An efficient germination substrate of anemone stellata based on bionic structure design and a preparation method thereof

Abstract

The application discloses a high-efficiency germination substrate of Puccinellia distans based on a bionic structure design and a preparation method thereof, and belongs to the field of agricultural technology, and aims to solve the problems of single substrate structure, insufficient dynamic demand matching and function space-time mismatching. The substrate constructs a bionic layered structure with differentiated functions and interlayer biochemical cascade response, and sequentially stacks a germination induction layer, a root system establishment transition layer and a dynamic aeration base layer. The germination induction layer contains enzyme microcapsules, the root system establishment transition layer contains a degradation framework and microbial microspheres, and the dynamic aeration base layer contains gas-producing particles. Each layer realizes function coupling through biochemical reactions triggered by plant secretions and dynamically optimizes the microenvironment. The preparation method comprises material preparation and molding and packaging. The application effectively improves the germination rate, root system establishment quality and seedling survival rate of Puccinellia distans under saline-alkali stress.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically, it relates to a high-efficiency germination substrate of *Imperata cylindrica* based on biomimetic structural design and its preparation method. Background Technology

[0002] Soil salinization is a major challenge to global ecosystems, hindering sustainable agricultural development and ecological health. The core of saline-alkali land restoration is vegetation recovery, and selecting highly salt-tolerant pioneer plants is an effective way to improve soil and promote positive ecological succession. *Imperata cylindrica*, a typical halophyte, has great potential in the remediation of saline-alkali land in northern China. However, its seeds are subjected to stresses such as high salt and high pH under natural conditions, resulting in extremely low germination and seedling survival rates. Therefore, artificially creating a highly efficient seedling substrate that precisely matches its initial growth dynamics is crucial for improving the success rate of ecological restoration.

[0003] To meet the needs of plant growth, technicians have developed various artificial seedling substrates. The mainstream approach is the homogeneous mixing method, which physically blends inert materials such as coconut shell biochar, vermiculite powder, and expanded perlite in a specific ratio to form a homogeneous medium, and may also include beneficial microbial agents. These methods have solved basic nutritional and physical environment problems in general seedling cultivation. However, for plants like *Imperata cylindrica*, which are highly sensitive to the initial microenvironment, the limitations of homogeneous substrates are extremely prominent. Plant growth is a dynamic, phased process, and the microenvironmental requirements at different stages exhibit significant "temporal heterogeneity": the seed germination stage requires a stable interface with high water retention and low salt ion activity to overcome osmotic stress, while the root formation stage requires a channel with high aeration and low water resistance to ensure respiration and prevent anaerobic rot. Homogeneous substrates cannot respond to these dynamic needs, leading to severe functional conflicts and spatiotemporal mismatches. Specifically, large particles used for deep aeration are indiscriminately distributed on the surface, easily causing seeds to be "elevated" and accelerating water evaporation, directly stressing seed germination. Simultaneously, fine particles used for surface water retention are abundant in the deeper layers, easily creating saturated water-holding zones after irrigation, leading to severe oxygen deficiency in the lower layers, directly conflicting with the core needs of root growth. Furthermore, bioactive components such as microorganisms are simply blended and diluted, failing to perform their precise functions at the right time and place.

[0004] To address these issues, existing technologies have attempted simple physical layering or the introduction of single-functional materials. For example, some schemes use pH-sensitive materials to encapsulate active substances, employ biodegradable frameworks, or add gas-producing particles to improve aeration. However, these schemes all suffer from fundamental flaws: the former acts on the seed itself, failing to construct a dynamic external matrix environment, and its release is immediate with no subsequent linkage; the latter relies on non-specific microbial enzymes or unstable concentrations of natural organic acids in the environment, and its degradation or gas production process is passive, slow, and uncontrollable, unable to be precisely synchronized with specific plant physiological events. Most critically, no existing technology has reported a solution that can effectively utilize plant root tip exudates as an initial trigger signal to drive a multi-level, time-sequential, functionally coupled biochemical reaction chain, thereby achieving dynamic and synergistic optimization of the matrix microenvironment.

[0005] Therefore, developing an intelligent germination substrate based on a biochemical cascade response mechanism is a long-standing and urgent problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of existing homogeneous substrates, which, due to their simple structure, cannot meet the dynamic and heterogeneous microenvironmental needs of plants at different developmental stages, leading to functional conflicts and spatiotemporal mismatches. To solve these problems, this invention provides a high-efficiency germination substrate for *Imperata cylindrica* based on a biomimetic structural design and its preparation method. This substrate, by constructing a biomimetic layered structure with functional differentiation and deterministic biochemical cascade response relationships between layers, provides a precisely time-matched optimized microenvironment for *Imperata cylindrica* throughout the entire process from seed germination to seedling establishment, thereby systematically improving its germination rate, root system quality, and seedling survival rate under salt-alkali stress.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] This invention provides a high-efficiency germination substrate for *Imperata cylindrica* based on a biomimetic structural design. The substrate is a composite with a preset hierarchical structure, which consists of a germination induction layer with a thickness of 0.5-1.0 cm, a root establishment transition layer with a thickness of 3.0-5.0 cm, and a dynamic aeration base layer with a thickness of 2.0-3.0 cm, stacked from top to bottom.

[0009] The germination induction layer contains pH-sensitive enzyme complex microcapsules;

[0010] The root system transition layer comprises a structural framework and functional microbial microspheres that can be degraded by enzymes released from the enzyme complex microcapsules;

[0011] The dynamic ventilation substrate layer contains slow-release gas-generating particles that can react with the degradation products of the structural framework.

