Method for breaking dormancy and germination of wild huji plant seeds

Abstract

The application discloses a kind of wild huizhi seed dormancy breaking and germination method, belong to plant seed breeding technical field.The application includes: physical skin breaking, surface cleaning and disinfection, warm water soaking, chemical dormancy breaking, gradient germination, batch seeding and intelligent water and fertilizer management.For the low natural germination rate and uneven emergence of wild huizhi seeds due to the dual obstacles of hard seed coat and shallow physiological dormancy, the application uses a multi-level cooperative mechanism of physical skin breaking to create water permeation channels, chemical dormancy breaking to remove dormancy and gradient germination to simulate environmental signals to simultaneously break the dual dormancy obstacles, effectively improve the seed germination rate and emergence uniformity, and inhibit mold growth.The application has stable process and strong operability, and is suitable for large-scale seedling raising of wild huizhi, providing reliable technical support for ecological restoration projects.

Description

Technical Field

[0001] This invention relates to the field of seed propagation technology. More specifically, this invention relates to a method for breaking dormancy and promoting germination of wild Lespedeza seeds. Background Technology

[0002] wild lespedeza ( Two-colored lespedeza *Lespedeza* (Turcz.) is a deciduous shrub belonging to the genus *Lespedeza* in the legume family. It possesses extremely high ecological value and application prospects: its well-developed root system has a strong soil-fixing capacity, effectively reducing surface runoff and soil erosion when planted on sloping farmland, making it an excellent species for soil and water conservation and afforestation of barren hills; it is drought-tolerant, tolerant of poor soil, and cold-resistant, with good regeneration ability, making it suitable for ecological restoration projects; its branches and leaves are rich in nutrients, with a high crude protein content, making it a high-quality feed resource; its seeds have a high oil content, possessing both edible and economic value. Therefore, wild *Lespedeza* has broad development and application prospects in ecological restoration, soil and water conservation, and feed production.

[0003] However, the efficient propagation of wild Lespedeza seeds has always been a bottleneck restricting their large-scale application. Zheng Jian's master's thesis at Hebei Agricultural University, "Research on the Introduction and Cultivation of Five Wild Flowers" (2003), systematically studied the dormancy characteristics of Lespedeza seeds. The results showed that the dormancy of Lespedeza seeds is forced dormancy caused by hardening of the seed, rather than embryo dormancy; its optimal germination temperature is 20-25℃; and light has a significant inhibitory effect on Lespedeza seed germination, classifying it as a light-sensitive seed. This study clarified the germination characteristics of Lespedeza seeds and provided a scientific basis for the development of seed treatment methods.

[0004] Existing methods for treating Lespedeza seeds mainly include: high-temperature water soaking, concentrated sulfuric acid etching, and single-hormone soaking. However, these methods have the following drawbacks in practical applications:

[0005] (1) Incomplete dormancy breaking: Although a single physical treatment (such as soaking seeds in hot water) can soften the seed coat, it has limited effect on some seeds with a high hard seed rate; a single chemical treatment (such as soaking in gibberellin) does not target the hard dormancy characteristics of Lespedeza bicolor and cannot solve the core obstacle of the seed coat being impermeable to water, resulting in a large number of seeds still failing to germinate after treatment.

[0006] (2) High seed damage rate: Although concentrated sulfuric acid etching can effectively break the hard kernel, the treatment time and concentration are difficult to control precisely, which can easily damage the embryo and reduce seed vigor. Some methods involve physical friction after soaking the seeds. At this time, the embryo has absorbed water and become soft. Friction can easily cause mechanical damage, which can reduce the germination rate.

[0007] (3) Uneven germination and severe mold: Existing methods do not effectively regulate the seed germination process, and the problem of asynchronous seed germination is common. Seeds that germinate first are easily damaged in the seedbed due to competition for water and nutrients, while seeds that germinate later may become moldy and inactive. The lack of effective antibacterial and water-retaining measures leads to a high rate of seed mold.

[0008] (4) Difficult to scale up application: Existing methods are mostly single-batch treatments under laboratory conditions, lacking a systematic process flow, and cannot meet the batch demand for high-quality seedlings in ecological restoration projects.

[0009] In summary, there is currently a lack of a wild Lespedeza seed treatment method based on seed germination characteristic diagnosis, step-by-step precise dormancy breaking, and consideration of both germination rate and seedling uniformity. This method would address the problems of low germination rate, uneven seedling emergence, and low seed utilization in existing technologies, and provide technical support for the large-scale propagation of wild Lespedeza. Summary of the Invention

[0010] This invention provides a method for breaking dormancy and promoting germination of wild Lespedeza seeds. It can simultaneously break dual dormancy barriers through a multi-level synergistic mechanism of physical skin breaking to construct water penetration channels, chemical dormancy breaking to remove dormancy, and gradient germination to simulate environmental signals. This effectively improves seed germination rate and seedling uniformity, inhibits mold growth, and is stable and highly operable. It is suitable for large-scale seedling cultivation of wild Lespedeza and provides reliable technical support for ecological restoration projects.

[0011] To achieve these objectives and other advantages according to the present invention, a method for breaking dormancy and promoting germination of wild Lespedeza seeds is provided, comprising the following steps:

[0012] Physical peel breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:3 to 1:5 and rolled and rubbed in a rotating container for 15 to 30 minutes to break the hard structure of the seed coat. After the treatment, the seeds are sieved and recovered.

[0013] Seed pretreatment: The wild Lespedeza seeds after friction treatment are cleaned and disinfected.

[0014] Warm water soaking: Soak the disinfected seeds in warm water at 35℃~45℃ for 6 h~12 h;

[0015] Chemical dormancy breaking: After soaking, the seeds are immersed in a chemical dormancy breaking agent solution at 18℃~28℃ for 4 h~10 h. The chemical dormancy breaking agent solution is composed of gibberellin (0.02%~0.08%, w / v) and osmotic regulator (0.2%~0.8%, w / v).

[0016] Gradient germination: Chemically treated seeds are mixed with a moist germination substrate with a humidity of 60%~80% at a volume ratio of 1:2~1:5 to form a mixed germination bed. The mixture is then cultured in a constant temperature environment of 18℃~25℃ for 48 h~96 h. During the germination period, the seeds are kept in the dark, and the substrate humidity of the mixed germination bed is maintained at 60%~80%.

[0017] First batch of sowing: Select the seeds that have germinated in the gradient germination process along with some substrate, spread them evenly on the seedbed, with a thickness of 2 cm to 5 cm, cover with moisture-retaining material and manage in the dark after sowing to complete the first batch of sowing.

[0018] Secondary germination and second batch sowing: The ungerminated seeds remaining from the first batch of sowing are mixed with the original substrate to form a secondary mixed germination bed. Germination is continued for 36 h to 60 h in a dark environment with a temperature of 20℃~28℃ and a substrate humidity of 65%~85%, before the second batch of sowing is carried out.

[0019] Reseeding treatment: Soak the secondary germinated seeds and the remaining seeds that have not germinated after the second batch of sowing in a 0.01%~0.03% (w / v) gibberellin solution for 3 h~9 h, and then sow them in the reseeding area of ​​the seedbed.

[0020] Seedling management: After all batches of sowing are completed, cover the sowing surface with a 1 cm to 3 cm thick layer of sterilized and moisturizing covering material, and implement drip irrigation and micro-spraying in coordination based on soil moisture sensor data to maintain substrate moisture at 40% to 80%, and manage the temperature and light environment until seedling emergence is completed.

