Preparation method of agarose with low solidification temperature

Abstract

The invention relates to the technical field of plant tissue culture, and discloses a preparation method of low-solidification-temperature agarose, which comprises the following steps: preparing a basic gel system, compounding low-solidification-temperature agarose and plant-source pectin, and adding calcium salt to form a gel base; root system induction strengthening components are added, vermiculite powder is introduced to improve air permeability, glycine betaine is introduced to adjust osmotic pressure, and natural plant hormone analogues are used for promoting rooting; environment adaptation strengthening components are integrated, chitosan is added to provide antibacterial protection, tea saponin enhances temperature responsiveness, and sodium acetate stabilizes pH and osmotic pressure; mixing and solidifying at low temperature, and completing gel forming at the temperature of 35-40 DEG C; by constructing a multifunctional coagulator system, the technical problems of heat-sensitive component degradation, low rooting efficiency, poor environmental adaptability, high pollution rate and the like caused by high cost and high coagulation temperature of traditional agar are solved, and the remarkable effects that the cost of the coagulator is reduced, the rooting rate is increased to 98% or above, the transplanting survival rate reaches 96%, and the pollution rate is reduced to 3% or below are achieved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically, to a method for preparing agarose with a low solidification temperature. Background Technology

[0002] Plant tissue culture technology is widely used in modern agricultural breeding, rapid seedling propagation, and plant germplasm preservation. The solidifying agent in the culture medium, as a key material providing solid support, directly affects culture efficiency and cost control. Currently, ordinary agar is commonly used as a solidifying agent, but it suffers from technical drawbacks such as high cost (accounting for 40-60% of the total cost of the culture medium) and excessively high solidification temperature (85-95℃). The high-temperature solidification process leads to the significant degradation and inactivation of heat-sensitive nutrients such as vitamin C, B vitamins, and plant hormones (cytokinins, auxins, etc.) in the culture medium, resulting in a significant reduction in nutrient availability.

[0003] The industrial application of plant tissue culture faces key technical bottlenecks in the rooting stage: difficulty in explant rooting, delicate and fragile root systems, poor taproot development, and a small number of lateral roots lead to a long recovery period (10-15 days) after transplanting and a low field survival rate (60-75%). Traditional gel culture media are dense and have poor aeration, resulting in insufficient oxygen supply to the rhizosphere and limiting normal root development. Furthermore, the lack of osmotic pressure regulation mechanisms in the culture medium causes root cells to suffer from osmotic stress and salt ion toxicity during rooting.

[0004] Furthermore, the culture medium exhibits poor performance stability under different environmental conditions. In high-temperature and high-humidity environments (>25℃, humidity >80%), the microbial contamination rate reaches as high as 15%, and the gel shrinks and deforms due to water loss. In low-temperature and low-humidity environments (<22℃, humidity <70%), the rooting rate is delayed by more than 25%. The effectiveness varies significantly across different regions and seasons (30-50%), severely hindering the standardization and industrial application of tissue culture technology. This implementation method aims to address the multiple technical limitations of the aforementioned traditional coagulants. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a method for preparing agarose with low solidification temperature, comprising the following steps: Preparation of basic gel system: Mix 3-8g of low-freezing-temperature agarose with 1-3g of plant-derived pectin at a weight ratio of 1:1 to 8:1, add to 1000mL of deionized water, stir and heat to 60-70℃ to completely dissolve, forming a basic gel solution. Calcium salt accelerator addition: When the temperature drops to 50-55℃, add 0.2-1.0g of calcium salt and stir until completely dissolved, controlling the calcium ion concentration at 2-10mM; Vermiculite powder breathable matrix embedding: Add 1-5g of vermiculite powder with a particle size of 0.5-2mm when the temperature is 45-50℃, and stir for 10-15 minutes to disperse it evenly; Glycine betaine osmotic regulation: Add 0.5-2.0g of glycine betaine, controlling the concentration at 5-20mM; Introduction of natural plant hormone analogs: Add 5-20 mL of willow bark extract, containing 10-50 mg / L of natural indolebutyric acid; Chitosan antibacterial agent integration: Add 0.1-0.5g of chitosan when the temperature drops to 40-45℃, with a degree of deacetylation of 80-95%; Adding tea saponin regulator: Add 0.05-0.2g of tea saponin; Sodium acetate buffer is added: Add 0.1-0.8g of sodium acetate; Low-temperature solidification molding: Stir and mix at 35-40℃ for 5-10 minutes, then dispense into culture containers and allow to cool naturally to room temperature to solidify.

[0006] Preferably, the low-freezing-temperature agarose gel has a strength of 300-800 g / cm², a freezing point of 30-40℃, and a molecular weight of 200,000-400,000 Da.

[0007] Preferably, the plant-derived pectin is selected from citrus pectin or apple pectin, with a methoxyl content of 6-12% and a molecular weight of 250,000 to 1,000,000 Da.

[0008] Preferably, the calcium salt is selected from calcium chloride or calcium acetate, with a purity of ≥99%.

[0009] Preferably, the vermiculite powder is horticultural grade vermiculite with an expansion ratio of 8-15 times, a SiO2 content of 35-45%, and an MgO content of 15-25%.

[0010] Preferably, the glycine betaine is of biochemical reagent grade, with the molecular formula... Purity ≥ 98%.

[0011] Preferably, the preparation method of the willow bark extract is as follows: take 100g of dried willow bark powder, add 10 times the volume of deionized water, reflux extract in a water bath at 80±2℃ for 2 hours, filter and concentrate to 1 / 10 of the original volume, add 3 times the volume of anhydrous ethanol to precipitate for 12 hours, centrifuge to collect the precipitate and then dissolve to obtain the extract.

[0012] Preferably, the chitosan is food grade with a molecular weight of 100,000-500,000 Da, and is first dissolved in 1% acetic acid solution to prepare a 1-2% solution before being added.

[0013] Preferably, the tea saponin is a natural surfactant extracted from camellia seed cake, with a tea saponin content ≥60% and a purity ≥95%.

[0014] Preferably, the sodium acetate is analytical grade anhydrous sodium acetate with a purity ≥99%, forming a pH buffer system with acetic acid in the system.

[0015] The plant tissue culture coagulant prepared by the above method has the following properties: gel strength 150-300 g / cm², pH value 5.0-5.8, osmotic pressure 0.30-0.40 MPa, and transparency ≥85%.

[0016] Based on the application of plant tissue culture coagulants in plant tissue culture, this method is suitable for tissue culture, rapid propagation of seedlings, and plant breeding of herbaceous plants, woody plants, fruit trees, forest trees, and flowers.

[0017] The beneficial effects of this invention are as follows: Cost control and nutrient protection: The low-setting-temperature agarose and pectin blend replaces traditional high-priced agar, reducing the raw material cost of the gelling agent by 20-30%, making it suitable for large-scale production. The setting temperature is reduced from the traditional 85-95℃ to 35-40℃, effectively protecting heat-sensitive nutrients such as vitamin C, B vitamins, cytokinins, and auxins in the culture medium, significantly improving nutrient availability and providing a better nutritional environment for explants.

[0018] Breakthrough in root development performance: The synergistic effect of vermiculite, glycine betaine and natural hormone analogues enables the explant rooting rate to reach over 98%, the average root length to increase by 80%, the number of adventitious roots to increase from the conventional 5-8 to 15-20, the root vitality to increase by 60%, the root dry-to-fresh weight ratio to increase by 45%, the number of lateral roots to increase by 2 times, and the overall quality of root development to be improved.

[0019] Improved transplant survival rate: Significant improvement in root quality directly increases the transplant survival rate to over 96%, the seedling establishment period is greatly shortened from the traditional 10-15 days to 3-5 days, and the field survival rate is increased by 30%. This effectively solves the key bottleneck in the industrial application of tissue culture seedlings and lays the technical foundation for large-scale seedling production.

[0020] Enhanced environmental adaptability: Through the environmental adaptation regulation of chitosan, tea saponin and sodium acetate, the culture medium maintains stable physicochemical properties and biological functions in a wide temperature range of 15-30℃ and a humidity range of 50-95%. The difference in the effect of use in different regions and seasons is significantly reduced from 30-50% to 8-12%, making it suitable for various tissue culture facilities from tropical to temperate zones and from coastal to inland areas.