[0012] Each adjacent layer is designed to achieve functional coupling through a sequential biochemical cascade reaction triggered by plant root tip exudates. The cascade reaction begins with the pH-sensitive dissociation of the enzyme complex microcapsule and is sequentially transmitted to subsequent layers to dynamically match the microenvironmental needs of different plant growth stages.

[0013] Furthermore, the germination induction layer is composed of a first matrix component and a first functional component:

[0014] The first matrix component is coconut shell biochar particles with polyγ-glutamic acid surface functionalization, with a particle size of 0.1-0.5 mm, a specific surface area of ​​≥500 m² / g, and the surface-grafted polyγ-glutamic acid has a molecular weight of 500,000-700,000 Daltons and a grafting density of 0.05-0.08 g / g biochar.

[0015] The first functional component is a germination-responsive enzyme complex microcapsule, which is dispersed in the first matrix component in a spherical structure with a particle size of 50-100 micrometers. The amount of the complex microcapsule added is 0.1%-0.2% of the dry weight of the matrix. Its core is a freeze-dried powder co-immobilized with β-glucosidase and cellulase in a 1:1 activity unit ratio. The outer wall is a pH-sensitive chitosan-sodium alginate bilayer complex shell material. The bilayer complex shell material dissociates at pH < 6.5 and maintains structural integrity at neutral to weakly alkaline conditions.

[0016] Furthermore, the root system transition layer is composed of a second matrix component and a second functional component:

[0017] The second matrix component is a three-dimensional network structure framework of controlled biodegradation, which is formed by cross-linking sugarcane bagasse fibers with an average length of 200-300 micrometers after alkali treatment and deligation with glutaraldehyde. Its interior is filled with and cross-linked carboxymethyl starch to regulate the water-holding capacity and degradation rate of the structure.

[0018] The second functional component is a binuclear-shell structured functional microbial hydrogel microsphere with a diameter of 0.8-1.2 mm and a density of 1×10⁻⁶. 9 The density of CFU / g dry matrix is ​​embedded in the pores of the three-dimensional network structure framework. Its innermost core is a mixed freeze-dried fungal powder of arbuscular mycorrhizal fungi (Glomus etunicatum) spores and dormant nitrogen-fixing bacteria (Azospirillum brasilense). The inner shell surrounding this core contains a concentration of 10⁻ 8 The outermost shell is a layer of calcium alginate gel containing mol / L of strigolactone analogue (GR24); the outermost shell is a layer of hydroxypropyl methylcellulose.

[0019] Furthermore, the germination induction layer and the root formation transition layer achieve functional coupling through an enzymatic reaction;

[0020] The enzymatic reaction is triggered by a stage-specific product of the plant. Specifically, when the seeds of *Imperata cylindrica* germinate, the root tip exudate lowers the local pH, triggering the disintegration of the enzyme complex microcapsules to release β-glucosidase and cellulase. The enzymes permeate to the transition layer with water, catalyzing the hydrolysis of the β-1,4-glycosidic bonds between cellulose and carboxymethyl starch in the three-dimensional framework. This causes the framework to gradually deconstruct within 72-96 hours, while simultaneously exposing the internally embedded binuclear-shell structured functional microbial hydrogel microspheres.

[0021] Furthermore, the dynamic ventilation substrate layer is composed of a third matrix component and a third functional component:

[0022] The third matrix component consists of hydrophobic expanded perlite particles with a particle size of 3-5 mm;

[0023] The third functional component is a slow-release gas-producing particle, the core of which is a calcite microparticle with a particle size of 0.5-1.0 mm, and the surface is coated with an ethyl cellulose membrane with a thickness of 0.1 micrometers. The ethyl cellulose membrane is water-insoluble and selectively permeates to small molecule organic acids.

[0024] The volume ratio of the slow-release gas-producing particles to perlite is 1:5.

[0025] Furthermore, the root system forms a transition layer and a dynamically aerated base layer, achieving functional coupling through the transfer of chemical reaction products;

[0026] The chemical reaction is triggered by plant stage products. Specifically, under enzymatic hydrolysis, the main products of cellulose and carboxymethyl starch in the transition layer skeleton, such as glucose and other reducing sugars, are partially converted into organic acids such as glucuronic acid by the metabolism of rhizosphere microorganisms or by the catalysis of mild bio-oxidants pre-placed in the skeleton. These acidic degradation products migrate to the basal layer and undergo an acid-base neutralization reaction with calcite through the ethyl cellulose membrane to generate carbon dioxide. The gas production rate is 0.01-0.05 ml / (g matrix·24h), forming a network of interconnected gas channels in the deep layer of the matrix, and maintaining a porosity of ≥20% at the bottom of the matrix.

[0027] Furthermore, the release mechanism of the dual-core-shell structured functional microbial hydrogel microspheres is as follows: after the transition layer skeleton degrades and exposes the microspheres, irrigation water causes the outer hydroxypropyl methylcellulose shell to rupture and release GR24 to induce the germination of mycorrhizal fungal spores. Subsequently, root contact or its secretions cause the calcium alginate inner shell to rupture, releasing mycorrhizal fungi and nitrogen-fixing bacteria, which then colonize the root system.

[0028] This invention also provides a method for preparing a high-efficiency germination substrate for *Imperata cylindrica* based on biomimetic structural design, comprising the following steps:

[0029] S1. Preparation of germination induction layer material:

[0030] (1) Surface functionalization modification of coconut shell biochar:

[0031] Coconut shell biochar, ball-milled to 0.1 to 0.5 mm, was mixed with a 2% polyγ-glutamic acid solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) catalyst were added at 60°C and pH 4.0 and reacted for 4 hours. The mixture was then washed, dried, and set aside for later use.

