A pH-responsive coating agent and its application in alkaligrass seed germination

Abstract

The application discloses a pH response type coating agent and application thereof in seed germination of alkali grass. The application comprises the following steps: pretreating alkali grass seeds with a mixed salt alkali solution with a specific ratio, coating the seeds with a double-layer coating agent composed of an outer layer of dimethylaminoethyl methacrylate-acrylic acid copolymer and an inner layer of sodium alginate microspheres loaded with Bacillus subtilis GB03, and culturing the seeds under alternating light and darkness and variable temperature conditions; when the environmental pH reaches a critical value, the coating agent precisely swells and ruptures to release live bacteria, and the salt alkali solution activates the bacteria, thereby significantly improving the seed germination rate of the alkali grass and effectively solving the technical problem of low seed germination rate in the reconstruction of vegetation in saline-alkali soil. The method for efficiently germinating alkali grass is simple and easy to implement, and does not require complex operations, and can quickly and effectively improve the seed germination rate of the alkali grass. The method can be applied to production, thereby solving the problem of low seed germination rate of the alkali grass in the production process, and has important significance for the construction of artificial grassland of the alkali grass.

Description

Technical Field

[0001] This invention relates to the field of forestry and grassland planting, specifically to a pH-responsive coating agent and its application in the germination of Imperata cylindrica seeds. Background Technology

[0002] *Puccinellia tenuiflora*, belonging to the class Monocotyledonous, family Poaceae, subfamily Pooideae, and genus *Puccinellia*, is an important halophyte species widely distributed in temperate saline habitats across Eurasia. As a pioneer plant for saline-alkali land ecological restoration, *Puccinellia tenuiflora* exhibits strong salt and alkali tolerance, drought resistance, and adaptability, enabling it to grow normally and form dominant communities in high-salt, high-pH soil environments. Its tender stems and leaves, high nutritional value, and excellent palatability make it an important resource for improving saline grasslands and constructing artificial forage bases. Furthermore, its well-developed root system effectively improves soil structure and reduces surface salt accumulation, demonstrating significant ecological value for saline land management and degraded ecosystem restoration. However, *Puccinellia tenuiflora* faces challenges in seed production, including fluctuating germination rates and difficulties in seedling establishment, particularly under abiotic stress conditions where seed germination is significantly inhibited. This severely restricts its large-scale application in the rapid restoration of vegetation in saline-alkali lands. Research on the germination mechanism and regulation technology of alkaline grass seeds is of great theoretical and practical significance for improving the utilization efficiency of halophyte resources and promoting the ecological management of saline-alkali land. Summary of the Invention

[0003] The purpose of this invention is to provide a pH-responsive coating agent and its application in the germination of *Imperata cylindrica* seeds, which can effectively: (1) solve the problem of inactivation of the inoculant in a saline-alkali environment: the outer copolymer precisely swells in saline-alkali soil, and the inner microspheres protect the live GB03 bacteria until targeted release. (2) achieve synergistic improvement in germination efficiency: combining pH solution pretreatment and controlled release of the coating agent, neutral to alkaline salt solutions (pH 8.5-10.2) break seed dormancy; the coating agent continuously releases live bacteria to degrade the seed coat inhibitor in the root microdomain.

[0004] The purpose of this invention is to provide a pH-responsive coating agent, comprising the following steps:

[0005] A pH-responsive coating agent comprising, from the outside to the inside, the following components:

[0006] Outer layer: Dimethylaminoethyl methacrylate-acrylic acid copolymer, with a swelling critical pH of 8.5-9.0;

[0007] Inner layer: Sodium alginate microspheres loaded with live Bacillus subtilis GB03 bacteria, with a microsphere diameter of 50-100 μm and a live bacterial load ≥1×10⁻⁶. 9 CFU / g.

[0008] The copolymer contains 35-40% by weight of dimethylaminoethyl methacrylate monomer.

[0009] The microspheres were cross-linked and cured by a 0.5±0.05 mol / L CaCl2 solution for 5±0.5 minutes.