[0021] Preferably, the surface cleaning and disinfection treatment includes the following steps: screening to remove light impurities, shriveled seeds, and seeds with a particle size of less than 1.2 mm; rinsing with clean water 2-3 times; immersing the seeds in a 0.5%-1.0% (w / v) sodium hypochlorite solution at 18℃-25℃ in the dark for 8-12 minutes; rinsing with sterile water until the outflow is neutral; and placing the seeds in a ventilated environment at 25℃-30℃ to drain the surface moisture for 30-60 minutes.

[0022] Preferably, the warm water soaking is carried out in a constant temperature water bath device, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at a speed of 30 r / min to 50 r / min for 15 min to 20 min every 2 hours, and isothermal sterile water is added every 3 hours, with the amount of water added being 5% to 8% of the initial soaking water volume. After the soaking is completed, chaff and inferior seeds floating on the liquid surface are removed.

[0023] Preferably, the physical friction medium is coarse river sand with a particle size of 2 mm to 5 mm, which is sterilized by dry heat at 120℃ to 130℃ for 20 min to 30 min before use; the rolling friction treatment is carried out in a horizontal drum with a rotation speed of 15 r / min to 25 r / min, and the seed breakage rate is monitored in real time to ensure that it does not exceed 5%; after the treatment, a double-layer vibrating screen with an upper sieve aperture of 4 mm to 6 mm and a lower sieve aperture of 1.5 mm is used to separate the seeds from the friction medium.

[0024] Preferably, the osmotic regulator in the chemical dormancy-breaking agent solution is potassium bicarbonate or potassium bisulfate, the pH value of the chemical dormancy-breaking agent solution is adjusted to 5.5~6.5, the soaking process is carried out under light-proof conditions, and the mixture is continuously stirred at a speed of 20 r / min~30 r / min, with the liquid level always 2 cm~3 cm higher than the seed surface.

[0025] Preferably, the osmotic regulator in the chemical sedative solution is a composite osmotic regulator, comprising potassium nitrate, potassium citrate, proline, and disodium ethylenediaminetetraacetate. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 40% to 60%, potassium citrate accounts for 20% to 35%, proline accounts for 5% to 15%, and disodium ethylenediaminetetraacetate accounts for 1% to 5%. The amount of the composite osmotic regulator added to the chemical sedative solution is 0.3% to 1.0% (w / v).

[0026] The preparation of the composite osmotic regulator includes: dissolving each component in a phosphate buffer solution with a pH of 6.0-6.8 in proportion, mixing them evenly under light-protected conditions at a temperature below 10°C to obtain a composite regulator stock solution; then mixing the composite regulator stock solution with the gibberellin stock solution in proportion to prepare a chemical sedative solution.

[0027] Preferably, the moisturizing and germination-promoting substrate is composed of sterilized humus, river sand and vermiculite in a volume ratio of 3:2:1; and composite particles are added thereto, the amount of composite particles being 0.4%~0.7% (w / w) of the total mass of the substrate, and the particle size of the composite particles being 0.1~0.5mm;

[0028] The composite particles have a core-shell structure: the core layer is porous starch loaded with carbendazim, accounting for 15% to 25% of the total mass of the composite particles; the middle layer is a polymer film formed by cross-linking sodium alginate and chitosan, accounting for 10% to 20%; and the shell layer is an acrylamide-acrylic acid copolymer grafted and cross-linked on a carboxymethyl cellulose backbone, accounting for 55% to 75%.

[0029] Preferably, the relative humidity of the environment during the constant temperature germination stage is maintained at 75%~80%, ventilation is carried out 3~4 times a day for 30~50 minutes each time, and the entire germination process is kept away from light.

[0030] Preferably, after forming the secondary mixed germination bed and before continuing germination, 10%–15% of the total mass of the secondary mixed germination bed is added with fresh moisturizing germination substrate, and the substrate humidity is adjusted to 65%–85%. During the continued germination process, the seeds are turned over every 12 hours, with a turning depth of 5–8 cm. For the remaining ungerminated seeds after the second batch of sowing, a second physical friction treatment is performed for 8–12 minutes before soaking in gibberellin solution. The second physical friction treatment uses the same friction medium and operating conditions as the physical peeling step. After soaking, the seeds are mixed with fresh moisturizing germination substrate at a volume ratio of 1:3 and sown in the replanting area of ​​the seedbed.

[0031] Preferably, in the seedling management step, the substrate moisture is dynamically maintained at 40% to 80% based on real-time monitoring data; the moisture-retaining covering material is crushed straw or rice husks that have been sterilized by dry heat at 120℃ to 130℃, and the covering thickness is 1 cm to 3 cm; the seedling environment temperature is controlled at 18℃ to 28℃, the day-night temperature difference is maintained at 5℃ to 8℃, and ventilation is carried out 2 to 3 times a day for 20 to 40 minutes each time.

[0032] The present invention has at least the following beneficial effects:

[0033] First, this invention, by integrating physical seed coat breaking, surface cleaning and disinfection, warm water soaking, chemical dormancy breaking, gradient germination, and batch sowing, synergistically overcomes the dual obstacles of hard seed coat and shallow physiological dormancy in wild Lespedeza seeds, increasing the seedling rate from less than 45% in conventional methods to over 88.5%, and reducing the coefficient of variation to below 0.12, thus solving the core problems of low germination rate, uneven emergence, and low seed utilization in the background technology.

[0034] Secondly, the composite permeability regulator of the present invention mainly includes potassium nitrate, potassium citrate, proline and EDTA-2Na, which work synergistically with gibberellin to achieve a more thorough breaking of seed dormancy through multiple pathways such as ion permeation, seed coat softening, membrane stabilization and protection, and free radical scavenging. Compared with a single regulator, the electrical conductivity of the seeds after soaking is reduced by 26.6% (from 65.7 μS / cm to 48.2 μS / cm), the water absorption rate is increased by 34.5% (from 42.3% to 56.9%), and the final germination rate is increased to 92.3% (Example 2), which significantly improves the dormancy breaking efficiency and seed vigor.

[0035] Third, the core-shell composite particles of this invention use a water-retaining polymer as the shell and a porous starch loaded with carbendazim as the core, combining physical water retention and chemical antibacterial functions. After addition, they can stably maintain the moisture content of the seedling substrate at a high level of 81.5% (Example 3), while simultaneously inhibiting the pathogen colony count to 8.6 × 10⁻⁶. 3The low level of CFU / g reduced the seed mold rate to 1.2% (Example 3), creating an optimized microenvironment with stable humidity and few pathogens for seed germination and seedling growth.

[0036] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0038] It should be understood that terms such as "having" and "comprising" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof. It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials described, unless otherwise specified, are commercially available and therefore should not be construed as limiting the invention.

[0039] This invention first systematically diagnosed the natural germination characteristics of wild Lespedeza seeds. Plump wild Lespedeza seeds were selected and germination tests were conducted under light-protected conditions at 25℃ and 60% substrate humidity, yielding a natural germination rate of 21.3% ± 2.1%. Further testing revealed that 52% ± 3% of the seeds did not absorb water after soaking for 48 hours (hard dormancy), and approximately 30% ± 4% of the seeds that did absorb water required hormone stimulation to germinate (indicating shallow physiological dormancy). Based on these diagnostic results, this invention designed a step-by-step, precise dormancy-breaking scheme: first, physical breaking of the hard seed coat; then, warm water soaking to promote water absorption; and finally, chemical treatment to enhance germination.

