swelling and permeability of the coating layer according to changes in soil acidity and alkalinity, thereby achieving on-demand control of the release of the inner active components.

5. The pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 1, characterized in that: The first active core layer also includes functional nanoparticles or carbon-based nanoparticles; The functional nanoparticles are nano-magnesium oxide.

6. The method for preparing a pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to any one of claims 1-5, comprising the steps of sequentially constructing an active core layer, a slow-release attachment layer, and a pH-responsive regulation layer on the seed surface, wherein the third layer achieves regulation of the release process of the inner active components by constructing a composite network structure with pH-responsive characteristics, characterized in that: (1) Preparation of the first active core layer: 1) Take sodium selenite aqueous solution as selenium precursor, add melatonin solution to sodium selenite aqueous solution, the melatonin solution is prepared by dissolving in a small amount of ethanol and then diluting; 2) Slowly add ascorbic acid solution as a reducing agent under stirring conditions to generate nano-selenium particles and simultaneously form a melatonin-nano-selenium composite system. Continue stirring for 30-60 min, adjust the pH of the system to 6-7, and obtain a stable dispersion of melatonin-nano-selenium composite nanomaterials. 3) Immerse the seeds in a melatonin-nano-selenium composite nanomaterial dispersion, ensuring that the seeds are completely immersed in the solution. After 5-30 minutes, remove the seeds and place them in a ventilated and dry place to air dry for 2-12 hours to obtain the first layer of seed coating. (2) Preparation of the second sustained-release adhesion layer: 1) A complex polysaccharide solution was prepared using pectin and sodium carboxymethyl cellulose as raw materials; 2) Add the mixed monosaccharide solution to the complex polysaccharide solution and stir thoroughly until completely dissolved; 3) Add calcium chloride solution to adjust the mechanical strength of the hydrogel and let it stand for 12 h; add 2 M NaOH solution with 70% of the pectin molar amount, and after 24~48 h, immerse the first layer of seed coating obtained in step (1) into the above system to form a hydrogel structure on the seed surface; after the hydrogel is formed, soak for 5 min, take it out, and air dry for 24~48 h to obtain the second layer of hydrogel seed coating with slow-release adhesion function. (3) Preparation of the third pH-responsive control layer: 1) Prepare a silk fibroin-polysaccharide composite solution according to the required ratio. Dissolve the polysaccharide in an aqueous acetic acid solution with a volume fraction of 0.5% to 1.5% as a solvent, and adjust the pH of the solution to 5.0 to 5.

5. Then mix the silk fibroin solution and the polysaccharide solution and stir thoroughly to obtain a homogeneous silk fibroin-polysaccharide composite solution. 2) Immerse the seeds with the second coating obtained in step (2) into the silk fibroin-polysaccharide composite solution to deposit and form a third outer coating structure on the seed surface. During or after the coating process, add sodium tripolyphosphate to the silk fibroin-polysaccharide composite solution to form an ionic cross-linking or covalent cross-linking network between the silk fibroin and chitosan or carboxymethyl chitosan molecules, thereby constructing a silk fibroin-polysaccharide composite regulatory layer structure with pH response characteristics.

7. The preparation method of the pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 6, characterized in that: The sodium selenite aqueous solution in (1) has a concentration of 0.5–5 mM and a melatonin concentration of 0.15 mg / mL. -1 The molar ratio of ascorbic acid to sodium selenite is 4:1; Alternatively, functional nanomaterials or carbon-based nanomaterials may be added to the melatonin-selenium nanocomposite nanomaterial dispersion of (1); the concentration of the functional nanomaterials in the melatonin-selenium nanocomposite nanomaterial dispersion is 10–100 mg / L. -1 .

8. The preparation method of the pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 6, characterized in that: In (2), the mass ratio of pectin to sodium carboxymethyl cellulose is 1:1, and the total mass fraction of the complex polysaccharide system is 5-10%. Alternatively, in (2), the mixed monosaccharides include D-(+)-xylose, L-(+)-arabinose, DL-arabinose, and L-rhamnose, with a molar ratio of 30:9:9:14, and the total mass fraction of the monosaccharides is 2-5%; Or, in (2), the final concentration of calcium chloride is 5 mM.

9. The preparation method of the pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance according to claim 6, characterized in that: In (3), the polysaccharide is chitosan or carboxymethyl chitosan, and the mass ratio of silk fibroin to polysaccharide is 1:3, 3:1 or 1:

1. Alternatively, in (3), the final concentration of sodium tripolyphosphate is 5 mM.

10. The application of the pH-biomimetic triggered multilayer smart seed coating agent for improving crop salt tolerance as described in any one of claims 1-5, characterized in that, The seed coating agent is used for pre-sowing treatment of crops under saline-alkali soil conditions. It regulates the release of salt-resistant active components in response to changes in soil pH, thereby improving seed germination rate and enhancing seedling salt tolerance.

Images