[0010] A method for preparing a pH-responsive coating agent, the preparation steps are as follows: (1) Bacillus subtilis GB03 bacterial solution and sodium alginate solution are mixed at a volume ratio of (1-2):(3-5); (2) CaCl2 solution is sprayed into the mixture by electrostatic spraying to form microspheres; (3) The microspheres in step (2) are dispersed in the copolymer precursor liquid and cured for 30±2s under ultraviolet light intensity of 10±0.5mW / cm².

[0011] In step (2), the voltage for electrostatic spraying is 18±0.5kV and the propulsion rate is 0.5±0.02mL / min.

[0012] In step (1), the concentration of sodium alginate in the microspheres is 2.0 ± 0.1 wt%.

[0013] A method for promoting the germination of *Imperata cylindrica* seeds: (1) treating *Imperata cylindrica* seeds with a mixed salt-alkali solution of pH 8.5-10.2; (2) coating the seeds with the above-mentioned pH-responsive coating agent at a weight gain rate of 10-15%; (3) when the environmental pH ≥ 8.5, the coating agent swells and ruptures to release live bacteria.

[0014] The mixed salt-alkali solution is prepared by mixing NaCl, Na2SO4, NaHCO3, and Na2CO3 in a molar ratio of (1-3):(1-2):(0.5-1):(0.5-1).

[0015] The culture conditions were 28°C under light for 12 hours, followed by 16°C in the dark for 12 hours.

[0016] The seeds of *Erigeron sarmentosum* are strains 23, 24, 46, or 59;

[0017] The culture was conducted under a light cycle of 12 hours of light and 12 hours of darkness per day.

[0018] The culture temperature was 28°C under light and 16°C in the dark.

[0019] The seeds of *Imperata cylindrica* are the seeds of the following varieties or strains: 23, 24, 46, and 59.

[0020] This invention provides a pH-controlled method for optimizing the germination of *Imperata cylindrica* seeds. By combining gradient pH solution pretreatment with salt-alkali stress adaptive activation technology, seed dormancy is precisely broken and germination resistance is enhanced. Addressing the key issue of germination inhibition in high-salt environments, this method selects a neutral to weakly alkaline treatment window, significantly improving seed germination rates in saline habitats. This technology overcomes the limitations of traditional *Imperata cylindrica* seed germination rates caused by drastic fluctuations in environmental salinity. Its unique pH-responsive seed coat permeability improvement mechanism synergistically enhances the conversion efficiency of endosperm energy storage substances, increasing seedling survival rates. This solution can be directly integrated into aerial seeding equipment for saline-alkali land, providing efficient seed source support for the rapid vegetation reconstruction of degraded saline grasslands and possessing significant engineering application value for the ecological restoration of "salt patch" areas.

[0021] Beneficial technical effects of the present invention:

[0022] This invention achieves a significant technological breakthrough through its innovatively designed pH-responsive coating agent and its precise application in the germination of *Imperata cylindrica* seeds. The coating agent uses a dimethylaminoethyl methacrylate-acrylic acid copolymer as its intelligent responsive outer layer, which rapidly swells when the pH of saline-alkali soil reaches a certain value, simultaneously triggering the rupture of the inner sodium alginate microspheres to release highly active *Bacillus subtilis* GB03 live bacteria, effectively solving the core problem of premature inactivation of the bacterial agent in an alkaline environment. Combined with pretreatment using a specific ratio of mixed saline-alkali solution (NaCl:Na2SO4:NaHCO3:Na2CO3=2:1:1:0.5) and variable-temperature cultivation at 28.0±0.5℃ light / 16.0±0.5℃ darkness, a dual-effect mechanism of "chemical activation + biological controlled release" is formed, overcoming the technical bottleneck of low seedling establishment rate in saline-alkali land vegetation reconstruction. Attached Figure Description

[0023] Figure 1 Seed germination rates under different treatments in the examples and comparative examples; Detailed Implementation

[0024] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0025] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or identical to those described in the embodiments of this invention may be used to implement this invention.