[0040] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0041] Physical peel breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:3 to 1:5 and rolled and rubbed in a rotating container for 15 to 30 minutes to break the hard structure of the seed coat. After the treatment, the seeds are sieved and recovered.

[0042] Seed pretreatment: The wild Lespedeza seeds after friction treatment are cleaned and disinfected.

[0043] Soaking seeds in warm water: Soak the disinfected seeds in warm water at 35℃~45℃ for 6 h~12 h;

[0044] Chemical dormancy breaking: After soaking, the seeds are immersed in a chemical dormancy breaking agent solution at 18℃~28℃ for 4 h~10 h. The chemical dormancy breaking agent solution is composed of gibberellin (0.02%~0.08%, w / v) and osmotic regulator (0.2%~0.8%, w / v).

[0045] Gradient germination: Chemically treated seeds are mixed with a moist germination substrate with a humidity of 60%–80% at a volume ratio of 1:2 to 1:5 to form a mixed germination bed. The mixture is then cultured in a constant temperature environment of 18℃–25℃ for 48 h–96 h. During the germination period, the mixture is kept in the dark, and the substrate humidity of the mixed germination bed is maintained at 60%–80%.

[0046] First batch of sowing: Select the seeds that have germinated in the gradient germination process along with some substrate, spread them evenly on the seedbed, with a thickness of 2 cm to 5 cm, cover with moisture-retaining material and manage in the dark after sowing to complete the first batch of sowing.

[0047] Secondary germination and second batch sowing: The ungerminated seeds remaining from the first batch of sowing are mixed with the original substrate to form a secondary mixed germination bed. Germination is continued for 36 h to 60 h in a dark environment with a temperature of 20℃~28℃ and a substrate humidity of 65%~85%, before the second batch of sowing is carried out.

[0048] Reseeding treatment: Soak the secondary germinated seeds and the remaining seeds that have not germinated after the second batch of sowing in a 0.01% to 0.03% (w / v) gibberellin solution for 3 to 9 hours, and then sow them in the reseeding area of ​​the seedbed.

[0049] Seedling management: After all batches of sowing are completed, cover the sowing surface with a 1 cm to 3 cm thick layer of sterilized and moisturizing covering material, and implement drip irrigation and micro-spraying in coordination based on soil moisture sensor data to maintain substrate moisture at 40% to 80%, and manage the temperature and light environment until seedling emergence is completed.

[0050] The above technical solution completely overcomes the seed hardness barrier through cleaning and disinfection, physical seed breaking (dry seed friction), warm water soaking, chemical dormancy breaking, gradient germination (constant temperature and light avoidance), batch sowing, and subsequent management, solving the problems of low germination rate and uneven germination in existing methods. The batch sowing strategy enables the secondary use of ungerminated seeds, reducing seed waste, improving seed utilization, increasing propagation efficiency, and ensuring uniform seedling emergence. It is suitable for large-scale seedling production, meeting the planting needs of wild Lespedeza vegetation restoration, soil and water conservation, and other fields. It is simple to operate, highly practical, and easy to promote and apply.

[0051] Specifically, the surface cleaning and disinfection treatment includes: first, removing light impurities, shriveled seeds, and seeds with a diameter of less than 1.2 mm to reduce the impact of inferior seeds on the breeding effect; then rinsing with clean water 2-3 times; subsequently, immersing the seeds in a 0.5%-1.0% (w / v) sodium hypochlorite solution at 18℃-25℃ for 8-12 minutes to effectively kill pathogens on the seed surface and prevent seed mold during subsequent germination and sowing; after disinfection, rinsing with sterile water until neutral to prevent disinfection residue from damaging the seeds; and finally, draining the seeds in a ventilated environment at 25℃-30℃ for 30-60 minutes.

[0052] Specifically, warm water soaking is carried out under constant temperature conditions, with temperature fluctuations controlled within ±1℃ to ensure that the seeds absorb water evenly in a stable temperature environment. During the soaking period, the seeds are stirred for 15 to 20 minutes every 2 hours to prevent them from sinking and piling up, ensuring that each seed is fully in contact with water. The stirring rate is 30 to 50 r / min, and isothermal sterile water is added every 3 hours, with the amount of water added being 5% to 8% of the initial water volume, to replenish the water evaporated during the soaking process and maintain a stable soaking water volume. After the soaking is completed, inferior seeds are removed.

[0053] Specifically, the physical desiccation treatment is carried out in a dry seed condition. The physical friction medium is coarse river sand with a particle size of 2 mm to 5 mm, which effectively abrades the seed coat. Before use, the seeds are sterilized at 120℃ to 130℃ for 20 min to 30 min. The rolling friction treatment is carried out in a horizontal drum with a rotation speed of 15 r / min to 25 r / min. During the treatment, the seed damage rate is controlled to not exceed 3% to 5% to maximize the protection of seed integrity. After treatment, a double-layer vibrating screen with an upper aperture of 4 mm to 6 mm and a lower aperture of 1.5 mm is used to separate the seeds from the medium, resulting in good separation effect and high efficiency.

[0054] Specifically, the osmotic regulator in the chemical dormancy-breaking agent solution is potassium bicarbonate or potassium bisulfate, which adjusts the seed osmotic pressure, promotes seed water absorption, and helps break down the seed coat barrier. The pH value of the chemical dormancy-breaking agent solution is adjusted to 5.5-6.5. The soaking process is carried out in the dark and under conditions of continuous stirring at a rate of 20 r / min-30 r / min. During soaking, the liquid level is 2 cm-3 cm higher than the seed surface to ensure that the seeds are evenly contacted with the dormancy-breaking agent, improve the dormancy-breaking effect, and accelerate the seed germination rate.

[0055] Preferably, the osmotic regulator in the chemical dormancy-breaking agent solution includes a composite regulator of potassium nitrate, potassium citrate, proline, and disodium EDTA. Potassium nitrate regulates osmotic pressure and promotes water absorption, potassium citrate softens the seed coat and assists in dormancy breaking, proline can enhance the seed's resistance to stress during the dormancy-breaking process, and disodium EDTA chelates metal ions and protects seed activity. This solution addresses multiple aspects such as seed water absorption, seed coat softening, and stress resistance protection, and its dormancy-breaking effect is superior to that of a single osmotic regulator. Based on the total weight of the osmotic regulator, potassium nitrate accounts for 40%–60%, potassium citrate accounts for 20%–35%, proline accounts for 5%–15%, and disodium EDTA accounts for 1%–5%. The amount of osmotic regulator added is 0.3%–1.0% (w / v) of the chemical dormancy-breaking agent solution.

[0056] Each component was dissolved in a phosphate buffer solution with a pH of 6.0–6.8 in a specific ratio and mixed thoroughly in a light-protected environment below 10°C to obtain a composite regulator stock solution. The composite regulator stock solution was then mixed with the gibberellin stock solution in a specific ratio to obtain a chemical sedative solution.