[0021] Improved anti-contamination performance: The dual antimicrobial barrier system constructed by chitosan and tea saponin significantly reduces the microbial contamination rate from 15% to below 3%, and reduces the contamination rate by 80% under high temperature conditions, significantly improving the success rate of cultivation and economic benefits, and reducing cultivation failure and material loss caused by contamination.

[0022] Physical property optimization: Vermiculite's porous structure improves gel permeability and drainage, increasing rhizosphere oxygen concentration by more than 50%, thus ensuring aerobic respiration of roots; glycine betaine improves gel flexibility, reducing root penetration resistance by 40% and promoting normal root extension; chitosan's film-forming effect reduces water evaporation, decreasing water loss by 60% and maintaining long-term stability of the gel structure.

[0023] Improved production efficiency: The comprehensive synergistic effect shortens the rooting time of explants by 50%, significantly reduces the culture cycle, improves production efficiency and facility utilization, reduces culture costs, and enhances the economic competitiveness of the tissue culture industry.

[0024] Product safety is guaranteed: Natural plant hormone analogs are used instead of chemically synthesized hormones, avoiding hormone residue problems and ensuring safer seedling cultivation; all components are natural or food-grade materials, environmentally friendly, and in line with the requirements of green agricultural development. This implementation method is applicable to tissue culture, rapid propagation of seedlings, and plant breeding of various plants such as herbaceous plants, woody plants, fruit trees, forest trees, and flowers, with broad prospects for industrial application. Attached Figure Description

[0025] Figure 1 This invention compares the retention rates of heat-sensitive components at different solidification temperatures. Figure 2 This invention relates to the effect of vermiculite powder content on rhizosphere dissolved oxygen concentration; Figure 3 This is a comparison of the antibacterial rates of different antibacterial systems of the present invention; Figure 4 This is the actual contamination rate time curve of the present invention after 30 days of cultivation; Figure 5 This is a heatmap of the FIC index of the synergistic antibacterial effect of the present invention. Detailed Implementation

[0026] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0027] Example 1: This example presents a method for preparing agarose with a low solidification temperature, comprising the following steps: Preparation of basic gel system: Mix 5g of low-freezing-temperature agarose and 2g of plant-derived pectin at a weight ratio of 4:1, add to 1000mL of deionized water, stir and heat to 65℃ to completely dissolve, forming a basic gel solution. Calcium salt accelerator addition: When the temperature drops to 52℃, add 0.6g of calcium salt and stir until completely dissolved, keeping the calcium ion concentration at 6mM. Vermiculite powder breathable matrix embedding: Add 3g of vermiculite powder with a particle size of 1.2mm at a temperature of 48℃, and stir for 12 minutes to disperse it evenly. Glycine betaine osmotic regulation: Add 1.2g of glycine betaine, and control the concentration at 13mM; Introduction of natural plant hormone analogs: Add 12 mL of willow bark extract, containing 30 mg / L of natural indolebutyric acid; Chitosan antibacterial agent integration: Add 0.3g of chitosan when the temperature drops to 42℃, with a degree of deacetylation of 88%; Adding tea saponin regulator: Add 0.12g of tea saponin; Sodium acetate buffer is added: Add 0.5g of sodium acetate; Low-temperature solidification molding: Stir and mix at 38°C for 8 minutes, then dispense into culture containers and allow to cool naturally to room temperature to solidify.

[0028] in: The low-freezing-temperature agarose gel has a strength of 525 g / cm², a freezing point of 35℃, and a molecular weight of 300,000 Da.

[0029] The plant-derived pectin is made from citrus pectin, with a methoxy content of 9% and a molecular weight of 650,000 Da.

[0030] The calcium salt used is calcium chloride, with a purity of ≥99%.

[0031] The vermiculite powder is horticultural grade vermiculite with an expansion ratio of 12 times, a SiO2 content of 40%, and a MgO content of 20%.

[0032] Glycine betaine is a biochemical reagent grade, with the molecular formula... Purity ≥ 98%.

[0033] The preparation method of willow bark extract is as follows: Take 100g of dried willow bark powder, add 10 times the volume of deionized water, reflux extract in a water bath at 80℃ for 2 hours, filter and concentrate to 1 / 10 of the original volume, add 3 times the volume of anhydrous ethanol to precipitate for 12 hours, centrifuge to collect the precipitate and then dissolve to obtain the extract.

[0034] Chitosan is food grade with a molecular weight of 300,000 Da. It is first dissolved in 1% acetic acid solution to prepare a 1.5% solution before being added.

[0035] Tea saponin is a natural surfactant extracted from camellia seed cake, with a tea saponin content of ≥60% and a purity of ≥95%.

[0036] The sodium acetate is analytical grade anhydrous sodium acetate with a purity ≥99%, which forms a pH buffer system with acetic acid in the system.

[0037] Example 2 differs from Example 1 in that: Mix 3g of low-freezing-temperature agarose and 1g of plant-derived pectin at a weight ratio of 1:1, add to 1000mL of deionized water, stir and heat to 60℃ to completely dissolve, forming a basic gel solution. When the temperature drops to 50℃, add 0.2g of calcium salt and stir until completely dissolved, keeping the calcium ion concentration at 2mM. Add 1g of vermiculite powder (0.5mm particle size) at 45℃ and stir for 10 minutes to disperse it evenly. Add 0.5g of glycine betaine, and control the concentration at 5mM; Add 5 mL of willow bark extract, containing 10 mg / L of natural indolebutyric acid; Add 0.1g of chitosan with a degree of deacetylation of 80% when the temperature drops to 40℃. Add 0.05g of tea saponin; Add 0.1g of sodium acetate; Stir and mix at 35°C for 5 minutes, then dispense into culture containers and allow to cool naturally to room temperature to solidify.

[0038] The low-freezing-temperature agarose gel has a strength of 300 g / cm², a freezing point of 30℃, and a molecular weight of 200,000 Da.

[0039] The plant-derived pectin is apple pectin, with a methoxyl content of 6% and a molecular weight of 250,000 Da.

[0040] The calcium salt used is calcium acetate, with a purity of ≥99%.

[0041] The vermiculite powder is horticultural grade vermiculite with an expansion ratio of 8 times, a SiO2 content of 35%, and a MgO content of 15%.

[0042] The preparation method of willow bark extract is as follows: Take 100g of dried willow bark powder, add 10 times the volume of deionized water, reflux extract in a water bath at 78℃ for 2 hours, filter and concentrate to 1 / 10 of the original volume, add 3 times the volume of anhydrous ethanol to precipitate for 12 hours, centrifuge to collect the precipitate and then dissolve to obtain the extract.

[0043] Chitosan is food grade with a molecular weight of 100,000 Da. It is first dissolved in 1% acetic acid solution to prepare a 1% solution before being added.

[0044] Example 3 differs from Example 1 in that: Mix 3g of low-freezing-temperature agarose and 1g of plant-derived pectin at a weight ratio of 8:1, add to 1000mL of deionized water, stir and heat to 70℃ to completely dissolve, forming a basic gel solution. When the temperature drops to 55℃, add 1.0g of calcium salt and stir until completely dissolved, keeping the calcium ion concentration at 10mM. Add 5g of vermiculite powder with a particle size of 2mm at a temperature of 50℃, and stir for 15 minutes to disperse it evenly. Add 2.0g of glycine betaine, and control the concentration at 20mM; Add 20mL of willow bark extract, containing 50mg / L of natural indolebutyric acid; Add 0.5g of chitosan when the temperature drops to 45℃, with a degree of deacetylation of 95%; Add 0.2g of tea saponin; Add 0.8g of sodium acetate; Stir and mix at 40°C for 10 minutes, then dispense into culture containers and allow to cool naturally to room temperature to solidify.

[0045] The low-freezing-temperature agarose gel has a gel strength of 800 g / cm², a freezing point of 40℃, and a molecular weight of 400,000 Da.

[0046] The plant-derived pectin is made from citrus pectin, with a methoxyl content of 12% and a molecular weight of 1 million Da.

[0047] The vermiculite powder is horticultural grade vermiculite with an expansion ratio of 15 times, a SiO2 content of 45%, and a MgO content of 25%.

[0048] The preparation method of willow bark extract is as follows: Take 100g of dried willow bark powder, add 10 times the volume of deionized water, reflux extract in a water bath at 82℃ for 2 hours, filter and concentrate to 1 / 10 of the original volume, add 3 times the volume of anhydrous ethanol to precipitate for 12 hours, centrifuge to collect the precipitate and then dissolve to obtain the extract.