[0026] Unless otherwise stated, the test methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] I. Collecting seeds of *Erigeron sarmentosum*

[0031] The seeds used in the experiment were 23, 24, 46, and 59 (from the photovoltaic field of the Datong District Energy Storage Demonstration Experiment Platform in Daqing City, Heping Ranch in Datong District, and Yinlang Ranch in Ranghulu District).

[0032] Mature seeds of the above four different types of *Imperata cylindrica* germplasm were taken, and seeds with plump kernels and basically the same size were selected for the following treatment.

[0033] II. Treatment of Germinating Alkali Grass Seeds

[0034] A method for promoting the germination of Imperata cylindrica seeds, characterized in that: (1) Imperata cylindrica seeds are treated with a mixed salt-alkali solution with pH 8.9; (2) a pH-responsive coating agent is prepared and coated with the seeds at a weight gain rate of 12%; (3) when the ambient pH is ≥ 8.9, the coating agent swells and ruptures to release live bacteria.

[0035] The mixed salt-alkali solution is prepared from NaCl, Na2SO4, NaHCO3, and Na2CO3 in a molar ratio of 2:1:1:0.5.

[0036] The culture conditions were 28°C under light for 12 hours, followed by 16°C in the dark for 12 hours.

[0037] A pH-responsive coating agent, characterized in that it comprises the following components from the outside to the inside:

[0038] Outer layer: Dimethylaminoethyl methacrylate-acrylic acid copolymer, with a swelling critical pH of 8.9;

[0039] Inner layer: Sodium alginate microspheres loaded with live Bacillus subtilis GB03 bacteria, with a microsphere diameter of 90 μm and a live bacterial load ≥1×10⁻⁶. 9 CFU / g.

[0040] The copolymer contains 38% by weight of dimethylaminoethyl methacrylate monomer.

[0041] The microspheres were cross-linked and cured using a 0.5 mol / L CaCl2 solution for 5 minutes.

[0042] A pH-responsive coating agent is prepared by the following steps: (1) Bacillus subtilis GB03 bacterial solution and sodium alginate solution are mixed at a volume ratio of 1:3; (2) CaCl2 solution is sprayed into the mixture by electrostatic spraying to form microspheres; (3) the microspheres in step (2) are dispersed in the copolymer precursor liquid and cured for 30s under ultraviolet light intensity of 10mW / cm².

[0043] In step (2), the voltage for electrostatic spraying is 18kV and the propulsion rate is 0.5mL / min.

[0044] In step (1), the concentration of sodium alginate in the microspheres is 2.0 wt%.

[0045] The experiment employed a completely randomized design, using double-layered filter paper as the germination bed. Seeds of uniform maturity, plumpness, and size were selected, and each treatment was set up with 3 biological replicates, with 50 seeds per replicate.

[0046] Seeds were placed under fluctuating temperatures of 16-28℃ for germination experiments, with a diurnal temperature variation cycle of 12h / 12h. Observations were conducted continuously for 20 days from the date of bed placement, with daily recording of the number of seeds in each petri dish whose radicles had broken through the seed coat by ≥1mm. During the experiment, the filter paper humidity was maintained by quantitatively replenishing the treatment solution, and germination criteria were strictly followed according to the ISTA protocol. The germination rate (GR) was finally calculated based on the cumulative number of germinated seeds using the formula: GR (%) = (Total number of germinated seeds / Number of tested seeds) × 100%.

[0047] Example 2

[0048] The difference between Example 2 and Example 1 is that in Example 2, the seeds of *Imperata cylindrica* were treated with a mixed salt-alkali solution with a pH of 8.6.

[0049] Example 3

[0050] The difference between Example 3 and Example 1 is that in Example 3, the seeds of *Imperata cylindrica* were treated with a mixed salt-alkali solution with a pH of 9.6.

[0051] Example 4

[0052] The difference between Example 4 and Example 1 is that in Example 4, the seeds of *Imperata cylindrica* were treated with a mixed salt-alkali solution with a pH of 10.0.

[0053] Example 5

[0054] The difference between Example 5 and Example 1 is that in Example 5, the seeds of *Imperata cylindrica* were treated with a mixed salt-alkali solution with a pH of 10.2.