[0057] Preferably, the moisture-retaining germination substrate is composed of humus, river sand and vermiculite in a volume ratio of 3:2:1. It has a loose texture and good water retention and air permeability, which can provide a suitable substrate environment for seed germination. All components are sterilized. Composite particles are added to achieve water retention and antibacterial functions. The amount of composite particles added is 0.4% to 0.7% (w / w) of the substrate, and the particle size is 0.1 to 0.5 mm.

[0058] The composite granules have a core-shell structure: the core layer is porous starch loaded with carbendazim, accounting for 15%–25% of the total mass of the composite granules; the middle layer is a polymer film formed by cross-linking sodium alginate and chitosan, accounting for 10%–20%; and the shell layer is an acrylamide-acrylic acid copolymer grafted and cross-linked onto a carboxymethyl cellulose backbone, accounting for 55%–75%. The shell layer can lock in water through a three-dimensional network, improving and stabilizing the substrate moisture, providing a continuous and stable water supply for seed germination and seedling growth; the middle layer has both hydrophilicity and structural stability, which can both assist in water retention and enhance the overall strength of the granules; the core layer, loaded with carbendazim through porous starch, can achieve slow release of fungicide and long-term inhibition of pathogen reproduction in the substrate.

[0059] Preferably, the relative humidity of the environment during the gradient germination stage is maintained at 75% to 80%, and ventilation is carried out 3 to 4 times a day for 30 to 50 minutes each time. The entire germination process is kept in the dark to meet the light-sensitive characteristics of Lespedeza seeds and promote uniform germination.

[0060] Preferably, before continuing germination, the secondary mixed germination bed is supplemented with 10%–15% of its total mass of fresh moist germination substrate and the humidity is adjusted to 65%–85% to provide sufficient nutrition and suitable humidity for ungerminated seeds, thereby improving the secondary germination effect. During the germination process, the seeds are turned over every 12 hours to a depth of 5–8 cm to prevent substrate compaction and seed mold. For the remaining seeds that have not germinated after the second batch of sowing, a second physical friction treatment of 8–12 minutes is performed before soaking in gibberellin solution to break down the seed coat barrier of the ungerminated seeds and assist in breaking dormancy. After soaking, the seeds are mixed with fresh moist germination substrate at a volume ratio of 1:3 and sown to provide a good germination environment for the remaining seeds.

[0061] Specifically, the unified water management adopts a drip irrigation and micro-sprinkler irrigation system based on soil moisture sensors. The sensors monitor the substrate moisture in real time, automatically control the start and stop of irrigation, and precisely control the substrate moisture. Based on the sensor data, the substrate moisture is maintained at 40% to 80%, avoiding the impact of over- or under-watering on seedlings. The moisture-retaining covering material is sterilized crushed straw or rice husks, with a covering thickness of 1 cm to 3 cm. The ambient temperature after germination and sowing is maintained at 18℃ to 28℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out regularly to meet the growth needs of wild Lespedeza seedlings.

[0062] This invention constructs a step-by-step precise dormancy-breaking system to achieve complete dormancy breaking and uniform germination of wild Lespedeza seeds, thus solving the seed dormancy problem:

[0063] First, the seed coat is physically broken while the seeds are dry, creating micro-scars on the seed coat. This opens physical channels for subsequent water penetration and avoids damage to the embryo caused by friction after soaking.

[0064] Secondly, soaking seeds in warm water utilizes the thermal expansion and lubrication effects of water to allow water to quickly penetrate along the micro-wounds, softening the seed coat and activating the hydrolytic enzymes inside the seed.

[0065] Secondly, the osmotic regulator enters the seed through channels opened by physical friction, and promotes the seed to fully absorb water and swell by utilizing the ion osmotic pressure difference, thus chemically supplementing and breaking down the seed coat hardening barrier; at the same time, gibberellin and proline and other components can enhance the seed's stress resistance and strengthen the germination of some seeds with shallow physiological dormancy.

[0066] Finally, gradient germination (constant temperature and light avoidance) provides stable energy for the emergence of the radicle, avoids the inhibition of light on light-sensitive seeds, and ensures the synchronicity of population germination.

[0067] <Example 1>

[0068] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0069] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm. Before use, the seeds are sterilized at 130℃ for 30 min and then rolled and rubbed in a horizontal drum with a speed of 20 r / min for 20 min. The seed breakage rate is controlled to be no more than 5%. After treatment, the seeds and medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm.

[0070] (2) Seed pretreatment: First remove light impurities, shriveled seeds and seeds with a diameter of less than 1.2 mm, then rinse with clean water 3 times, then immerse the seeds in a 0.7% (w / v) sodium hypochlorite solution at 22℃ for 10 min, disinfect and rinse with sterile water until neutral, and finally drain the seeds in a ventilated environment at 25℃~30℃ for 45 min.

[0071] (3) Soaking seeds in warm water: Place the disinfected seeds in a constant temperature water bath at 40℃ for 9 hours, and control the temperature fluctuation within ±1℃. During the soaking period, stir at 30 r / min for 15 minutes every 2 hours, and add isothermal sterile water (6% of the initial water volume) every 3 hours. After the soaking is completed, remove the inferior seeds floating on the liquid surface.

[0072] (4) Chemical dormancy breaking: The seeds after soaking are immersed in a chemical dormancy breaking agent solution, which is composed of gibberellin (0.05%, w / v) and potassium bicarbonate (0.5%, w / v) as an osmotic regulator, and the pH value is adjusted to 6.0; under the condition of 25℃ in the dark, the seeds are continuously stirred and soaked for 6 h at 20 r / min, and the liquid level is always 2 cm higher than the seed surface.

[0073] (5) Gradient germination: The chemically treated seeds are mixed with a 70% moisture-retaining germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisture-retaining germination substrate is composed of humus, river sand and vermiculite at a volume ratio of 3:2:1 (all components are sterilized); the mixed germination bed is placed in a constant temperature environment of 22℃ for 72 h, kept in the dark during germination, the relative humidity of the environment is maintained at 75% to 80%, and ventilation is carried out 3 times a day for 30 min each time, and the substrate humidity is maintained at 60% to 80%.

[0074] (6) First batch of sowing: Select the seeds that have germinated in the gradient germination process along with some substrate, spread them evenly on the seedbed with a thickness of 3 cm, cover them with 1-2 cm of sterilized straw after sowing and manage them in the dark to complete the first batch of sowing.

[0075] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining from the first batch of sowing are mixed with the original substrate again, and 12% of the total mass of fresh moist germination substrate is added. The humidity is adjusted to 70% to form a secondary mixed germination bed. Germination continues for 48 hours in a dark environment at 22℃ and 70% humidity, and the seeds are turned over once every 12 hours (to a depth of 6 cm). Then the second batch of sowing is carried out.

[0076] (8) Replanting treatment: The remaining seeds that have not germinated after the second batch of sowing are subjected to a second physical friction treatment for 10 min (under the same conditions as step 1), then soaked in 0.02% (w / v) gibberellin solution for 6 h, and then mixed with fresh moist germination substrate at a volume ratio of 1:3, and sown in the seedbed replanting area.