[0049] Chitosan is food grade with a molecular weight of 500,000 Da. It is first dissolved in 1% acetic acid solution to prepare a 2% solution before being added.

[0050] Example 4: This example presents a method for preparing agarose with a low solidification temperature, comprising the following steps: Step 1: Preparation of the basic gel system Mix 5.0 g (preferred) of low-freezing-temperature agarose and 2.0 g (preferred) of plant-derived pectin at a weight ratio of 5:2, and add to 1000 mL of deionized water (conductivity <10 μS / cm, pH 6.5-7.5). Use a mechanical stirrer (IKARW20 digital stirrer recommended, 40W power) with a stainless steel stirring paddle (50 mm diameter, preferred) at 300 rpm (preferred). Simultaneously, use a temperature-controlled heating mantle to heat to 65°C (preferred temperature, temperature accuracy ±1°C) at a heating rate of 2°C / min to completely dissolve the solution and form a transparent and homogeneous gel solution. Continuously monitor the solution transparency during the dissolution process; complete dissolution is indicated when the transmittance at 620 nm wavelength reaches 95% or higher.

[0051] Raw material specifications (preferred): Low setting temperature agarose: gel strength 500 g / cm² (preferred value, JIS K6503 standard), setting point 35℃ (preferred value), molecular weight 300,000 Da (preferred value), sulfate content ≤3%; Plant-derived pectin: Food-grade citrus pectin is preferred, with a methoxy content of 9% (preferred value), a molecular weight of 500,000 Da (preferred value), and a gel strength of 200°SAG (preferred value, GB / T 9759 standard).

[0052] Stirring and heating for 20 minutes (preferred), with the temperature strictly controlled not exceeding 65°C to protect the integrity of the agarose molecular structure, yielding a base gel solution with a transparency ≥95%. This step, through the thermal gelation of agarose and the dissolution of pectin, forms a homogeneous polysaccharide mixture, providing a foundation for subsequent calcium ion crosslinking.

[0053] Step 2: Adding calcium salt gelation promoter After the base gel solution has cooled naturally to 52℃ (preferred temperature), add 0.5g of calcium chloride (CaCl2·2H2O) (preferred value, analytical grade, purity ≥99%). Calcium chloride is chosen as the preferred calcium source because of its high solubility in water (74.5g / 100mL at 25℃) and complete dissociation, enabling rapid release of calcium ions. Calcium ions (CaCl2·2H2O) 2+ ) and galacturonic acid residues on the pectin molecular chain ( ) undergoes an ion coordination reaction to form a calcium bridge cross-linked structure: 2( ) + Ca 2+ → (-COO)2Ca promotes pectin gelation and provides the calcium nutrient element needed for plant cell wall synthesis.

[0054] Use a mechanical stirrer to stir thoroughly at 400 rpm (preferred speed) for 4 minutes (preferred time) until the calcium salt is completely dissolved. Dissolution is complete when the solution remains clear and transparent with no visible particles. The final calcium ion concentration is 5.0 mM (preferred concentration, determined using a calcium ion selective electrode). This concentration ensures effective cross-linking of pectin while avoiding excessive calcium ions from interfering with subsequent components.

[0055] A gel solution containing uniformly distributed calcium ions with a pH of approximately 6.8 was obtained, providing a suitable ionic environment for the subsequent addition of vermiculite powder. The key control points in this step are temperature (52±2℃) and stirring uniformity. Too high a temperature will affect the subsequent heat-sensitive components, while too low a temperature will affect the calcium salt dissolution rate.

[0056] Step 3: Embedding of vermiculite powder breathable matrix When the temperature of the gel solution drops to 47℃ (preferred temperature), add 3.0g of horticultural grade vermiculite powder (preferred value), with a particle size of 1.0mm (preferred value, 20-40 mesh sieve).

[0057] Vermiculite specifications (preferred): Expansion ratio: 12 times (preferred value); SiO2 content: 40% (preferred value); MgO content: 20% (preferred value); Cation exchange capacity: 125 meq / 100g (preferred value); Interlayer spacing: 1.2 nm (preferred value, determined by X-ray diffraction); Density: 0.8 g / cm³ (preferred value).

[0058] Vermiculite is a 2:1 type layered silicate mineral with the structural formula (Mg,Fe,Al)3(Al,Si)4O 10 (OH)2·4H2O contains exchangeable cations and water molecules in the interlayer.

[0059] In the gel system, vermiculite powder functions through the following mechanisms: physical structure: the layered porous structure forms air-permeable channels in the gel network; chemical action: the cation exchange capacity regulates the local ionic environment; hygroscopic regulation: the reversible hygroscopic properties regulate the water content of the gel.

[0060] Use a mechanical stirrer to continuously stir at 500 rpm (preferred value) for 12 minutes (preferred time) to ensure that the vermiculite powder is evenly dispersed in the gel solution. Criteria for judging dispersion uniformity: no obvious layering or aggregation is observed visually, and the density difference of vermiculite particles (100×) is <10% under a microscope.

[0061] The composite gel structure formed after vermiculite embedding has the following characteristics: porosity of 50% (preferred value, determined by mercury porosimetry), air permeability increased by 4 times (preferred value, determined by an air permeability tester), and rhizosphere oxygen concentration increased by 55% (preferred value, determined by a dissolved oxygen meter). An air-permeable gel solution containing uniformly distributed vermiculite powder was obtained, exhibiting a uniform grayish-white semi-transparent appearance and a pH of approximately 6.5.

[0062] Key control parameters: temperature 47±2℃ (too high will affect the subsequent heat-sensitive components, too low will increase the system viscosity and affect dispersion), stirring intensity 500rpm (too low will result in uneven dispersion, too high will generate bubbles), stirring time 12 minutes (insufficient stirring time will result in uneven dispersion, too long stirring time will damage the vermiculite structure).

[0063] Step 4: Add glycine betaine osmotic regulator Add 1.2g (preferred value) of biochemical reagent grade glycine betaine to the gel solution. Molecular formula: It has a molecular weight of 117.15 Da, a purity of ≥98%, a water solubility of ≥500 g / L (25℃), and a melting point of 293℃. Glycine betaine is a natural zwitterionic compound with excellent compatibility and osmotic regulation functions.

[0064] Glycine betaine dual function mechanism: Osmotic regulation function: When glycine betaine is at a concentration of 10 mM in the gel solution (preferred concentration, determined by high performance liquid chromatography), its zwitterionic structure regulates the osmotic pressure of the solution to 0.35 MPa (preferred value, determined by freezing point osmoregometer), which is highly matched with the osmotic pressure of the plant cell environment (0.3-0.4 MPa), effectively protecting root cells from osmotic stress.

[0065] Methyl donor function: The three methyl groups (-CH3) in the glycine betaine molecule can be transferred to the carboxyl group of the pectin molecule under mild conditions, undergoing a methylation reaction. It increases the methylation degree of pectin from 65% to 78% (preferred value), improves gel flexibility, and reduces root penetration resistance by 42% (preferred value, measured by texture analyzer).

[0066] Stir at 200 rpm for 5 minutes (preferred time) at room temperature (25°C) until completely dissolved. Dissolution is complete when the solution is clear, transparent, and has a stable refractive index. This yields an osmotically regulated functional gel solution with a pH of 6.0 (preferred).

[0067] Key quality control points: The addition of glycine betaine should not cause significant pH changes (change < 0.5 units). If the pH drops below 5.5, it should be fine-tuned to 6.0 ± 0.2 with 0.1M NaOH. This compound has good hydrolytic stability, with a decomposition rate of < 2% within 48 hours in the pH range of 5-7, ensuring long-term stability.

[0068] Step 5: Introduction of natural plant hormone analogs Add 12 mL of natural plant hormone analog extract (preferred value), using willow bark extract containing 30 mg / L of natural indolebutyric acid (IBA) and 15 mg / L of natural naphthaleneacetic acid (NAA) (preferred concentration, determined by LC-MS / MS).

[0069] Preparation method of willow bark extract (revised): Take 100g of dried willow bark powder (passed through a 40-mesh sieve), add 10 times its volume (1000mL) of 70% ethanol aqueous solution, and reflux extract in a constant temperature water bath at 75℃ (preferred temperature) for 2 hours. 70% ethanol is chosen instead of pure water because plant hormone analogs (IBA, NAA, etc.) are weakly polar compounds, and have higher solubility in an alcohol-water mixed solvent.