[0055] Comparative Example 1

[0056] The main difference between Comparative Example 1 and Example 1 is:

[0057] Treatment conditions: No pH solution treatment, routine culture (distilled water, constant temperature 28℃);

[0058] Objective: To verify the necessity of saline-alkali solution treatment with pH 8.5-10.2 for germination.

[0059] Comparative Example 2

[0060] The main difference between Comparative Example 2 and Example 1 is:

[0061] Treatment conditions: pH 7.0 solution treatment (non-saline-alkali environment), other culture conditions as in Example 1;

[0062] Objective: To verify the irreplaceable role of the neutral-alkaline salt solution system in germination.

[0063] Comparative Example 3

[0064] The main difference between Comparative Example 3 and Example 1 is:

[0065] Treatment conditions: pH 8.9 solution treatment, constant temperature 25℃ incubation;

[0066] Objective: To verify the necessity of light / dark temperature alternating culture cycles for germination.

[0067] Comparative Example 4

[0068] The main difference between Comparative Example 4 and Example 1 is:

[0069] Treatment conditions: no coating agent, pH 8.9 solution treatment;

[0070] Objective: To verify the independent synergistic effect of pH-responsive coating agents on improving germination rate.

[0071] Comparative Example 5

[0072] The main difference between Comparative Example 5 and Example 1 is:

[0073] Treatment conditions: Conventional chitosan coating (non-pH responsive), pH 8.9 solution treatment;

[0074] Objective: To verify the specific effect of pH-responsive outer layer materials on precise bacterial release.

[0075] Comparative Example 6

[0076] The main difference between Comparative Example 6 and Example 1 is:

[0077] Treatment conditions: CaCl2 solution concentration was 0.8 mol / L;

[0078] Objective: To verify the effect of high concentration CaCl2 cross-linking on microsphere formation and live bacteria release;

[0079] Comparative Example 7

[0080] The main difference between Comparative Example 7 and Example 1 is:

[0081] Treatment conditions: CaCl2 solution concentration was 0.3 mol / L;

[0082] Objective: To verify the effect of low-concentration CaCl2 crosslinking on microsphere formation and live bacteria release.

[0083] Test Item 1: Determining the germination rate of *Imperata cylindrica* seeds under different conditions

[0084] The germination rate of the seeds was recorded daily after sowing. Data were statistically analyzed using SPSS 15.0. A one-way ANOVA was performed on the germination rate after 20 days of growth. The results are shown in Table 1.

[0085] Table 1. Average germination rate (%) of *Imperata cylindrica* seeds under different treatments.

[0086] Processing method / number 23（%） 24（%） 46（%） 59（%） Example 1 95.2±1.9 82.0±2.5 89.6±2.3 93.5±1.7 Example 2 88.6±3.2 70.0±4.1 74.3±3.8 85.0±2.9 Example 3 84.3±4.0 65.3±5.2 76.0±4.5 79.8±3.3 Example 4 76.5±5.1 48.6±6.8 63.2±5.9 71.3±4.7 Example 5 68.7±6.3 42.0±7.5 57.8±6.8 65.4±5.8 Comparative Example 1 38.4±5.2 33.6±6.1 18.3±4.3 36.2±5.0 Comparative Example 2 62.1±4.8 54.7±5.6 42.5±5.2 58.9±4.3 Comparative Example 3 71.5±4.1 58.2±5.0 52.7±4.8 66.3±3.9 Comparative Example 4 76.0±3.8 64.6±4.7 68.3±4.2 72.6±3.1 Comparative Example 5 68.9±4.5 57.1±5.3 48.5±5.0 63.7±4.6 Comparative Example 6 72.3±4.3 60.8±5.1 55.2±4.9 67.9±4.0 Comparative Example 7 59.8±5.6 50.4±6.2 39.7±5.8 54.6±5.3

[0087] The analysis results in Table 1 above show that there are significant differences in the germination rate of the four different types of *Imperata cylindrica* seeds under varying temperature conditions of 16-28℃. Among them, the germination rate of seed 23 is relatively high, reaching 95.2±1.9%, indicating that the germination rate varies due to the genetic characteristics of *Imperata cylindrica* itself.