[0077] (9) Seedling management: After all batches of sowing are completed, the surface is covered with 2 cm of sterilized crushed straw; drip irrigation and micro-spraying are coordinated and controlled according to soil moisture sensor data to maintain substrate moisture of 40% to 80%; the ambient temperature is controlled at 22℃, the day-night temperature difference is 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0078] <Example 2>

[0079] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0080] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0081] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0082] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0083] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) composite osmotic regulator. The composite osmotic regulator consists of potassium nitrate, potassium citrate, proline, and disodium EDTA. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 54.4%, potassium citrate accounts for 29.1%, proline accounts for 12.7%, and disodium EDTA accounts for 3.8%. During preparation, each component is first dissolved in a phosphate buffer solution with a pH of 6.5 in proportion and mixed evenly under light-protected conditions at a temperature below 10°C to obtain a composite regulator stock solution. Then, the composite regulator stock solution and the gibberellin stock solution are mixed in proportion to prepare the chemical dormancy breaking agent solution, and the pH value is adjusted to 6.0. The soaking process is carried out under light-protected conditions, with the temperature controlled at 25°C and continuous stirring at a speed of 20 r / min to ensure that the liquid level is always higher than the seed surface by 2 mm. cm, soaking time is 6 h.

[0084] (5) Gradient germination: The seeds after chemical dormancy breaking treatment are mixed with the moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate is composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component is sterilized before use, and the substrate humidity is adjusted to 70%; the mixed germination bed is placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds are kept in the dark, and the relative humidity of the environment is maintained at 75% to 80%. The seeds are ventilated 3 times a day for 30 minutes each time. The substrate humidity of the mixed germination bed is always maintained within the range of 60% to 80%.

[0085] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0086] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment of 22℃ and 70% humidity for 48 hours to continue germination. During the germination process, the seeds are turned over once every 12 hours, and the turning depth is 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0087] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0088] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0089] <Example 3>

[0090] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0091] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0092] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the following steps are taken: First, light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm are removed by wind selection or manual screening. Then, the seeds are rinsed three times with clean water. Next, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0093] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0094] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) potassium bicarbonate osmotic regulator. The pH value of the solution is adjusted to 6.0 with dilute acid or dilute alkali. The soaking process is carried out under light-proof conditions, the temperature is controlled at 25℃, and the stirring is continuously carried out at a speed of 20r / min to ensure that the liquid level is always 2 cm higher than the seed surface. The soaking time is 6 h.

[0095] (5) Gradient germination: The seeds after chemical dormancy breaking treatment were mixed with a moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate was composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component was sterilized before use. The substrate humidity was adjusted to 70%, and 0.5% (w / w) of core-shell structured composite particles were added. The particle size of the composite particles was 0.1-0.5 mm. The composite particles were core-shell structured. The core layer was porous starch loaded with carbendazim, accounting for 20% of the total mass of the composite particles. The middle layer was a polymer film formed by cross-linking sodium alginate and chitosan, accounting for 15%. The shell layer was acrylamide-acrylic acid copolymer grafted and cross-linked on a carboxymethyl cellulose backbone, accounting for 65%. The mixed germination bed was placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds were kept in the dark, and the relative humidity was maintained at 75%-80%. The soil was ventilated 3 times a day for 30 minutes each time. The substrate moisture content of the mixed germination bed is maintained within the range of 60% to 80% throughout the process.

[0096] The method for preparing composite particles is as follows: porous starch is immersed in a carbendazim aqueous solution for adsorption and drying to obtain a core layer, then coated with a mixed layer of sodium alginate and chitosan, and finally coated with an acrylamide water-retaining layer. After drying and sieving, core-shell structured composite particles are obtained, specifically including:

[0097] 1) Core layer preparation: Porous starch and 8% (w / v) carbendazim aqueous solution were mixed at a mass ratio of 1:4, stirred and adsorbed at 25℃ for 3h, then filtered and dried at 60℃ to obtain carbendazim-loaded core layer particles.

[0098] 2) Intermediate layer coating: The core layer particles obtained in step 1) are immersed in a mixed aqueous solution of sodium alginate with a concentration of 2% (w / v) and chitosan with a concentration of 1.2% (w / v). The mass ratio of core layer particles to mixed aqueous solution is 1:4. The mixture is stirred and crosslinked at 25°C for 30 min. After filtration, it is dried at 45°C to obtain particles coated with an intermediate layer.

[0099] 3) Shell construction: The particles obtained in step 2) are immersed in a mixed solution containing acrylamide, acrylic acid, N-isopropylacrylamide and carboxymethyl cellulose, wherein the molar ratio of acrylamide to acrylic acid is 1:1.2, the amount of N-isopropylacrylamide added is 15% of the total mass of acrylamide and acrylic acid, and the amount of carboxymethyl cellulose added is 10% of the total mass of acrylamide, acrylic acid and N-isopropylacrylamide. Potassium persulfate is used as an initiator, and its amount is 1.2% of the total mass of acrylamide, acrylic acid and N-isopropylacrylamide. The reaction is stirred at 66°C for 2 hours. After the reaction, the mixture is filtered, dried at 66°C, and sieved to obtain the core-shell structured composite particles with a particle size of 0.1~0.5 mm.

[0100] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0101] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate (with 0.5% of composite particles added) is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment of 22℃ and 70% humidity to continue germination for 48 h, and is turned over once every 12 h during the germination process, with a turning depth of 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0102] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate (with added composite particles) at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0103] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0104] <Example 4>

[0105] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0106] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0107] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0108] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0109] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) composite osmotic regulator. The composite osmotic regulator consists of potassium nitrate, potassium citrate, proline, and disodium EDTA. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 54.4%, potassium citrate accounts for 29.1%, proline accounts for 12.7%, and disodium EDTA accounts for 3.8%. During preparation, each component is first dissolved in a phosphate buffer solution with a pH of 6.5 in proportion and mixed evenly under light-protected conditions at a temperature below 10°C to obtain a composite regulator stock solution. Then, the composite regulator stock solution and the gibberellin stock solution are mixed in proportion to prepare the chemical dormancy breaking agent solution, and the pH value is adjusted to 6.0. The soaking process is carried out under light-protected conditions, with the temperature controlled at 25°C and continuous stirring at a speed of 20 r / min to ensure that the liquid level is always higher than the seed surface by 2 mm. cm, soaking time is 6 h.

[0110] (5) Gradient germination: The seeds after chemical dormancy breaking treatment were mixed with a moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate was composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component was sterilized before use. The substrate humidity was adjusted to 70%, and 0.5% (w / w) of core-shell structured composite particles were added. The particle size of the composite particles was 0.1-0.5 mm. The composite particles were core-shell structured. The core layer was porous starch loaded with carbendazim, accounting for 20% of the total mass of the composite particles. The middle layer was a polymer film formed by cross-linking sodium alginate and chitosan, accounting for 15%. The shell layer was acrylamide-acrylic acid copolymer grafted and cross-linked on a carboxymethyl cellulose backbone, accounting for 65%. The mixed germination bed was placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds were kept in the dark, and the relative humidity was maintained at 75%-80%. The soil was ventilated 3 times a day for 30 minutes each time. The substrate moisture content of the mixed germination bed is maintained within the range of 60% to 80% throughout the process.

[0111] The method for preparing composite particles is as follows: porous starch is immersed in a carbendazim aqueous solution for adsorption and drying to obtain a core layer, then coated with a mixed layer of sodium alginate and chitosan, and finally coated with an acrylamide water-retaining layer. After drying and sieving, core-shell structured composite particles are obtained, specifically including:

[0112] 1) Core layer preparation: Porous starch and 8% (w / v) carbendazim aqueous solution were mixed at a mass ratio of 1:4, stirred and adsorbed at 25℃ for 3h, then filtered and dried at 60℃ to obtain carbendazim-loaded core layer particles.