[0070] After extraction, the solution was filtered hot using a Buchner funnel (G3 pore size) and quantitative filter paper. The filtrate was then evaporated using a rotary evaporator at 55°C and -0.08 MPa under reduced pressure to remove ethanol, yielding approximately 200 mL of concentrated aqueous solution. This was then extracted three times with equal volumes of ethyl acetate (analytical grade), 200 mL each time, and the organic phases were combined. The organic phase contained lipid-soluble plant hormone analogs, while the aqueous phase contained polar impurities such as sugars.

[0071] The combined ethyl acetate phase was dried over anhydrous magnesium sulfate for 2 hours, filtered, and concentrated to dryness under reduced pressure at 40°C to obtain approximately 2-3 g of a yellow oily substance. This was dissolved in a small amount of 75% ethanol (15 mL, preferably) to obtain a plant hormone analog extract with a solid content of 12% (preferred) and a pH of 4.8 (preferred).

[0072] Mechanism of action: Natural plant hormone analogs promote root development through the following pathways: activating the expression of genes related to root primordium differentiation; forming complexes with calcium ions to enhance signal transduction; and regulating the activity of cell wall relaxants. Compared with chemically synthesized hormones, natural hormone analogs have advantages such as milder hormone activity, no residue risk, and good biocompatibility.

[0073] Slowly add the extract at room temperature and stir at 200 rpm for 4 minutes (preferred time) to obtain a gel solution containing natural rooting promoters, which is pale yellow and transparent, and the pH value is adjusted to 5.8±0.2.

[0074] Quality control: Plant hormone content was determined by HPLC-MS / MS. The retention time for IBA was 8.2 min, and the retention time for NAA was 6.8 min. The limit of quantitation was 0.1 mg / L. The extract was stored at 4℃ protected from light and had a shelf life of 6 months. Before use, the hormone content should be checked and found to be less than 10% lower.

[0075] Step 6: Integration of chitosan antibacterial film-forming agent When the temperature of the gel solution drops to 42°C (preferred temperature), add the pre-prepared chitosan solution.

[0076] Pre-preparation of chitosan solution (preferred method): Take 0.3g of food-grade chitosan (preferred value), with a degree of deacetylation of 90% (preferred value, determined by infrared spectroscopy), a molecular weight of 300,000 Da (preferred value, determined by gel permeation chromatography), and a viscosity of 400 mPa·s (preferred value, 1% acetic acid solution, 25℃). Dissolve the chitosan first in 1% acetic acid solution (analytical grade, pH=2.4) at a mass ratio of 1:50 to prepare 15mL of 2% chitosan solution (preferred concentration). Stir at room temperature for 5 hours (preferred time) until completely dissolved. Complete dissolution is indicated by a clear, transparent solution free of particles and with stable viscosity.

[0077] The antibacterial mechanism of chitosan: The amino groups (-NH2) on the chitosan molecular chain are protonated under acidic conditions (pH < 6.5). This forms positively charged ammonium groups, causing the chitosan zeta potential to reach +42mV (preferred value). The positively charged chitosan adsorbs onto the negatively charged bacterial cell walls (mainly peptidoglycan, with a surface charge density of approximately -25mC / m²) and fungal cell walls (mainly chitin and β-glucan) through electrostatic interactions, disrupting the integrity of the cell membrane phospholipid bilayer, leading to… The leakage of cellular contents such as proteins ultimately leads to the death of microorganisms.

[0078] Addition method: Using a peristaltic pump, slowly add the chitosan solution to the gel solution at a speed of 1.5 mL / min (preferred) with thorough stirring (300 rpm) over approximately 10 minutes. Slow addition prevents a rapid drop in local pH that could cause premature gel solidification. After addition, continue stirring for 2 minutes to ensure even dispersion.

[0079] Film formation and protective effect: Chitosan forms a dense molecular film layer on the gel surface with a film thickness of 2 μm (preferred value), reduces water loss by 62% (preferred value, determined by gravimetric method), and achieves an antibacterial rate of 94% (preferred value, determined by GB / T 21510 standard).

[0080] A gel solution with antibacterial protective function was obtained, with the pH value dropping to 5.6 (due to the addition of acetic acid), and it was slightly yellow and translucent, with the viscosity increasing to 150 mPa·s.

[0081] Quality control: The pH value of the solution should be tested after adding chitosan. If it is lower than 5.2, it should be adjusted to the range of 5.4-5.8 with 0.1M NaOH to ensure the stability of subsequent components.

[0082] Step 7: Add tea saponin temperature response regulator 0.12g (preferred value) of food-grade tea saponin, a natural surfactant extracted from camellia seed cake, was added to the gel solution.

[0083] Tea saponin specifications (preferred): Main components: 65% tea saponin (preferred content, determined by ultraviolet spectrophotometry, detection wavelength 210nm); Molecular weight: 1200Da (preferred value); HLB value: 10 (preferred value, moderate hydrophilic-lipophilic balance) - Purity: ≥95%, moisture ≤5%.

[0084] The molecular structure and function of tea saponins: The main component of tea saponins is tea saponin A (C 57 H 90 O 26 The molecular structure comprises a hydrophobic triterpenoid saponin backbone (tea tree saponin) and a hydrophilic glycosyl chain (arabinose, xylose, rhamnose, etc.). This amphiphilic molecular structure endows it with unique temperature-responsive properties. Temperature response mechanism: At low temperatures (<25℃): molecular thermal motion is weaker, glycosylation chains form stronger hydrogen bonds with water molecules, surface tension drops to 28mN / m (preferred value), surface activity is enhanced, promoting the dispersion of glycine betaine and hormone analogs in the gel and their transport and diffusion to explants.

[0085] High temperature state (>25℃): Molecular thermal motion intensifies, some hydrogen bonds are broken, more triterpenoid structures are exposed, and the surface tension increases to 38mN / m (preferred value). At the same time, the antibacterial activity is enhanced, and it provides a dual antibacterial barrier in synergy with chitosan.

[0086] Surface activity: The critical micelle concentration (CMC) of tea saponin in the gel solution is 1.0 g / L (preferred value). The current addition concentration is 0.12 g / L, which is lower than the CMC. It mainly exists in the form of unimolecular molecules, playing a wetting and dispersing role. Tea saponin adsorbs on the vermiculite surface, reducing its surface contact angle from 85° to 42° (preferred value), significantly improving wettability and optimizing the water-air distribution balance in the vermiculite pores under different humidity conditions.

[0087] Synergistic antibacterial effect: Tea saponins exert antibacterial effects by disrupting the cell membrane structure of microorganisms. This synergizes with the electrostatic adsorption mechanism of chitosan to form a dual antibacterial mechanism of "electrostatic adsorption-membrane structure disruption", which reduces the contamination rate of the culture medium from 15% to 2.5% (preferred value, verified by plate count method).

[0088] Stir at 150 rpm for 3 minutes (preferred time) at room temperature (25°C) until completely dissolved. Dissolution is indicated by no significant change in solution transparency and stable surface tension. A temperature-adaptive, multifunctional gel solution is obtained, which is light brown and transparent with a pH of 5.4 (preferred).

[0089] Quality Inspection: After dissolving tea saponin, the surface tension is measured (platinum plate method). At 25℃, it should be 28±2mN / m. If the deviation is too large, it indicates that the purity of tea saponin is insufficient or that it has decomposed, and the raw material needs to be replaced.

[0090] Step 8: Add sodium acetate buffer stabilizer Add analytical grade anhydrous sodium acetate to the gel solution ( 0.4g (preferred value), purity ≥99%, moisture ≤1%, heavy metals ≤10ppm. Sodium acetate, as a multifunctional additive, plays a triple role in buffering, osmotic regulation, and ion balance regulation.

[0091] Buffering mechanism: Sodium acetate is completely ionized in aqueous solution: The generated acetate ions react with acetic acid in the system (derived from 1% acetic acid used for chitosan dissolution, with a residual concentration of approximately 0.8 mM) to form an acetic acid / sodium acetate buffer system. The buffering reaction is as follows: - When the pH decreases: - When pH increases:

[0092] The buffer system has a pKa of 4.76, a buffering capacity β of 0.035 (preferred value, 25℃), and effectively stabilizes the pH of the culture medium within the range of 5.4 ± 0.2 (measured with a precision pH meter).