[0032] (2) Preparation of enzyme complex microcapsules:

[0033] After freeze-drying a mixture of β-glucosidase and cellulase with equal activity, the mixture was coated with chitosan-sodium alginate bilayer material under a pH gradient of 4.5-6.0. The resulting microcapsules were obtained by centrifugation, washing, and freeze-drying.

[0034] The prepared functionalized biochar and microcapsules were mixed at a dry weight ratio of 99.8:0.2 for 15 minutes to obtain the finished induction layer material;

[0035] S2. Preparation of root system transition layer material:

[0036] (1) Fabrication of the three-dimensional mesh structure framework:

[0037] Sugarcane bagasse fiber was treated with 1 mol / L sodium hydroxide at 80°C for 2 hours to remove lignin, washed, and then crosslinked with glutaraldehyde at pH 3.0. After being impregnated with 5% carboxymethyl starch solution, it was freeze-dried.

[0038] (2) Preparation of binucleate-shell microbial hydrogel microspheres:

[0039] The bacterial powder and GR24 sodium alginate solution are extruded and solidified to form the core, and then coated with a hydroxypropyl methylcellulose outer shell through a coaxial nozzle.

[0040] Hydrogel microspheres and structural framework are cold-mixed at -20℃ to make the microspheres uniformly embedded in the pores of the framework to obtain the transition layer finished material.

[0041] S3. Preparation of dynamic ventilation substrate material:

[0042] (1) Preparation of slow-release gas-producing particles:

[0043] Calcite microparticles with a particle size of 0.5 to 1.0 mm were screened and coated with ethyl cellulose ethanol solution to a weight gain of 5% under conditions of inlet air temperature of 70℃ and atomization pressure of 0.2 MPa.

[0044] Slow-release gas-generating particles are mixed with 3-5 mm expanded perlite at a volume ratio of 1:5 for 20 minutes to obtain the finished base layer material.

[0045] S4. Molding and Packaging:

[0046] The finished product is laid in sequence from bottom to top: a dynamic aeration base layer, a root establishment transition layer, and a germination induction layer. It is then slightly compacted with a pressure not exceeding 0.1 kPa and vacuum-sealed.

[0047] The beneficial effects of this invention are:

[0048] This invention overcomes the design limitations of traditional homogenized or statically layered matrices by constructing a biomimetic structural matrix with an interlayer biochemical cascade response mechanism. Instead of passively providing growth elements, it actively and intelligently responds to specific physiological signals from plants at different developmental stages. Through a series of precisely pre-programmed enzymatic and chemical reactions, it dynamically and sequentially optimizes the germination interface, root channels, and deep aeration environment. This microenvironment regulation method, highly coordinated with plant life rhythms, fundamentally solves the functional spatiotemporal mismatch problem faced by halophytes during germination and seedling establishment in artificial matrices, providing disruptive technical support for saline-alkali land ecological restoration projects. Detailed Implementation

[0049] The core technical idea of ​​this invention lies in abandoning the traditional homogenized design concept of substrates in terms of physical structure and chemical composition, and instead starting from the perspective of plant physiological ecology, simulating the heterogeneity of the microenvironment in natural soil that is coordinated with the plant growth rhythm. By constructing a multi-layered complex with a deterministic biochemical cascade response relationship, consisting of a germination induction layer, a root formation transition layer, and a dynamically aerated base layer, it provides precisely timed and functionally coupled optimized microenvironmental support for the key developmental stages of the halophyte *Eragrostis thunbergii*, such as seed germination, root formation, and seedling growth.

[0050] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0051] Example 1

[0052] S1. Preparation of germination induction layer material

[0053] (1) Surface functionalization modification of coconut shell biochar

[0054] Coconut shells sourced from specific production areas (such as Wenchang, Hainan) were heated to 650°C in a tube furnace at a heating rate of 10°C / min and pyrolyzed for 2 hours under nitrogen protection. After cooling, they were mixed with KOH at a mass ratio of 1:4 and activated in a muffle furnace at 800°C for 1 hour. The activated carbon was washed until neutral and dried. The resulting activated carbon was then ground using a planetary ball mill and graded using standard sieves, collecting particles ranging from 0.1 to 0.5 mm.

[0055] Weigh 100 grams of the biochar granules and place them in a 2-liter reactor. Add 1 liter of MES buffer solution (0.1M, pH 4.0) containing 2% γ-PGA (purchased from Bloomage Biotechnology, molecular weight 600,000 Daltons).

[0056] While stirring continuously, 6.0 g of EDC and 3.6 g of NHS were added sequentially. The reaction system was heated to 60°C and maintained for 4 hours.

[0057] After the reaction, the functionalized biochar was collected by vacuum filtration using a Buchner funnel and repeatedly washed with deionized water until the conductivity of the filtrate was close to that of pure water. Finally, it was dried in an oven at 60°C for 24 hours for later use. At this point, the grafting density of surface-grafted polyγ-glutamic acid reached 0.05-0.08 g / g biochar, and the specific surface area of ​​the functionalized coconut shell biochar was ≥500 m² / g.

[0058] (2) Preparation of enzyme complex microcapsules

[0059] β-glucosidase (Sigma-Aldrich, C6105) and cellulase (Sigma-Aldrich, C2730) were precisely weighed to achieve an activity unit ratio of 1:1, dissolved in phosphate buffer (PBS, pH 7.0), and then spray-dried into micron-sized lyophilized powder to serve as capsule cores.