[0088] Under different pH conditions, compared with pH 8.6, pH 9.6, pH 10.0, and pH 10.2, the germination rate of all four *Eragrostis stenoptera* species was significantly increased under pH 8.9, with germination rates of 23 and 59 reaching 95.2±1.9% and 93.5±1.7%, respectively. Seeds treated with pH 8.9 can rapidly achieve a higher germination rate compared with other treatments, thus rapidly improving the germination rate of *Eragrostis stenoptera* seeds.

[0089] Comparing Examples 1-5, the possible reasons affecting the germination of *Imperata cylindrica* seeds are analyzed as follows: Example 1 shows the optimal synergy between pH and coating agent: the outer copolymer swells precisely at pH 8.9, and metabolism is activated during temperature cycles. Example 2 shows pH 8.6, close to the lower limit of swelling, resulting in insufficient release of the inoculant. Example 3 shows high pH inhibiting bacterial activity, but strain 59 exhibits strong alkali tolerance. Example 4 shows pH 10.0 exceeding the copolymer's tolerance limit, inhibiting radicle growth. Example 5 shows strong alkalinity leading to inactivation of the inoculant and deterioration of seed coat permeability.

[0090] Comparing Example 1 with Comparative Examples 1-7, the possible reasons affecting the germination of *Imperata cylindrica* seeds were analyzed as follows: Comparative Example 1: Lack of pH activation and microbial agent assistance resulted in unbroken dormancy. Comparative Example 2: pH 7.0 did not trigger coating agent swelling, and the microbial agent was not released. Comparative Example 3: Constant temperature disrupted physiological rhythms, affecting endosperm energy storage and conversion efficiency. Comparative Example 4: Treatment with only pH solution lacked the continuous degradation inhibitor of the seed coat by the microbial agent. Comparative Example 5: Chitosan coating showed no pH response, resulting in low microbial agent release rate under alkaline conditions. Comparative Example 6: High cross-linking concentration led to excessively hard microspheres, delaying microbial release. Comparative Example 7: Low cross-linking concentration resulted in insufficient microsphere strength.

[0091] Test Item 2: Effect of light / shade on the germination rate of *Imperata cylindrica* seeds

[0092] I. Collecting seeds of *Erigeron sarmentosum*

[0093] Same as Test Example 1 above.

[0094] II. Treatment of Alkali Grass Seeds Before Sowing

[0095] The effect of light on the germination rate of *Imperata cylindrica* seeds was investigated using two methods: 12 hours of low temperature (dark conditions), 12 hours of high temperature (light conditions, Sylvaniacool white fluorescent lamps, 200 μmol / m⁻² s⁻¹, 400-700 nm), and germination was performed by wrapping the seeds in aluminum foil in complete darkness. Fifty seeds from each group were evenly spread in a 9cm diameter petri dish with double-layered filter paper for germination experiments.

[0096] III. Sowing and Cultivation of the Treated Alkali Grass Seeds

[0097] Seeds were placed under fluctuating temperatures of 16-28℃ for germination experiments, with a diurnal temperature variation cycle of 12 h / 12 ​​h. Observations were conducted continuously for 20 days from the date of bed placement, with daily recording of the number of seeds in each petri dish whose radicles had broken through the seed coat by ≥1 mm. During the experiment, the filter paper humidity was maintained by quantitatively replenishing the treatment solution, and germination criteria were strictly followed according to the ISTA protocol. The germination rate (GR) was finally calculated based on the cumulative number of germinated seeds using the formula: GR (%) = (Total number of germinated seeds / Number of tested seeds) × 100%.

[0098] III. Determining the germination rate of *Imperata cylindrica* seeds

[0099] The germination rate of the seeds was recorded daily after sowing. Data were statistically analyzed using SPSS 15.0. A one-way ANOVA was performed on the germination rate after 20 days of growth. The results are shown in Table 2.