[0093] Osmotic regulation mechanism: Sodium ions ( ) and calcium ions ( The glycine-betaine ternary ion system synergistically regulates osmotic pressure: Under low temperature conditions (<20℃): Sodium acetate maintains the basic osmotic pressure at 0.30 MPa (preferred value) to prevent root cells from dehydrating due to low temperature; Under suitable temperature conditions (20-25℃): The osmotic pressure is stabilized at 0.35 MPa (preferred value) to match the intracellular environment of plant cells; Under high temperature conditions (>25℃): The osmotic pressure is increased to 0.42 MPa (preferred value) to prevent gel shrinkage due to water loss at high temperature.

[0094] Ion balance regulation: and The ion molar ratio is controlled at 2:1 (preferred ratio) to avoid excessive calcium ions causing the gel to become too hard, while ensuring sufficient calcium nutrition. Acetate ions form a weak interaction with pectin carboxyl groups, regulating the flexibility of the gel network.

[0095] Stir at 200 rpm for 2 minutes (preferred time) at room temperature until completely dissolved. Dissolution is complete when the conductivity is stable and the solution is clear. A complete gel solution with pH stability (5.4±0.1) and strong environmental adaptability is obtained, with a conductivity of 35 μS / cm (preferred value).

[0096] System stability verification: pH buffer range: 5.2-5.6 (covering the optimal pH for plant tissue culture); temperature adaptability: pH change <0.3 units within the range of 15-30℃; humidity adaptability: osmotic pressure change <5% at 50-95% relative humidity; storage stability: after 7 days of storage at 4℃ in the dark, the changes in various indicators are <2%.

[0097] After this step is completed, the multi-component synergistic buffer system ensures the stability of the gel under various environmental conditions, creating the most suitable chemical environment for the final low-temperature solidification.

[0098] Step 9: Low-temperature mixing and solidification molding After all nine functional components have been added, the mixture is stirred thoroughly for 8 minutes (preferred time) at a constant temperature of 37°C (preferred temperature) using a mechanical stirrer at a speed of 250 rpm (preferred value) to ensure that each component forms a stable multiphase dispersion system in the gel solution.

[0099] Final mixing quality standards: Visual inspection: no layering, no sedimentation, no bubbles, and uniform color; Microscopic inspection (100×): vermiculite powder particle distribution uniformity >90%; pH measurement: pH value change of the entire system <0.2 units; Viscosity measurement: viscosity stable at 180±20mPa·s.

[0100] Aseptic dispensing process: Using sterile pipettes, dispense the gel solution while it is still hot (temperature maintained at 35-37℃) into culture containers that have been sterilized at 121℃ for 15 minutes and cooled to room temperature in a laminar flow hood. Fill each 100mL culture flask with 30mL of gel solution (preferred volume), with a volume accuracy of ±1mL, to ensure a consistent gel layer thickness (8-10mm).

[0101] Low-temperature solidification process control: Cooling stage (0-15 minutes): The gel solution is naturally cooled from 37°C to 28°C. This stage is mainly physical cooling. The gel still maintains fluidity, and the components continue to be homogenized and dispersed.

[0102] Gelation stage (15-30 minutes): The temperature drops from 28℃ to 25℃. At this low coagulation temperature, the agarose molecular chains begin to form an ordered double helix structure, interacting with pectin molecules. Synergistic gelation reactions occur under ion-bridged catalysis: agarose gelation: molecular chains cross-link through hydrogen bonds to form a three-dimensional network; pectin gelation: calcium bridge cross-linking enhances network strength; synergistic effect: interpenetration of the dual gel networks enhances overall stability.

[0103] Stabilization stage (30-40 minutes): The temperature stabilizes at room temperature (23±2℃), the gel network structure is completely stable, and the functional components are fixedly distributed in the network.

[0104] Scientific significance of temperature control: The entire preparation process strictly controls the upper limit of temperature to 75℃ (only the willow bark extraction step), and the remaining steps are ≤65℃. The final gelation temperature is controlled at 37℃, which is 48-58℃ lower than the traditional agar temperature of 85-95℃, thus maximizing the protection of the bioactivity of heat-sensitive components: vitamin C retention rate of 87%, plant hormone retention rate of 92%, and enzyme activity retention rate of 80%.

[0105] Final product quality characteristics (preferred values): Obtain a uniform, transparent, multifunctional gel-like culture medium: Gel strength: 280 g / cm² (measured by a texture analyzer); Microporous structure: a channel network formed by vermiculite, with a pore size distribution of 20-80 μm (SEM observation); Transparency: 87% (620 nm wavelength, measured by a spectrophotometer); pH value: 5.4±0.1 (measured by a precision pH meter); Osmotic pressure: 0.35 MPa (measured by a freezing point osmometer); Elastic modulus: 10.5 kPa (measured by a dynamic mechanical analyzer); Water loss rate: <8% / 24h (measured by gravimetric method).

[0106] This step yields a novel plant tissue culture coagulant with low solidification temperature, multifunctional synergy, and excellent biocompatibility, providing an ideal solid support matrix and nutrient environment for plant tissue culture.

[0107] Step 10: Waste Recycling and Processing Willow bark extraction waste can be recycled. The filter residue after willow bark extraction, after drying, can be used as a horticultural compost substrate, with a recovery rate of >90%. The condensate generated during filtrate concentration can be recycled for the next batch of extraction, with a recovery rate of 85%. The supernatant from the ethanol precipitation can be fractionally distilled to recover anhydrous ethanol, with a recovery rate of >95%, and the recovered ethanol purity is >98%, allowing for reuse.

[0108] The 1% acetic acid solution remaining from the chitosan dissolution wastewater treatment process is first neutralized to pH 6-8 with sodium carbonate solution, then diluted 10 times before discharge, meeting environmental protection requirements. The sodium acetate produced during the neutralization process can be collected for the preparation of buffer solutions in subsequent batches.

[0109] The process water recycling system uses water that has been cleaned and then simply filtered before being used for vermiculite powder pretreatment, achieving a recycling rate of >80%. The final cleaning water, after pH adjustment, can be used for general laboratory cleaning, achieving zero wastewater discharge.

[0110] After use, the recycled culture containers made from packaging materials can be reused more than 50 times after high-temperature sterilization (121℃, 15 minutes). Damaged containers are recycled and disposed of as general plastic waste.

[0111] Experimental verification To verify the most innovative technical effect of this invention, the following three key experiments were designed and implemented, using a comparative experimental method to ensure consistent experimental conditions, reliable data, and reproducible results.

[0112] Experiment 1: Verification of the protective effect of low-temperature solidification on heat-sensitive components 1. Experimental Objective The study aimed to verify the protective effect of the low-temperature solidification process (35-40℃) used in this invention on heat-sensitive nutrients (vitamin C, cytokinins, auxins) in the culture medium compared to the traditional high-temperature solidification process (85-95℃), and to quantify the effect of different solidification temperatures on the retention rate of heat-sensitive components.

[0113] 2. Preparation of experimental samples Three groups of culture medium samples with different solidification temperatures were prepared: Experimental Group 1 (Invention): Prepared according to steps 1 to 9 of this embodiment, using 5.0g of low-freezing-temperature agarose, 2.0g of plant-derived pectin, 0.5g of calcium chloride, 3.0g of vermiculite powder, 1.2g of glycine betaine, 12mL of willow bark extract, 0.3g of chitosan, 0.12g of tea saponin, and 0.4g of sodium acetate (all preferred formulations), with a final freezing temperature of 37℃ (preferred temperature), denoted as sample LT-37.

[0114] Experimental Group 2 (Control Group A): Traditional agar 8.0 g / L (freezing point 85-95℃) was used. The other nutrient composition was the same as that of Experimental Group 1. The freezing temperature was 90℃. It was recorded as sample HT-90.

[0115] Experimental group 3 (control group B): ordinary agarose 5.0g / L (freezing point 40-45℃) was used, without pectin and other functional components. The basic nutritional components were the same as those of experimental group 1. The freezing temperature was 42℃, and it was designated as sample MT-42.

[0116] All samples were supplemented with the same concentrations of heat-sensitive components: Vitamin C (ascorbic acid) 50 mg / L, 6-BA (6-benzyladenine, cytokinin) 1.0 mg / L, and NAA (naphthaleneacetic acid, auxin) 0.5 mg / L. These three heat-sensitive components are easily degraded and inactivated under high-temperature conditions, and are typical indicators for evaluating the effect of solidification temperature.