[0060] Prepare a 1% (w / v) chitosan solution (dissolved in 1% acetic acid) and a 1% (w / v) sodium alginate solution. Disperse 10 g of enzyme powder in 200 mL of sodium alginate solution, and slowly add 200 mL of chitosan solution dropwise under high-speed shear (1000 rpm). During the addition, dynamically adjust the pH of the system with 0.1 M hydrochloric acid solution, slowly decreasing it from approximately 6.0 to 4.5, during which time aggregates form. After continuing stirring for 30 minutes, collect the microcapsules by centrifugation, wash three times with pure water, and finally freeze-dry to obtain the finished microcapsules.

[0061] The enzyme complex microcapsules were added at a rate of 0.15% (dry weight of the matrix). The prepared functionalized biochar and microcapsules were placed in a V-type mixer at a dry weight ratio of 99.85:0.15 and mixed at a low speed of 20 rpm for 15 minutes to ensure uniform mixing without damaging the microcapsule structure, thus obtaining the germination induction layer.

[0062] S2. Preparation of root system transition layer material

[0063] (1) Preparation of three-dimensional mesh structure skeleton

[0064] 500 g of dried bagasse fiber was placed in 5 L of 1 M NaOH solution and magnetically stirred in an 80 °C water bath for 2 hours. The mixture was filtered and rinsed thoroughly with tap water until the pH of the filtrate was neutral. It was then washed three times with pure water and dried. Bagasse fiber with an average length of 200-300 micrometers was collected.

[0065] Weigh 100g of collected, delignified fibers with an average length of 200-300 micrometers, disperse them in 2 liters of acetate buffer solution at pH 3.0, add 100 ml of 5% (v / v) glutaraldehyde aqueous solution, and react at 40°C for 3 hours to carry out cross-linking.

[0066] The preliminary skeleton was collected by filtration and washed with pure water to remove unreacted glutaraldehyde. The moistened skeleton was then immersed in 1 liter of 5% (w / v) carboxymethyl starch (DS=0.7) solution under vacuum for 30 minutes to ensure complete penetration of the solution.

[0067] The entire system was then pre-frozen at -80°C for 12 hours, and then transferred to a freeze dryer and dried at -50°C and 10 Pa for 48 hours to obtain the final three-dimensional mesh structure skeleton.

[0068] (2) Preparation of binucleate-shell microbial hydrogel microspheres

[0069] Arbuscular mycorrhizal fungal spore powder and nitrogen-fixing bacteria dormant lyophilized mycorrhizal powder were mixed at a 1:1 mass ratio to prepare a mixture containing 1% (w / v) of the mixed mycorrhizal powder and 10⁻ 8 A 2% (w / v) sodium alginate solution was used as the core fluid for M GR24. A 3% (w / v) HPMC solution was prepared as the outer shell fluid.

[0070] A coaxial dual-channel microfluidic device was used. The core liquid was pumped out from a nozzle with an inner diameter of 0.2 mm at a flow rate of 1 mL / h, and the outer shell liquid was pumped out from a coaxial nozzle with an outer diameter of 0.5 mm at a flow rate of 2 mL / h. The liquid was droplets added to a crosslinking bath containing 2% (w / v) CaCl2 and 0.5% (v / v) Tween-20, and stirred and cured for 15 minutes. The microspheres were collected and washed with sterile water to obtain the final product.

[0071] The prepared hydrogel microspheres and structural framework were cold-mixed in a -20°C cold storage by gentle stirring, so that the microspheres were evenly embedded in the pores of the framework without breaking, thus obtaining the finished material of the root system transition layer.

[0072] S3. Preparation of dynamic ventilation substrate material

[0073] (1) Preparation of slow-release gas-producing particles

[0074] Industrial-grade calcite powder was classified using standard analytical sieves, and the fraction with a particle size between 0.5 and 1.0 mm was collected.

[0075] Weigh 1 kg of sieved calcite microparticles as the core and place them in a bottom-spray fluidized bed coating machine (GlattGPCG-1).

[0076] Prepare a 5% (w / v) 95% ethanol solution of ethyl cellulose (Ethocel™ 10 cP) as the coating solution.

[0077] Set the inlet air temperature to 70℃, the product temperature to 45℃, and the atomization pressure to 0.2 MPa.

[0078] Continuous spray coating is performed, with online weighing monitoring until the coating weight gain reaches 5% of the core dry weight. After drying, the material is discharged, yielding slow-release gas-generating particles.

[0079] The prepared slow-release gas-generating particles are mixed with screened hydrophobic expanded perlite with a particle size of 3 to 5 mm at a volume ratio of 1:5 in a drum mixer at a speed of 15 rpm for 20 minutes to ensure uniformity, thus obtaining the finished dynamic aeration base layer material.

[0080] S4. Molding and Packaging

[0081] A stainless steel mold with an internal depth profile of 2.5 cm for the bottom layer, 4.0 cm for the middle layer, and 0.75 cm for the top layer was used; that is, a germination induction layer of 0.75 cm, a root system establishment transition layer of 4.0 cm, and a dynamic aeration base layer of 2.5 cm were set.

[0082] Following the bottom-up order, first, the dynamic aeration base layer finished material is evenly laid and lightly compacted with a pressure of 0.1 kPa; then, the root system transition layer finished material is laid on top and lightly compacted in the same way; finally, the germination induction layer finished material is laid on the top layer.