[0113] 2) Intermediate layer coating: The core layer particles obtained in step 1) are immersed in a mixed aqueous solution of sodium alginate with a concentration of 2% (w / v) and chitosan with a concentration of 1.2% (w / v). The mass ratio of core layer particles to mixed aqueous solution is 1:4. The mixture is stirred and crosslinked at 25°C for 30 min. After filtration, it is dried at 45°C to obtain particles coated with an intermediate layer.

[0114] 3) Shell construction: The particles obtained in step 2) are immersed in a mixed solution containing acrylamide, acrylic acid, N-isopropylacrylamide and carboxymethyl cellulose, wherein the molar ratio of acrylamide to acrylic acid is 1:1.2, the amount of N-isopropylacrylamide added is 15% of the total mass of acrylamide and acrylic acid, and the amount of carboxymethyl cellulose added is 10% of the total mass of acrylamide, acrylic acid and N-isopropylacrylamide. Potassium persulfate is used as an initiator, and its amount is 1.2% of the total mass of acrylamide, acrylic acid and N-isopropylacrylamide. The reaction is stirred at 66°C for 2 hours. After the reaction, the mixture is filtered, dried at 66°C, and sieved to obtain the core-shell structured composite particles with a particle size of 0.1~0.5 mm.

[0115] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0116] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate (with 0.5% of composite particles added) is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment of 22℃ and 70% humidity to continue germination for 48 h, and is turned over once every 12 h during the germination process, with a turning depth of 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0117] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate (with added composite particles) at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0118] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0119] <Comparative Example 1>

[0120] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0121] (1) Seed pretreatment: Wild Lespedeza seeds were cleaned and disinfected. Specifically, the following steps were taken: first, light impurities, shriveled seeds and inferior seeds with a diameter of less than 1.2 mm were removed by winnowing or manual screening; then, the seeds were rinsed three times with clean water; then, the seeds were soaked in a 0.7% (w / v) sodium hypochlorite solution at 22°C in the dark for 10 min for disinfection; after disinfection, the seeds were rinsed repeatedly with sterile water until the outflow was neutral; finally, the seeds were placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0122] (2) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent the seeds from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0123] (3) Direct sowing: Without physical skin breaking, chemical dormancy breaking and gradient germination treatment, the seeds soaked in warm water are directly and evenly spread on the seedbed with a thickness of 3 cm. After sowing, the surface is covered with 1-2 cm of sterilized crushed straw as a moisture-retaining covering material to complete the first batch of sowing.

[0124] (4) Seedling management: After sowing, water management is carried out using a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor. The substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data. The seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0125] <Comparative Example 2>

[0126] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0127] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0128] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0129] (3) Direct sowing: Without soaking seeds in warm water, chemical dormancy breaking and gradient germination treatment, the pretreated seeds are directly and evenly spread on the seedbed with a thickness of 3 cm. After sowing, the surface is covered with 1-2 cm of sterilized crushed straw as a moisture-retaining covering material to complete the first batch of sowing.

[0130] (4) Seedling management: After sowing, water management is carried out using a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor. The substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data. The seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0131] <Comparative Example 3>

[0132] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0133] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0134] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0135] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0136] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) composite osmotic regulator. The composite osmotic regulator consists of potassium nitrate, potassium citrate, and disodium EDTA, and does not contain proline. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 62.1%, potassium citrate accounts for 33.2%, and disodium EDTA accounts for 4.7%. During preparation, each component is first dissolved in a phosphate buffer solution with a pH of 6.5 in proportion and mixed evenly under light-protected conditions at a temperature below 10°C to obtain a composite regulator stock solution. Then, the composite regulator stock solution and the gibberellin stock solution are mixed in proportion to prepare the chemical dormancy breaking agent solution, and the pH value is adjusted to 6.0. The soaking process is carried out under light-protected conditions, with the temperature controlled at 25°C and continuous stirring at a speed of 20 r / min to ensure that the liquid level is always 2 cm above the seed surface. The soaking time is 6 minutes. h.

[0137] (5) Gradient germination: The seeds after chemical dormancy breaking treatment are mixed with the moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate is composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component is sterilized before use, and the substrate humidity is adjusted to 70%; the mixed germination bed is placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds are kept in the dark, and the relative humidity of the environment is maintained at 75% to 80%. The seeds are ventilated 3 times a day for 30 minutes each time. The substrate humidity of the mixed germination bed is always maintained within the range of 60% to 80%.

[0138] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0139] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment of 22℃ and 70% humidity for 48 hours to continue germination. During the germination process, the seeds are turned over once every 12 hours, and the turning depth is 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0140] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0141] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0142] <Comparative Example 4>

[0143] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0144] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0145] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0146] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0147] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) composite osmotic regulator. The composite osmotic regulator consists of potassium nitrate, potassium citrate, and disodium EDTA, and does not contain proline. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 62.1%, potassium citrate accounts for 33.2%, and disodium EDTA accounts for 4.7%. During preparation, each component is first dissolved in a phosphate buffer solution with a pH of 6.5 in proportion and mixed evenly under light-protected conditions at a temperature below 10°C to obtain a composite regulator stock solution. Then, the composite regulator stock solution and the gibberellin stock solution are mixed in proportion to prepare the chemical dormancy breaking agent solution, and the pH value is adjusted to 6.0. The soaking process is carried out under light-protected conditions, with the temperature controlled at 25°C and continuous stirring at a speed of 20 r / min to ensure that the liquid level is always 2 cm above the seed surface. The soaking time is 6 minutes. h.

[0148] (5) Gradient germination: The seeds after chemical dormancy breaking treatment are mixed with the moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate is composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component is sterilized before use, and the substrate humidity is adjusted to 70%; the mixed germination bed is placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds are kept in the dark, and the relative humidity of the environment is maintained at 75% to 80%. The seeds are ventilated 3 times a day for 30 minutes each time. The substrate humidity of the mixed germination bed is always maintained within the range of 60% to 80%.

[0149] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0150] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment of 22℃ and 70% humidity for 48 hours to continue germination. During the germination process, the seeds are turned over once every 12 hours, and the turning depth is 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0151] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0152] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0153] <Comparative Example 5>

[0154] Methods for breaking dormancy and promoting germination of wild Lespedeza seeds include the following steps:

[0155] (1) Physical breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:4. The physical friction medium is coarse river sand with a particle size of 3.5 mm, which is sterilized at 130℃ for 30 min before use. The mixture is placed in a horizontal drum with a rotation speed of 20 r / min for 20 min of rolling friction treatment. During the treatment, the seed breakage rate is monitored in real time and controlled to not exceed 5%. After the treatment, the seeds and friction medium are separated by a double-layer vibrating screen with an upper pore size of 5 mm and a lower pore size of 1.5 mm, and the seeds are recovered.

[0156] (2) Seed pretreatment: After physical cracking, the seeds are cleaned and disinfected. Specifically, the seeds are first removed by wind selection or manual screening to remove light impurities, shriveled seeds and inferior seeds with a particle size of less than 1.2 mm. Then, the seeds are rinsed with clean water 3 times. Then, the seeds are immersed in a 0.7% (w / v) sodium hypochlorite solution and disinfected at 22°C in the dark for 10 min. After disinfection, the seeds are rinsed repeatedly with sterile water until the outflow is neutral. Finally, the seeds are placed in a ventilated environment at 25°C to 30°C to drain the surface moisture for 45 min.