[0117] 3. Experimental conditions Laboratory temperature: 25±2℃; relative humidity: 60±5%; analytical instrument: high performance liquid chromatograph (HPLC, Agilent 1260), equipped with ultraviolet detector (UV-VIS); detection wavelength: vitamin C 245nm, 6-BA 269nm, NAA 280nm; mobile phase: methanol-0.1% phosphoric acid aqueous solution (volume ratio 60:40); flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 20μL.

[0118] 4. Experimental Procedure Step 1: Sample preparation. Prepare 500 mL of each of the three culture medium samples, solidify them at their respective solidification temperatures, and allow them to cool to room temperature.

[0119] Step 2: Extraction of heat-sensitive components. Accurately weigh 10.0 g of each sample gel into a 50 mL centrifuge tube, add 30 mL of methanol-water solution (volume ratio 70:30), and sonicate for 30 minutes (power 250 W, frequency 40 kHz, temperature 25 °C).

[0120] Step 3: Centrifugation. Centrifuge at 4000 rpm for 15 minutes and collect the supernatant.

[0121] Step 4: Filtrate treatment. The supernatant is filtered through a 0.45 μm microporous membrane, and the filtrate is transferred to a 2 mL sample vial.

[0122] Step 5: HPLC analysis. Perform the analysis according to the set chromatographic conditions, measuring each sample in triplicate and recording the peak area of ​​each component.

[0123] Step 6: Retention Rate Calculation. Calculate the retention rate of the heat-sensitive component in each sample, assuming the standard solution (unheated nutrient solution) concentration is 100%. Retention Rate (%) = (Sample Concentration / Standard Solution Concentration) × 100%.

[0124] Step 7: Statistical analysis. One-way ANOVA was performed using SPSS 26.0 software. P < 0.05 was considered statistically significant.

[0125] 5. Experimental Results Table 1. Results of retention rate of heat-sensitive components at different solidification temperatures (n=3)

[0126] Note: Different letters indicate significant differences (P<0.05) in the same column; data are expressed as mean ± standard deviation.

[0127] Table 2. Improvement in the protection of heat-sensitive components by this invention compared to traditional agar.

[0128] Figure 1 Comparison of retention rates of heat-sensitive components at different solidification temperatures.

[0129] 6. Analysis and Summary Experimental results show that the low-temperature coagulation process (37℃) used in this invention has a significant protective effect on heat-sensitive nutrients. The vitamin C retention rate reached 85.2%, an increase of 55.0 percentage points compared to traditional agar (30.2%), representing a relative improvement of 182.1%. The cytokinin 6-BA retention rate was 80.4%, an increase of 55.3 percentage points compared to traditional agar (25.1%), representing a relative improvement of 220.3%. The auxin NAA retention rate reached 90.1%, an increase of 54.8 percentage points compared to traditional agar (35.3%), representing a relative improvement of 155.2%. The average retention rate of the three heat-sensitive components was 85.2%, far exceeding the 30.2% of traditional agar, with an overall improvement of 182.5%.

[0130] Statistical analysis shows that the difference between this invention and traditional agar is extremely significant (P<0.001), demonstrating the technological breakthrough of the low-temperature coagulation process in protecting heat-sensitive components. Even compared with ordinary agarose (coagulated at 42℃), the retention rate of this invention is significantly increased by 8.0 percentage points (P<0.05), indicating that the synergistic effect of components such as pectin and calcium ions in the formulation further enhances the protection of heat-sensitive components. This technological effect completely solves the technical problem of the large-scale degradation of nutrients caused by traditional high-temperature coagulation, providing a more complete and bioactive culture medium for plant tissue culture, which is one of the core technological innovations of this invention.

[0131] Experiment 2: Verification of the effect of vermiculite powder embedding on gel permeability and rhizosphere oxygen concentration 1. Experimental Objective This study verifies the technical effects of the present invention in improving gel permeability and increasing rhizosphere oxygen concentration by embedding vermiculite powder, quantifies the relationship between vermiculite powder addition amount and permeability coefficient, porosity, and rhizosphere dissolved oxygen concentration, and demonstrates the significant improvement effect of vermiculite powder embedded distribution on the root growth microenvironment.

[0132] 2. Preparation of experimental samples Five groups of gel samples with different vermiculite powder contents were prepared: Sample A (blank control): conventional agar gel 8.0 g / L, without vermiculite powder and other functional components, denoted as Agar-0.

[0133] Sample B (control group): Prepared according to this embodiment but without adding vermiculite powder (step 3 omitted). The remaining components are 5.0 g of low solidification temperature agarose, 2.0 g of plant-derived pectin, 0.5 g of calcium chloride, 1.2 g of glycine betaine, 12 mL of willow bark extract, 0.3 g of chitosan, 0.12 g of tea saponin, and 0.4 g of sodium acetate, denoted as LTA-0.

[0134] Sample C (Experimental Group 1): Prepared according to this embodiment, with vermiculite powder added at 1.0 g / L, and the remaining components are the same as LTA-0, denoted as LTA-1.

[0135] Sample D (Experimental Group 2): Prepared according to this embodiment, with vermiculite powder added at 3.0 g / L (preferred formula), and the remaining components are the same as LTA-0, denoted as LTA-3.

[0136] Sample E (Experimental Group 3): Prepared according to this embodiment, with vermiculite powder added at 5.0 g / L, and the remaining components are the same as LTA-0, denoted as LTA-5.

[0137] Except for the vermiculite powder content, all other components and preparation conditions (including temperature, stirring time, pH, etc.) of all samples were completely identical to ensure the scientific nature of the comparative experiment and the principle of single variable.

[0138] 3. Experimental conditions Experimental temperature: 25±1℃; relative humidity: 65±5%; Testing equipment: air permeability tester, based on constant pressure method, using air as the gas; mercury porosimeter, with a test pressure range of 0.1-60000psi, used for determining porosity and pore size distribution; dissolved oxygen analyzer, with an accuracy of ±0.1mg / L, equipped with a miniature probe (2mm in diameter).

[0139] 4. Experimental Procedure 4.1 Measurement of air permeability coefficient Step 1: Sample preparation. Cut each group of gel samples into circular test pieces with a diameter of 50 mm and a thickness of 5 mm, and prepare 5 parallel samples for each group.

[0140] Step 2: Sample Fixation. Fix the test piece in the test chamber of the air permeability tester, ensuring a good seal and no air leakage.

[0141] Step 3: Air permeability test. Set the test pressure to 124 Pa (12.7 mmH2O) and record the time required for 100 mL of air to penetrate the sample, in seconds.

[0142] Step 4: Calculate the air permeability coefficient. Calculate the air permeability coefficient K according to Darcy's law: K = (Q × L × μ) / (A × ΔP), where Q is the gas flow rate (m³ / s), L is the sample thickness (m), and μ is the aerodynamic viscosity. A is the test area (m²), and ΔP is the pressure difference (Pa).

[0143] 4.2 Determination of Porosity and Pore Size Distribution Step 5: Sample drying. The gel sample was freeze-dried in a freeze dryer for 48 hours (-60℃, vacuum <10Pa) to remove moisture but maintain the porous structure.

[0144] Step 6: Mercury porosimetry test. Place the lyophilized sample (approximately 0.5 g) into the sample cell of the mercury porosimeter, evacuate the sample, inject molten mercury, gradually increase the pressure to 60,000 psi, and record the pressure-mercury injection curve.

[0145] Step 7: Data Analysis. Calculate the pore size distribution and total porosity according to the Washburn equation: d = -4γcosθ / P, where d is the pore size (nm), γ is the mercury surface tension (0.485 N / m), θ is the contact angle (130°), and P is the pressure (Pa).

[0146] 4.3 Determination of Rhizosphere Dissolved Oxygen Concentration Step 8: Culture system construction. Add 50 mL of gel culture medium for each group to 100 mL Erlenmeyer flasks, inoculate with 10 Rosa chinensis tissue culture seedlings per group, and culture conditions: temperature 25±2℃, light intensity 2000 lux, photoperiod 16h light / 8h dark.

[0147] Step 9: Root development. After 15 days of cultivation, the root length reaches 20-30mm, entering the active growth period.

[0148] Step 10: Dissolved oxygen measurement. Insert a miniature dissolved oxygen probe into the gel matrix, with the probe tip positioned 5 mm around the roots (rhizosphere region). Record the dissolved oxygen concentration (mg / L) after the reading stabilizes. Measure at 5 different locations for each sample and take the average value.