[0083] After the laying is completed, the formed substrate block is removed from the mold and vacuum-sealed using a high-barrier aluminum-plastic composite film to maintain the chemical stability and biological activity of its internal components until final use.

[0084] Example 2

[0085] Structural layer thickness: germination induction layer 0.5cm, root establishment transition layer 3.0cm, dynamic aeration base layer 2.0cm;

[0086] Functional component: 0.1% enzyme complex microcapsule addition (matrix dry weight).

[0087] Preparation method: The preparation steps for each layer of material are the same as in Example 1, only the layer thickness and the amount of enzyme microcapsules added are adjusted.

[0088] Example 3

[0089] Structural layer thickness: germination induction layer 1.0 cm, root establishment transition layer 5.0 cm, dynamic aeration base layer 3.0 cm;

[0090] Functional component: 0.2% enzyme complex microcapsule addition (matrix dry weight).

[0091] Preparation method: The preparation steps for each layer of material are the same as in Example 1, only the layer thickness and the amount of enzyme microcapsules added are adjusted.

[0092] Example 4

[0093] The ratio of β-glucosidase to cellulase activity units in the enzyme complex microcapsules was 1:1.2 (deviating from 1:1 in Example 1).

[0094] The weight gain of the sustained-release gas-producing particles was 4% (lower than 5% in Example 1), and the gas production rate was reduced to 0.01-0.03 ml / (g matrix·24h).

[0095] Preparation method: Except for the enzyme ratio and coating weight gain, the other steps are the same as in Example 1.

[0096] Comparative Example 1: Homogeneous structure (no functional components)

[0097] For comparison, a conventional homogeneous matrix was prepared. Coconut shell biochar (unfunctionalized with γ-PGA), bagasse fiber (delignin-only, without cross-linking and CMS filling), and expanded perlite, used in Example 1, were physically mixed at the same total mass ratio as in Example 1 to form a homogeneous mixed matrix. This matrix contained no functional components (i.e., no enzyme microcapsules, no microbial microspheres, no slow-release gas-producing particles) and had no layered structure.

[0098] Comparative Example 2: Only the layered structure is retained (no functional components).

[0099] The preparation method is the same as in Example 1, but only the three-layer structure of Example 1 is retained, and all functional components are removed:

[0100] (1) The germination induction layer contains only polyγ-glutamic acid-functionalized coconut shell biochar (enzyme-free microcapsules).

[0101] (2) The root system forms a transition layer containing only a three-dimensional network of sugarcane bagasse skeleton (without microbial microspheres);

[0102] (3) The dynamic aeration base layer contains only hydrophobic expanded perlite (without slow-release gas-producing particles).

[0103] Comparative Example 3: Homogeneous structure (containing functional components)

[0104] All matrix components (functionalized biochar, bagasse fiber, expanded perlite) of Example 1 were mixed with functional components (enzyme microcapsules, microbial microspheres, gas-producing particles) at a mass ratio of 3:5:2 to form a homogeneous matrix without stratification.

[0105] Comparative Example 4

[0106] The three-layer structure is retained, but the enzyme in the germination induction layer is replaced with non-catalytically active protein powder, and the rest is the same as in Example 1.

[0107] Experimental Design

[0108] Take seedling pots with a diameter of 10 cm and a height of 10 cm, and fill them with the substrates of Examples 1-4 and Comparative Examples 1-4 respectively.

[0109] 100 selected seeds of *Imperata cylindrica* were evenly sown on the surface of each pot.

[0110] All seedling pots were placed in a light incubator to simulate a saline-alkali soil environment (14h / 10h light day and night, 25℃ / 18℃ temperature, 70% humidity).

[0111] Seedling pots sown with *Imperata cylindrica* seeds and filled with the substrates of Examples 1, 4, and Comparative Examples 1-4 were irrigated with a 300 mmol / L NaCl solution every 3 days to keep the substrate moist. Each treatment was replicated in triplicate.

[0112] Seedling pots sown with *Imperata cylindrica* seeds and filled with the substrate from Example 2 were irrigated with a 100 mmol / L NaCl solution every 3 days to keep the substrate moist. Each treatment was replicated in triplicate.

[0113] Seedling pots sown with *Imperata cylindrica* seeds and filled with the substrate from Example 3 were irrigated with a 200 mmol / L NaCl solution every 3 days to keep the substrate moist. Each treatment was replicated in triplicate.

[0114] Performance index measurement:

[0115] On the 7th day after sowing, count the number of germinated seeds in each pot and calculate the germination rate.

[0116] On the 14th day after sowing, 10 seedlings were randomly selected, their roots were carefully washed, the length of the taproot was measured, and the average value was calculated. At the same time, a micro oxygen electrode (Unisense OX-100) was inserted into the bottom of the substrate (7 cm from the surface) to measure the oxygen concentration.

[0117] On the 21st day after sowing, 10 seedlings were randomly selected, their above-ground parts were cut off, dried in an 80℃ oven to constant weight, and their dry weight was measured.

[0118] On the 28th day after sowing, the number of surviving seedlings in each pot was counted, and the survival rate was calculated. At the same time, root samples were taken for trypan blue staining, and the root colonization rate of arbuscular mycorrhizal fungi was observed and calculated under a microscope.

[0119] Analysis of Experimental Results

[0120] The experimental data of Examples 1-4 and Comparative Examples 1-4 were statistically analyzed, and the results are shown in Table 1.