[0157] (3) Soaking seeds in warm water: The pretreated seeds are placed in a constant temperature water bath at 40℃ for soaking for 9 hours, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at 30 r / min for 15 minutes every 2 hours to prevent them from sinking and accumulating. Every 3 hours, isothermal sterile water is added, and the amount of water added is 6% of the initial soaking water volume to maintain the stability of the soaking liquid surface. After the soaking is completed, the chaff and inferior seeds floating on the liquid surface are removed.

[0158] (4) Chemical dormancy breaking: After soaking in warm water, the seeds are immersed in a chemical dormancy breaking agent solution. The chemical dormancy breaking agent solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) potassium bicarbonate osmotic regulator. The pH value of the solution is adjusted to 6.0 with dilute acid or dilute alkali. The soaking process is carried out under light-proof conditions, the temperature is controlled at 25℃, and the stirring is continuously carried out at a speed of 20 r / min to ensure that the liquid level is always 2 cm higher than the seed surface. The soaking time is 6 h.

[0159] (5) Gradient germination: The seeds after chemical dormancy breaking treatment are mixed with the moisturizing germination substrate at a volume ratio of 1:5 to form a mixed germination bed; the moisturizing germination substrate is composed of humus, river sand and vermiculite at a volume ratio of 3:2:1. Each component is sterilized before use. The substrate humidity is adjusted to 70%, and a 0.4% (w / v) acrylamide-acrylic acid copolymer water-retaining agent is added directly to it at a concentration of 2.5% (w / w) of the total substrate mass; the mixed germination bed is placed in a constant temperature environment of 22℃ for 72 h for gradient germination. During the germination period, the seeds are kept in the dark, and the relative humidity of the environment is maintained at 75% to 80%. Ventilation is carried out 3 times a day for 30 minutes each time. The substrate humidity of the mixed germination bed is always maintained within the range of 60% to 80%.

[0160] (6) First batch of sowing: Select the seeds that have germinated after gradient germination along with some moisturizing germination substrate, spread them evenly on the seedbed, and spread them to a thickness of 3 cm; after sowing, cover the surface with 1-2 cm of sterilized crushed straw as a moisturizing covering material, and manage it under the dark to complete the first batch of sowing.

[0161] (7) Secondary germination and second batch sowing: The ungerminated seeds remaining in the seedbed after the first batch of sowing are collected together with the original moisturizing germination substrate to form a secondary mixed germination bed; 12% of the total mass of fresh moisturizing germination substrate (with water-retaining agent added) is added to the secondary mixed germination bed, and the substrate humidity is adjusted to 70%; the secondary mixed germination bed is placed in a dark environment at 22℃ and 70% humidity for 48 hours to continue germination, and is turned over once every 12 hours during the germination process, with a turning depth of 6 cm; after the germination is completed, the second batch of sowing is carried out, and the sowing method is the same as step (6).

[0162] (8) Replanting treatment: Collect the remaining seeds that have not germinated after the second batch of sowing, and perform a second physical friction treatment. The treatment conditions are the same as in step (1), and the treatment time is 10 min. Then soak them in a 0.02% (w / v) gibberellin solution for 6 h. The soaking conditions are the same as in step (4). After soaking, mix the seeds with fresh moisturizing germination substrate (with added water-retaining agent) at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

[0163] (9) Seedling management: After all batches of sowing are completed, a 2 cm thick layer of sterilized crushed straw is evenly covered on the sowing surface as a moisture-retaining covering material; a drip irrigation and micro-spraying coordinated control system based on soil moisture sensor is used for water management, and the substrate moisture is dynamically maintained at 40% to 80% according to real-time monitoring data; the seedling environment temperature is controlled at 22℃, the day-night temperature difference is controlled at 5℃ to 8℃, and ventilation is carried out twice a day for 30 minutes each time until the seedlings emerge.

[0164] <Comparative Experiment on Overall Breeding Effects>

[0165] To verify the comprehensive advantages of the systematic breeding process of this invention in terms of germination potential, seedling rate, uniformity, and mold resistance, 1000 plump wild Lespedeza seeds were selected from each of Examples 1-4 and Comparative Examples 1-2. Seed treatment, germination, sowing, and management were carried out according to the methods of each group. The number of germinated seeds in each group was counted daily (germination was defined as the radicle breaking through the seed coat ≥ 2 mm), and detailed records were kept. Based on the daily germination records, the germination potential on day 7 (percentage of germinated seeds on day 7 out of the total number of seeds), coefficient of variation (expressed as the coefficient of variation of germination time, recording the average X and standard deviation S of the germination time for each seed, coefficient of variation = (S / X) × 100%), and germination rate on day 21 (percentage of cumulative germinated seeds on day 21 out of the total number of seeds) were calculated. When the seedlings grew to 4 true leaves, the seedling rate (number of healthy seedlings / number of germinated seeds) was calculated, and the average plant height and average ground diameter (diameter measured 1 cm above the base) were measured. Throughout the experiment (from seed treatment to the seedling stage with 4 true leaves), the mold growth of seeds in each group was observed and recorded daily, and the mold rate was finally calculated. The experimental results are shown in Table 1.

[0166] Table 1

[0167]

[0168] As shown in Table 1, Example 1 showed a comprehensive improvement in all indicators compared to Comparative Example 1. Water absorption tests on the ungerminated seeds of Comparative Example 1 revealed that approximately 85% of the seeds did not absorb water after 48 hours of soaking (hard seed dormancy), and 15% of the seeds failed to germinate after water absorption (possibly due to shallow physiological dormancy or impaired viability). This indicates that the low germination rate of Comparative Example 1 was mainly due to the lack of physical seed coat breaking, resulting in the seed coat remaining hard and unable to absorb water. A small number of seeds that absorbed water also failed to germinate due to a lack of chemical stimulation.

[0169] Compared to Comparative Example 2, Example 1 showed a significant advantage in germination vigor and seedling survival rate. Testing of the ungerminated seeds in Comparative Example 2 revealed that approximately 60% of the seeds absorbed water after soaking (indicating that physical breaking of the seed coat effectively opened the seed coat channels), but failed to germinate after swelling, indicating that physical breaking alone cannot resolve the physiological dormancy issue in some seeds; the remaining 40% of seeds did not absorb water, suggesting that the intensity of the physical breaking treatment may be insufficient or there may be individual differences. Comparative Example 2 relied solely on physical breaking, without warm water soaking to activate enzyme activity or chemical dormancy breaking to enhance germination, resulting in insufficient seed germination momentum.

[0170] Compared to Example 1, Example 2 showed further improvements in germination potential and seedling survival rate. This is because in its compound regulator, potassium nitrate and potassium citrate synergistically regulate osmotic pressure and soften the seed coat, proline enhances the seed's resistance to stress during dormancy breaking, and EDTA-2Na chelates metal ions, together achieving a more thorough dormancy breaking effect than potassium bicarbonate alone.

[0171] Compared with Example 1, Example 3 showed a significantly reduced mold rate and better seedling growth. This is because the core-shell composite particles simultaneously provide water retention and slow-release antibacterial functions, creating a more stable and healthier rhizosphere microenvironment for the seedlings.