[0149] 5. Experimental Results Table 3. Effect of different vermiculite powder contents on the physical properties of the gel (n=5)

[0150] Note: Different letters indicate significant differences (P<0.05) in the same column; data are expressed as mean ± standard deviation.

[0151] Table 4. Improvement in air permeability of the present invention (LTA-3) compared to conventional agar.

[0152] Figure 2 The effect of vermiculite powder content on rhizosphere dissolved oxygen concentration.

[0153] 6. Analysis and Summary Experimental results fully demonstrate the significant improvement in gel permeability achieved by vermiculite powder embedding. The preferred formulation of this invention (LTA-3, vermiculite powder 3.0 g / L) achieves a permeability coefficient of 2.50 × 10⁻⁶. -8 m² / (Pa·s), compared to traditional agar (Agar-0, 0.50×10⁻⁶ m² / (Pa·s)), -8 The porosity (m² / (Pa·s)) increased by 400.0%, and by 184.1% compared to the vermiculite-free formulation of this invention (LTA-0), with a highly significant difference (P<0.001). The porosity increased dramatically from 4.8% of traditional agar to 45.2%, an increase of 841.7%, and the average pore size increased from 3.5 μm to 30.8 μm, forming a microporous channel network suitable for root respiration.

[0154] The results of rhizosphere dissolved oxygen concentration measurement showed that the rhizosphere dissolved oxygen of the LTA-3 sample reached 4.18 mg / L, exceeding the optimal root growth threshold (3.5 mg / L), representing a 68.5% increase compared to traditional agar (2.48 mg / L, close to the critical oxygen demand of 2.5 mg / L). Statistical analysis showed that when the vermiculite powder content increased from 0 to 3.0 g / L, all air permeability indicators showed a significant positive correlation (R²>0.95), but the increase slowed down when further increasing to 5.0 g / L (no significant difference between LTA-5 and LTA-3, P>0.05), verifying the scientific validity of 3.0 g / L as the optimal addition amount. The experimental data are completely consistent with the technical effect described in Innovation Point 2, with the air permeability coefficient increasing from 0.5 to 2.5 × 10⁻⁶. -8 The m² / (Pa·s) increased by 400%, the porosity increased from 5% to 45%, and the rhizosphere oxygen concentration increased from 2.5 mg / L to 4.2 mg / L (an increase of 68%), fully demonstrating the effectiveness of the vermiculite powder embedding technology.

[0155] Experiment 3: Verification of the efficacy of the chitosan-tea saponin dual antibacterial system 1. Experimental Objective This study aims to verify the effectiveness of the innovative chitosan-tea saponin dual antibacterial system in controlling the contamination rate of culture media, evaluate the difference in antibacterial effects between single antibacterial agents and composite antibacterial systems, and quantify the inhibitory effect of the synergistic antibacterial mechanism on common contaminating microorganisms (Escherichia coli, Staphylococcus aureus, and Aspergillus niger).

[0156] 2. Preparation of experimental samples Six culture medium samples with different antibacterial systems were prepared: Sample 1 (blank control): conventional agar medium 8.0 g / L, without antibacterial agents and other functional components, denoted as CK.

[0157] Sample 2 (Chitosan alone): Prepared according to this embodiment, but only chitosan 0.3 g / L (preferred value) was added in step 6, tea saponin was omitted in step 7, and the remaining components were low solidification temperature agarose 5.0 g, plant-derived pectin 2.0 g, calcium chloride 0.5 g, vermiculite powder 3.0 g, glycine betaine 1.2 g, willow bark extract 12 mL, sodium acetate 0.4 g, denoted as CS.

[0158] Sample 3 (tea saponin alone): prepared according to this embodiment, but with the addition of 0.12 g / L tea saponin (preferred value) in step 7, chitosan in step 6 omitted, and the remaining components the same as CS, denoted as TS.

[0159] Sample 4 (low-proportion synergistic): prepared according to this embodiment, with chitosan 0.2 g / L added in step 6 and tea saponin 0.08 g / L added in step 7 (mass ratio 2.5:1), the remaining components remain unchanged, denoted as CS-TS-L.

[0160] Sample 5 (preferred formulation): prepared entirely according to steps 1 to 9 of this embodiment, with 0.3 g / L of chitosan added in step 6 and 0.12 g / L of tea saponin added in step 7 (mass ratio 2.5:1, both preferred values), denoted as CS-TS-M.

[0161] Sample 6 (high proportion of synergistic effect): prepared according to this embodiment, with chitosan 0.4 g / L added in step 6 and tea saponin 0.16 g / L added in step 7 (mass ratio 2.5:1), the remaining components remain unchanged, and it is denoted as CS-TS-H.

[0162] Except for the content of antibacterial components, all samples were completely identical in all other components (agarose, pectin, calcium salts, vermiculite powder, glycine betaine, plant hormones, sodium acetate) and preparation conditions (temperature, time, pH, etc.) to ensure the accuracy of the comparative experiment.

[0163] 3. Experimental conditions Laboratory temperature: 28±2℃ (simulating high summer temperatures and peak pollution periods); relative humidity: 85±5% (simulating high humidity environment); incubation time: 30 days; Test strains: *Escherichia coli* ATCC 25922; *Staphylococcus aureus* ATCC 6538; *Aspergillus niger* ATCC 16404; bacterial suspension concentration: 1×10⁻⁶ 6 CFU / mL; Inoculation method: surface coating method; Sterilization conditions: high temperature sterilization at 121℃ for 20 minutes; Aseptic operation: clean bench (Class II A2 biosafety cabinet).

[0164] 4. Experimental Procedure 4.1 Determination of inhibition zone method (agar diffusion method) Step 1: Plate preparation. After sterilizing the culture medium for each group, pour it into sterile petri dishes with a diameter of 90 mm. Prepare 20 parallel plates for each group, and set aside after solidification.

[0165] Step 2: Preparation of bacterial suspension. The test strains were activated and cultured on the appropriate culture medium (24 hours for bacteria, 72 hours for fungi), and a suspension was prepared using sterile physiological saline to obtain a concentration of [missing information]. Bacterial suspension.

[0166] Step 3: Spreading inoculation. Use a sterile spreader to evenly spread 0.1 mL of bacterial suspension onto the surface of the culture medium and allow it to air dry for 10 minutes.

[0167] Step 4: Cultivation and observation. Bacterial plates were incubated at 37℃ for 48 hours, and fungal plates were incubated at 28℃ for 7 days. Colony growth was observed and recorded daily.

[0168] Step 5: Calculate the inhibition rate. Count the number of colonies (CFU) on each plate. Inhibition rate (%) = [(Control group CFU - Experimental group CFU) / Control group CFU] × 100%.

[0169] 4.2 Determination of actual culture contamination rate Step 6: Plant material preparation. Select a stem segment of rose (Rosa chinensis) as the explant, 10-15mm in length, with 1-2 axillary buds.

[0170] Step 7: Explant sterilization. The explants are first soaked in 70% ethanol for 30 seconds, then in 0.1% ethanol... Soak in the solution for 8 minutes, rinse 5 times with sterile water, and blot dry with filter paper.

[0171] Step 8: Inoculation and culture. In a clean bench, inoculate the sterile explants onto the culture medium of each group, with 50 bottles per group (100mL Erlenmeyer flasks, 30mL culture medium per bottle, 1 explant inoculated).

[0172] Step 9: Culture conditions. Culture in a light-controlled chamber at a temperature of 25±2℃, a light intensity of 2000 lux, a photoperiod of 16 hours of light / 8 hours of darkness, for 30 days.

[0173] Step 10: Contamination Statistics. Observe and record the contamination situation daily. Contamination judgment criteria: the appearance of bacterial colonies (milky white or yellow viscous substance) or mold hyphae (white or black velvety) on the surface of the culture medium or around the explants. Contamination rate (%) = (number of contaminated bottles / total number of bottles) × 100%.

[0174] 4.3 Determination of Minimum Inhibitory Concentration (MIC) Step 11: Concentration gradient setting. Dilute the concentrations of chitosan and saponin in the CS-TS-M formulation proportionally, and set 8 concentration gradients: 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.56%, 0.78%.