[0121] Table 1

[0122] Germination rate (day 7, %) Average taproot length (day 14, cm) Oxygen concentration at the bottom of the substrate (day 14) %) Average dry weight of aboveground parts of seedlings (day 21, mg / plant) Seedling survival rate (day 28, %) Root AMF colonization rate (day 28, %) Example 1 92.3 ±3.1 8.1 ± 0.6 18.5 ± 1.2 15.6 ± 1.8 88.7 ± 2.5 76.4 ± 5.2 Example 2 86.5 ±2.8 7.3 ± 0.5 17.2 ± 1.1 13.8 ± 1.5 83.5 ± 2.3 71.3 ± 4.8 Example 3 87.1 ±0.8 7.1 ± 0.3 17.6 ± 1.5 13.6 ± 1.3 84.2 ± 2.1 72.1 ± 4.2 Example 4 81.2 ±3.2 6.8 ± 0.4 16.5 ± 1.0 12.5 ± 1.3 79.6 ± 2.6 68.5 ± 5.1 Comparative Example 1 45.7 ±4.5 2.4 ± 0.3 6.2 ± 0.9 5.1 ± 0.7 31.3 ± 3.8 < 5 Comparative Example 2 51.3 ±4.1 3.2 ± 0.3 8.5 ± 0.8 5.8 ± 0.6 39.2 ± 3.3 < 5 Comparative Example 3 58.7 ±3.6 4.0 ± 0.4 9.8 ± 0.9 6.3 ± 0.7 46.5 ± 3.5 22.4 ± 3.7 Comparative Example 4 91.8 ±2.9 3.1 ± 0.4 6.5 ± 0.8 5.3 ± 0.7 32.1 ± 4.0 < 5

[0123] The comparative data in the table above clearly show that, using Examples 1-4 of the present invention, the growth indicators of *Erigeron tongde* under simulated saline-alkali stress are significantly improved, and are generally superior to those of the comparative examples.

[0124] The germination rate of Example 1 reached 92.3±3.1%, Example 2 was 86.5±2.8%, Example 3 was 87.1±0.8%, and Example 4 was 81.2±3.2%, all significantly higher than that of Comparative Example 1 (45.7±4.5%), Comparative Example 2 (51.3±4.1%), and Comparative Example 3 (58.7±3.6%). This indicates that regardless of how parameters such as layer thickness and enzyme microcapsule addition are adjusted within a reasonable range, the germination induction layer can effectively overcome germination obstacles through its moisturizing and salt ion chelation functions.

[0125] Regarding the average taproot length, Example 1 was 8.1±0.6 cm, Example 2 was 7.3±0.5 cm, Example 3 was 7.1±0.3 cm, and Example 4 was 6.8±0.4 cm, significantly higher than Comparative Example 1 (2.4±0.3 cm), Comparative Example 2 (3.2±0.3 cm), and Comparative Example 3 (4.0±0.4 cm). This directly demonstrates the effectiveness of the enzyme-driven dynamic channel construction mechanism in the root system transition layer, which can create favorable conditions for root growth. Even with adjustments to the enzyme ratio and coating weight gain parameters as in Example 4, although the effect is not as good as in Examples 1-3, it still maintains a superior effect compared to Comparative Examples 1-3.

[0126] Regarding the oxygen concentration at the bottom of the substrate, Example 1 had 18.5±1.2%, Example 2 had 17.2±1.1%, Example 3 had 17.6±1.5%, and Example 4 had 16.5±1.0%, which is significantly different from the 6.2±0.9% of Comparative Example 1, 8.5±0.8% of Comparative Example 2, and 9.8±0.9% of Comparative Example 3. This irrefutably confirms the unique function of the dynamically aerated substrate layer in actively improving aeration through in-situ gas production. All examples were able to maintain a high oxygen concentration, ensuring root respiration.

[0127] Ultimately, these synergies are reflected in the biomass and survival rate of seedlings. The average dry weight of the aboveground parts of the seedlings in Example 1 was 15.6±1.8 mg / plant, in Example 2 it was 13.8±1.5 mg / plant, in Example 3 it was 13.6±1.3 mg / plant, and in Example 4 it was 12.5±1.3 mg / plant, all of which were more than twice that of Comparative Example 1 (5.1±0.7 mg / plant), Comparative Example 2 (5.8±0.6 mg / plant), and Comparative Example 3 (6.3±0.7 mg / plant). In terms of seedling survival rate, Example 1 was 88.7±2.5%, Example 2 was 83.5±2.3%, Example 3 was 84.2±2.1%, and Example 4 was 79.6±2.6%, which were about 1.7-2.8 times that of Comparative Example 1 (31.3±3.8%), Comparative Example 2 (39.2±3.3%), and Comparative Example 3 (46.5±3.5%).

[0128] Regarding the root AMF colonization rate, Example 1 achieved 76.4±5.2%, Example 2 71.3±4.8%, Example 3 72.1±4.2%, and Example 4 68.5±5.1%, while Comparative Examples 1 and 2 were both <5%, and Comparative Example 3 was only 22.4±3.7%. This indicates that the roots in each example were able to successfully coexist with beneficial microorganisms, laying a solid foundation for subsequent long-term growth.