[0100] Table 2. Average germination rate of *Imperata cylindrica* seeds under light / shade conditions.

[0101] Processing method / number 23（%） 24（%） 46（%） 59（%） Example 1 (Illumination) 95.2±1.9 82.0±2.5 89.6±2.3 93.5±1.7 Example 1 (Shaded) 50.3±4.8 44.1±5.2 16.5±3.1 45.2±4.5

[0102] As shown in Table 2, shading treatment significantly inhibited the germination of all four seed varieties (23, 24, 46, and 59) of *Imperata cylindrica*. Compared to the full-light control, the germination rate under shading conditions decreased dramatically. The sensitivity of different seed varieties to shading varied significantly: seed variety 46 was the most sensitive, with a germination rate of only 16.5 ± 3.1% under shading, a decrease of over 70%; seed variety 23 maintained the highest absolute germination rate (50.3 ± 4.8%) under shading, but its decrease was still significant; the responses of varieties 24 and 59 were in between. These results fully demonstrate that light is a key environmental factor for the germination of *Imperata cylindrica* seeds, and shading severely hinders its germination process.

[0103] Comparing the effects of light and shade treatments in Example 1 on the germination rate of Imperata cylindrica seeds, the possible reasons are as follows: 660nm red light activates phytochromes and promotes the expression of the GA20ox gene, while shade inhibits the transmission of photosensitized signals, and the high expression of the PIF1 gene in strain 46 leads to deeper dormancy.

[0104] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pH-responsive coating agent, characterized in that, From the outside to the inside, it includes the following components: Outer layer: Dimethylaminoethyl methacrylate-acrylic acid copolymer, with a swelling critical pH of 8.5-9.0; Inner layer: Sodium alginate microspheres loaded with live Bacillus subtilis GB03 bacteria, with a microsphere diameter of 50-100 μm and a live bacterial load ≥1×10⁻⁶. 9 CFU / g.

2. The pH-responsive coating agent according to claim 1, characterized in that: The copolymer contains 35-40% by weight of dimethylaminoethyl methacrylate monomer.

3. The pH-responsive coating agent according to claim 1, characterized in that: The microspheres were cross-linked and cured by a 0.5±0.05mol / L CaCl2 solution for 5±0.5 minutes.

4. A method for preparing a pH-responsive coating agent according to any one of claims 1-3, characterized in that, The preparation steps are as follows: (1) Bacillus subtilis GB03 bacterial solution and sodium alginate solution are mixed at a volume ratio of (1-2):(3-5); (2) CaCl2 solution is sprayed into the mixture by electrostatic spraying to form microspheres; (3) The microspheres in step (2) are dispersed in the copolymer precursor liquid and cured for 30±2s under ultraviolet light intensity of 10±0.5mW / cm².

5. The method for preparing a pH-responsive coating agent according to claim 4, characterized in that: In step (2), the voltage for electrostatic spraying is 18±0.5kV and the propulsion rate is 0.5±0.02mL / min.

6. The method for preparing a pH-responsive coating agent according to claim 4, characterized in that: In step (1), the concentration of sodium alginate in the microspheres is 2.0 ± 0.1 wt%.

7. A method for promoting the germination of *Imperata cylindrica* seeds, characterized in that: (1) Treat the seeds of *Imperata cylindrica* with a mixed salt-alkali solution of pH 8.5-10.2; (2) Coat the seeds with a pH-responsive coating agent according to any one of claims 1-4 at a weight gain rate of 10-15%; (3) When the ambient pH is ≥ 8.5, the coating agent swells and ruptures to release live bacteria.

8. The method for promoting the germination of *Imperata cylindrica* seeds according to claim 7, characterized in that: The mixed salt-alkali solution is prepared by mixing NaCl, Na2SO4, NaHCO3, and Na2CO3 in a molar ratio of (1-3):(1-2):(0.5-1):(0.5-1).

9. The method for promoting the germination of *Imperata cylindrica* seeds according to claim 7, characterized in that: The culture conditions were 28°C under light for 12 hours, followed by 16°C in the dark for 12 hours.

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