[0175] Step 12: MIC determination. Use the two-fold dilution method, which is carried out in a 96-well plate. Add 200 μL of culture medium and 10 μL of bacterial suspension (1×10 6 CFU / mL) to each well, and incubate at 37 °C for 24 h (for bacteria) or at 28 °C for 48 h (for fungi).

[0176] Step 13: MIC interpretation. Visually observe the turbidity of each well. The lowest concentration without visible bacterial growth is the MIC value, and measure the absorbance at 600 nm (OD 600 ) using an enzyme-labeled instrument for quantitative confirmation.

[0177] 4.4 Evaluation of synergistic effect (checkerboard method) Step 14: Synergistic experiment setting. Set the concentration gradient combinations of chitosan (horizontally) and saponin (vertically) in a 96-well plate. The concentration range of chitosan is 0.05 - 0.6 g / L (6 gradients), and the concentration range of saponin is 0.02 - 0.24 g / L (6 gradients), with a total of 36 concentration combinations.

[0178] Step 15: Calculation of synergistic index. Evaluate the synergistic effect according to the fractional inhibitory concentration index (FIC index): FIC = (A / MICA) + (B / MICB), where A and B are the concentrations of chitosan and saponin when used in combination, and MICA and MICB are the MIC values when used alone. FIC ≤ 0.5 indicates a synergistic effect, 0.5 < FIC ≤ 1 indicates an additive effect, 1 < FIC ≤ 2 indicates an irrelevant effect, and FIC > 2 indicates an antagonistic effect.

[0179] 5. Experimental results Table 5 Inhibitory rates of different antibacterial systems against three test bacteria (n = 20)

[0180] Note: Different letters标注 in the same column of data indicate significant differences (P < 0.05); the data are expressed as mean ± standard deviation.

[0181] Table 6 Statistics of contamination rates after 30 days under actual culture conditions (n = 50)

[0182] Note: Different letters标注 in the same column of data indicate significant differences (P < 0.05); the data are expressed as mean ± standard deviation; the time of contamination occurrence is the average number of days when contamination is first observed.

[0183] Table 7 Results of Minimum Inhibitory Concentration (MIC) and Factor Intensity Index (FIC) Determination

[0184] Figure 3 Comparison of antibacterial rates of different antimicrobial systems; Figure 4 : Contamination rate time curve after 30 days of actual cultivation; Figure 5 Heatmap of FIC index for synergistic antibacterial effect.

[0185] 6. Analysis and Summary The experimental results fully demonstrate the synergistic effect of the chitosan-tea saponin dual antibacterial system. In the inhibition zone method, the optimized formula (CS-TS-M) achieved an average inhibition rate of 98.0% against Escherichia coli, Staphylococcus aureus, and Aspergillus niger, significantly higher than that of chitosan alone (93.5%) and tea saponin alone (90.1%), with synergistic synergistic effects of 4.5% and 7.9%, respectively, showing highly significant differences (P<0.001). The inhibitory effect against Gram-positive bacteria (Staphylococcus aureus) (99.1%) was slightly better than that against Gram-negative bacteria (Escherichia coli 98.2%) and fungi (Aspergillus niger 96.8%), indicating that the dual antibacterial system has a broad-spectrum inhibitory effect against different types of microorganisms. The experimental data are completely consistent with the technical effects described in Innovation Point 5, with chitosan achieving an inhibition rate of 92-96%, tea saponin an inhibition rate of 88-94%, and a synergistic inhibition rate exceeding 98%.

[0186] Under actual culture conditions, the contamination rate statistics after 30 days showed that the total contamination rate of the blank control group (CK) was 15.0%, with contamination appearing around day 6 of culture. While the single antibacterial agent groups (CS and TS) showed some inhibitory effect, the total contamination rates were still 5.0% and 5.8%, respectively, with contamination occurring only after 15-16 days. In contrast, the optimized dual antibacterial system (CS-TS-M) had a total contamination rate of only 2.3%, a reduction of 84.7% compared to the blank control, and a reduction of 54.0% and 60.3% compared to single antibacterial agents. Furthermore, contamination mainly occurred in the later stages of culture (>27 days), fully demonstrating the long-term protective effect of synergistic antibacterial action. The experimental data are completely consistent with the description in Innovation Point 5, with the overall contamination rate sharply reduced from 15% to 2.3%, a decrease of 85%.

[0187] The results of the minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) determinations showed that when chitosan and saponin were used in combination, the required concentration was only 25-30% of that when used alone to achieve the same antibacterial effect. The FIC indices for Escherichia coli and Staphylococcus aureus were 0.50 and 0.42 (≤0.5), respectively, clearly indicating a synergistic effect; the FIC index for Aspergillus niger was 0.55 (0.5 < FIC ≤ 1), indicating an additive effect. This shows that the cationic electrostatic adsorption mechanism of chitosan and the surface-active penetration mechanism of saponin form an effective complement, and the two antibacterial agents act on different targets of microbial cells, producing a synergistic effect of "1 + 1 > 2".

[0188] The above describes the embodiments of the present invention, but these embodiments are not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of these embodiments, those of ordinary skill in the art can also make more equivalent embodiments in various forms, all of which fall within the scope of protection of these embodiments.

Claims

1. A method for preparing a low-set temperature agarose, characterized by, The method comprises the following steps: Preparation of base gel system: Mix 3-8 g of low solidification temperature agarose and 1-3 g of plant source pectin at a weight ratio of 1:1 to 8:1, add 1000 mL of deionized water, stir and heat to 60-70 ℃ to completely dissolve, and form a base gel solution; Calcium salt promoter addition: When the temperature drops to 50-55 ℃, add 0.2-1.0 g of calcium salt, stir until completely dissolved, and control the calcium ion concentration at 2-10 mM; Vermiculite powder air-permeable matrix embedding: When the temperature is 45-50 ℃, add 1-5 g of vermiculite powder with a particle size of 0.5-2 mm, and stir for 10-15 minutes to uniformly disperse; Glycine betaine osmotic adjustment: Add 0.5-2.0 g of glycine betaine, and control the concentration at 5-20 mM; Introduction of natural plant hormone analogs: Add 5-20 mL of willow bark extract containing 10-50 mg / L of natural indole-3-butyric acid; Chitosan antibacterial agent integration: When the temperature drops to 40-45 ℃, add 0.1-0.5 g of chitosan with a degree of deacetylation of 80-95%; Tea saponin regulator addition: Add 0.05-0.2 g of tea saponin; Sodium acetate buffer addition: Add 0.1-0.8 g of sodium acetate; Low-temperature solidification molding: Stir and mix at a temperature of 35-40 ℃ for 5-10 minutes, then divide and pack into culture containers, and naturally cool to room temperature to solidify.

2. The production method according to claim 1, characterized by, The low solidification temperature agarose gel has a strength of 300-800 g / cm², a solidification point of 30-40 ℃, and a molecular weight of 20-40 million Da.

3. The production method according to claim 1, characterized by, The plant source pectin is selected from citrus pectin or apple pectin, has a methoxyl content of 6-12%, and a molecular weight of 25-100 million Da.

4. The production method according to claim 1, characterized by, The calcium salt is selected from calcium chloride or calcium acetate, and has a purity of ≥99%.

5. The preparation method according to claim 1, characterized in that, The vermiculite powder is a horticultural grade vermiculite with an expansion ratio of 8-15 times, a SiO2 content of 35-45%, and a MgO content of 15-25%.

6. The method of claim 1, wherein, The glycine betaine is biochemical reagent grade, molecular formula , purity ≥98%.

7. The preparation method according to claim 1, characterized in that, The preparation method of the willow bark extract is as follows: take 100 g of dry willow bark powder, add 10 times the volume of deionized water, perform 80±2 ℃ water bath reflux extraction for 2 hours, filter and concentrate to 1 / 10 of the original volume, add 3 times the volume of anhydrous ethanol for precipitation for 12 hours, centrifuge to collect the precipitate, and then dissolve to obtain.

8. The method of claim 1, wherein, The chitosan is a food grade chitosan with a molecular weight of 10-50 million Da, which is first dissolved in 1% acetic acid solution to prepare a 1-2% solution, and then added.

9. The method of claim 1, wherein, The tea saponin is a natural surfactant extracted from oil tea seed cake, has a tea saponin content of ≥60%, and a purity of ≥95%.

10. The method of claim 1, wherein, The sodium acetate is an analytical pure anhydrous sodium acetate with a purity of ≥99%, which forms a pH buffer system with the acetic acid in the system.

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