[0129] Furthermore, the germination rate of Comparative Example 4 (91.8±2.9%) was comparable to that of Example 1 (92.3±3.1%), demonstrating that the physical water retention and ion chelation functions of its germination induction layer remained effective. However, its subsequent growth indicators deteriorated sharply: the average taproot length was only 3.1±0.4 cm, the oxygen concentration at the bottom of the substrate was as low as 6.5±0.8%, the average dry weight of the aboveground seedlings was 5.3±0.7 mg / plant, the final survival rate was only 32.1±4.0%, and the root AMF colonization rate was less than 5%. These data were even worse than those of Comparative Example 1, which only used a homogeneous structure. This result demonstrates that the excellent overall performance of this invention does not stem from a simple physical layered structure, but rather depends on a biochemical cascade reaction chain triggered by plant physiological signals that runs through all three layers. Once this dynamic response mechanism is disrupted, even with the same physical configuration, the substrate cannot provide effective support for seedling root formation and deep aeration, ultimately leading to seedling failure.

[0130] It is particularly noteworthy that the biomimetic structural matrix with an interlayer biochemical cascade response mechanism triggered by plant root tip exudates constructed in this invention has been validated for its effectiveness under simulated saline-alkali stress environments of varying intensities, demonstrating excellent environmental adaptability and stability. As described in the experimental design section, Examples 2, 3, and 1 were tested under irrigation conditions with NaCl solutions of 100 mmol / L, 200 mmol / L, and 300 mmol / L, respectively. The experimental results (see Table 1) show that under the above-mentioned different salt concentration gradients, each example maintained a high germination rate (86.5%-92.3%), significant root development (average taproot length 7.1-8.1 cm), sufficient deep substrate oxygen supply (17.2%-18.5%), and excellent seedling survival rate (83.5%-88.7%).

[0131] This series of parallel controlled experiments strongly demonstrates that the core mechanism of this invention—the release of pH-sensitive enzymes triggered by plant root tip exudates, enzymatic skeleton degradation, and in-situ gas production driven by degradation products—can operate stably and orderly under a certain range of external environmental fluctuations (especially changes in salt ion concentration). The synergistic design of the system's functional components (such as pH-sensitive microcapsules, specific enzyme systems, controllable degradation skeletons, and selectively permeable gas-producing particles) ensures the reliable initiation and transmission of the cascade reaction chain, thus providing a solid guarantee for the successful germination and seedling establishment of *Erigeron tongdeensis* in the complex and variable microenvironment of typical saline-alkali land. This characteristic is crucial for the widespread application of this invention in practical ecological restoration projects.

[0132] In summary, this invention, by constructing a biomimetic structural matrix with an interlayer biochemical cascade response mechanism triggered by plant root tip exudates, achieves in-situ, dynamic, and synergistic optimization of the seed germination interface, root establishment channels, and deep aeration microenvironment of *Erigeron tongdeensis*. This invention achieves significant progress in key indicators such as germination rate, root development quality, deep aeration assurance, seedling biomass accumulation, survival rate, and beneficial microbial symbiosis. It effectively solves the problem of the functional conflict and spatiotemporal mismatch between "water retention" and "aeration" caused by the static nature of the matrix in the artificial seedling cultivation of halophytes. This invention not only provides a complete and reliable technical solution for the large-scale and efficient seedling cultivation of halophytes such as *Erigeron tongdeensis* in saline-alkali land ecological restoration projects, but also lays a solid foundation for the development of intelligent seedling substrates for other rare or high-value plants sensitive to dynamic microenvironmental needs (such as orchids and medicinal plant seedlings).

Claims

1. A high-efficiency germination substrate for *Imperata cylindrica* based on biomimetic structural design, characterized in that: The substrate is a composite with a preset hierarchical structure, which consists of a germination induction layer with a thickness of 0.5-1.0 cm, a root establishment transition layer with a thickness of 3.0-5.0 cm, and a dynamic aeration base layer with a thickness of 2.0-3.0 cm, stacked sequentially from top to bottom. The germination induction layer consists of a first matrix component and a first functional component: the first matrix component is coconut shell biochar particles with polyγ-glutamic acid surface functionalization, with a particle size of 0.1-0.5 mm, a specific surface area ≥500 m² / g, and polyγ-glutamic acid grafted on the surface with a molecular weight of 500,000-700,000 Daltons and a grafting density of 0.05-0.08 g / g biochar; the first functional component is germination-responsive enzyme complex microcapsules, dispersed in the first matrix component in a spherical structure with a particle size of 50-100 μm, the amount of the complex microcapsules added is 0.1%-0.2% of the dry weight of the matrix, the core of which is freeze-dried powder co-immobilized with β-glucosidase and cellulase in a 1:1 activity unit ratio, and the outer wall is a pH-sensitive chitosan-sodium alginate bilayer complex coagulated shell material, the bilayer complex coagulated shell material dissociates at pH<6.5 and maintains structural integrity at neutral to weakly alkaline conditions; The root system transition layer consists of a second matrix component and a second functional component: the second matrix component is a controlled biodegradable three-dimensional network structure framework, formed by alkali-treated, delignified sugarcane bagasse fibers with an average length of 200-300 micrometers cross-linked with glutaraldehyde, and filled with and cross-linked carboxymethyl starch to regulate the water-holding capacity and degradation rate of the structure; the second functional component is a binuclear-shell structured functional microbial hydrogel microsphere with a diameter of 0.8-1.2 mm and a density of 1×10⁻⁶. 9 The density of CFU / g dry matrix is ​​embedded in the pores of the three-dimensional network structure framework. Its innermost core is a mixed freeze-dried fungal powder of arbuscular mycorrhizal fungal spores and nitrogen-fixing dormant bodies. The inner shell surrounding this core contains a concentration of 10⁻ 8 A calcium alginate gel layer containing moles per liter of strigolactone analogues; the outermost shell is a hydroxypropyl methylcellulose layer; The dynamic ventilation substrate is composed of a third matrix component and a third functional component: the third matrix component is hydrophobic expanded perlite particles with a particle size of 3-5 mm; the third functional component is slow-release gas-generating particles, the core of which is calcite microparticles with a particle size of 0.5-1.0 mm, and the surface is covered with a 0.1 μm thick ethyl cellulose membrane, which is water-insoluble and selectively permeable to small molecule organic acids; the volume ratio of the slow-release gas-generating particles to perlite is 1:

5. Each adjacent layer is designed to achieve functional coupling through a sequential biochemical cascade reaction triggered by plant root tip exudates. The cascade reaction begins with the pH-sensitive dissociation of the enzyme complex microcapsule and is sequentially transmitted to subsequent layers to dynamically match the microenvironmental needs of different plant growth stages.

2. The high-efficiency germination substrate of *Imperata cylindrica* based on biomimetic structural design according to claim 1, characterized in that, The germination induction layer and the root system formation transition layer achieve functional coupling through an enzymatic reaction; The enzymatic reaction is triggered by a stage-specific product of the plant. Specifically, when the seeds of *Imperata cylindrica* germinate, the root tip exudate lowers the local pH, triggering the disintegration of the enzyme complex microcapsules to release β-glucosidase and cellulase. The enzymes permeate to the transition layer with water, catalyzing the hydrolysis of the β-1,4-glycosidic bonds between cellulose and carboxymethyl starch in the three-dimensional framework. This causes the framework to gradually deconstruct within 72-96 hours, while simultaneously exposing the internally embedded binuclear-shell structured functional microbial hydrogel microspheres.

3. The high-efficiency germination substrate of *Imperata cylindrica* based on biomimetic structural design according to claim 1, characterized in that, The root system forms a transition layer and a dynamically aerated base layer, achieving functional coupling through the transfer of chemical reaction products. The chemical reaction is triggered by plant stage products. Specifically, cellulose and carboxymethyl starch in the transition layer skeleton undergo enzymatic hydrolysis, and their main products are reducing sugars including glucose. These are partially converted into organic acids including glucuronic acid by the metabolism of rhizosphere microorganisms or by the catalysis of mild bio-oxidants pre-placed in the skeleton. These acidic degradation products migrate to the basal layer and undergo an acid-base neutralization reaction with calcite through the ethyl cellulose membrane to generate carbon dioxide. The gas production rate is 0.01-0.05 ml / g matrix·24 hours, forming a network of interconnected gas channels in the deep layer of the matrix, and maintaining a porosity of ≥20% at the bottom of the matrix.

4. The high-efficiency germination substrate of *Imperata cylindrica* based on biomimetic structural design according to claim 1, characterized in that, The release mechanism of the dual-core-shell structured functional microbial hydrogel microspheres is as follows: after the transition layer skeleton degrades and exposes the microspheres, irrigation water causes the outer hydroxypropyl methylcellulose shell to rupture and release GR24 to induce the germination of mycorrhizal fungal spores. Subsequently, root contact or its secretions cause the calcium alginate inner shell to rupture, releasing mycorrhizal fungi and nitrogen-fixing bacteria, which then colonize the root system.

5. A method for preparing a high-efficiency germination substrate for *Imperata cylindrica* based on biomimetic structural design, characterized in that... Includes the following steps: S1. Preparation of germination induction layer material: (1) Surface functionalization modification of coconut shell biochar: Coconut shell biochar, ball-milled to 0.1 to 0.5 mm, was mixed with a 2% polyγ-glutamic acid solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide catalyst were added at 60°C and pH 4.0 and reacted for 4 hours. The mixture was then washed, dried, and set aside for later use. (2) Preparation of enzyme complex microcapsules: After freeze-drying a mixture of β-glucosidase and cellulase in a 1:1 ratio of active units, the mixture was coated with chitosan-sodium alginate bilayer material under a pH gradient of 4.5-6.

0. The finished microcapsules were obtained by centrifugation, washing, and freeze-drying. The prepared functionalized biochar and microcapsules were mixed at a dry weight ratio of 99.8:0.2 for 15 minutes to obtain the finished induction layer material; S2. Preparation of root system transition layer material: (1) Fabrication of the three-dimensional mesh structure framework: Sugarcane bagasse fiber was treated with 1 mol / L sodium hydroxide at 80°C for 2 hours to remove lignin, washed, and then crosslinked with glutaraldehyde at pH 3.

0. After being impregnated with 5% carboxymethyl starch solution, it was freeze-dried. (2) Preparation of binucleate-shell microbial hydrogel microspheres: The bacterial powder and GR24 sodium alginate solution are extruded and solidified to form the core, and then coated with a hydroxypropyl methylcellulose outer shell through a coaxial nozzle. Hydrogel microspheres and structural framework are cold-mixed at -20℃ to make the microspheres uniformly embedded in the pores of the framework to obtain the transition layer finished material. S3. Preparation of dynamic ventilation substrate material: (1) Preparation of slow-release gas-producing particles: Calcite microparticles with a particle size of 0.5 to 1.0 mm were screened and coated with ethyl cellulose ethanol solution to a weight gain of 5% under conditions of inlet air temperature of 70℃ and atomization pressure of 0.2 MPa. Slow-release gas-generating particles are mixed with 3-5 mm expanded perlite at a volume ratio of 1:5 for 20 minutes to obtain the finished base layer material. S4. Molding and Packaging: The finished material is laid in sequence from bottom to top: a dynamic aeration base layer, a root establishment transition layer, and a germination induction layer. It is then slightly compacted with a pressure not exceeding 0.1 kPa and vacuum-sealed.