[0172] Example 4 showed the best performance across all metrics because the compound regulator maximized the seed dormancy potential, while the compound particles provided an ideal and stable microenvironment for the germination of highly vigorous seeds and the growth of seedlings, resulting in a synergistic effect.

[0173] <Chemical Sleep Disruptor Synergistic Validation Test>

[0174] To verify the synergistic advantages of the composite osmotic regulator over the single regulator in terms of conductivity, water absorption, and dormancy breaking efficiency, Examples 1 and 2, Comparative Example 3, and a blank group (soaked in water, with other operations the same as in Example 1) were selected for comparison. Each group used 1000 plump wild Lespedeza seeds (52% hard seed rate, 21% natural germination rate) and treated them according to the methods used in each group. Before and after soaking in the dormancy-breaking agent (or water), the conductivity of the seed extract was measured using a conductivity meter with an accuracy of 0.1 μS / cm; the seed mass was weighed before and after soaking, and the water absorption rate was calculated. After dormancy breaking, each group was cultured under the same germination conditions as in Example 1. The number of germinated seeds was counted on day 21, and the germination rate was calculated. A cutting test was performed on ungerminated seeds: seeds with hard seed coats that could not be cut were considered dormant; seeds with cut seed coats but no embryo viability were considered dormant. The results are shown in Table 2.

[0175] Table 2

[0176]

[0177] As shown in Table 2, Example 2 exhibited the lowest electrical conductivity (48.2 μS / cm), highest water absorption (56.9%), and highest germination rate (92.3%) after soaking. This is attributed to the synergistic effect of its components: K + The dominant osmotic regulation, citrate softens the seed coat, proline enhances cell membrane stability and improves seed stress resistance, and EDTA-2Na scavenge free radicals, together effectively breaking the hard seed dormancy and strengthening the seed with shallow physiological dormancy.

[0178] Comparative Example 3 (proline deficiency) was inferior to Example 2 in all aspects. This is because the lack of proline weakened the seeds' resistance during the dormancy breaking process, and the vitality of some seeds was damaged, resulting in incomplete breaking of hard dormancy.

[0179] Example 1 has limited effectiveness because potassium bicarbonate can only provide an alkaline environment and K. + Ions provide initial osmotic regulation and soften the seed coat, but have a weak effect on breaking the dormancy of hard seeds.

[0180] The increased conductivity of the blank group (water) after soaking indicates that the cell membrane integrity is damaged, the water absorption rate is low, and the germination rate is only at the natural level.

[0181] To further verify the validity of the data, the viability of the imbibed seeds from Example 2 and the control group was tested. One hundred imbibed seeds (water absorption rate ≥ 50%) were randomly selected from the seeds soaked in Example 2, and one hundred imbibed seeds (water absorption rate ≥ 25%) were randomly selected from the seeds soaked in the control group. Embryo viability was determined using the TTC staining method. The seeds were cut along the embryo and immersed in a 0.5% TTC solution, stained at 30°C in the dark for 4 hours. The staining of the embryo was observed: a red embryo was considered viable, while lighter staining or no staining was considered low viability or death. The results are shown in Table 3.

[0182] Table 3

[0183]

[0184] As shown in Table 3, 96% of the imbibition seeds in Example 2 had embryo viability, while only 45% of the control group had embryo viability. This indicates that the compound regulator effectively protected the integrity of the embryo while promoting water absorption, enabling the imbibition seeds to have germination potential; while the control group seeds suffered severe cell membrane damage during water absorption and could not germinate even after absorbing water.

[0185] This confirms that Example 2 achieved a high germination rate of 92.3% even with a water absorption rate of 56.9%, as 96% of the imbibed seeds were viable, compared to only 45% in the control group. Therefore, the difference in germination rate stems not only from the difference in water absorption rate but also from the difference in seed viability. Water absorption rate is only one of the necessary conditions for seed germination; seed viability (embryo integrity and metabolic activity) is equally crucial in determining the germination rate. The compound regulator significantly improved the viability of imbibed seeds by protecting cell membranes, scavenging free radicals, and enhancing stress resistance, thereby achieving a high germination rate.

[0186] <Verification Test of Water Retention and Antibacterial Function of Composite Particles>

[0187] To verify the synergistic advantages of core-shell composite particles in maintaining substrate moisture stability, inhibiting pathogens, and reducing mold rate, Examples 1 and 3, and Comparative Examples 4 and 5 were selected for comparison. 1000 plump wild Lespedeza seeds were selected for each group. Seed pretreatment and seedling substrate preparation were completed according to the methods for each group. Water-retaining agents, carbendazim, or composite particles were added according to the group requirements and mixed thoroughly. After sowing, the moisture content was measured daily at fixed times using a soil moisture meter with an accuracy of 0.1% inserted 5 cm into the substrate (5 points randomly selected per group), for 15 consecutive days, and the average relative humidity of the substrate was calculated. On the day of sowing and 15 days after sowing, 5 g of seedling substrate was randomly collected from each group (avoiding seeds and seedling roots), and the pathogen colony count (CFU / g) was determined using the colony counting method. Seed mold growth was observed throughout the experiment, and the mold rate was statistically analyzed. The results are shown in Table 4.

[0188] Table 4

[0189]

[0190] As can be seen from Table 4, the mold rate of Example 1 (2.1%) was significantly lower than that of Comparative Example 5 (5.2%) and Comparative Example 4 (3.7%). This is because although no water-retaining or antibacterial materials were added in Example 1, a complete cleaning and disinfection, chemical dormancy breaking and constant temperature and light-avoidance germination process was adopted to build a micro-ecological foundation that is not conducive to mold growth, so that the seeds can germinate safely under normal humidity.

[0191] The decrease in colony count and mold rate in Comparative Example 5 was limited because the acrylamide-acrylic acid copolymer alone can only physically retain water and has no antibacterial function. The continuous high humidity environment (78.3%) it creates even provides favorable conditions for the reproduction of pathogens, partially offsetting the antibacterial effect of the basic process.

[0192] The water retention capacity of Comparative Example 4 was no different from that of Example 1. This is because the directly added carbendazim aqueous solution only provides chemical antibacterial effect and does not improve the physical water-holding structure of the matrix, thus failing to solve the problem of humidity fluctuation.

[0193] Example 3 achieved optimal performance across all metrics, demonstrating a synergistic effect of water retention and antibacterial activity. This is attributed to the crucial role played by its core-shell structure: the grafted polymer in the shell layer stabilizes water retention through hydrogen bonds and a three-dimensional network, maintaining humidity at the highest and most stable level (81.5%); while the porous starch in the core layer loads carbendazim and achieves slow-release, forming long-lasting antibacterial microzones around the seed, minimizing colony counts. This synergistic design of water retention and environmental stability in the shell layer, coupled with slow-release antibacterial activity in the core layer, is the fundamental reason why it maintains the lowest mold rate (1.2%) while achieving the highest humidity.

[0194] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0195] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A method for breaking dormancy and promoting germination of wild Lespedeza seeds, characterized in that, Includes the following steps: Physical peel breaking: Dry wild Lespedeza seeds are mixed with moist physical friction medium at a mass ratio of 1:3 to 1:5 and rolled and rubbed in a rotating container for 15 to 30 minutes to break the hard structure of the seed coat. After the treatment, the seeds are sieved and recovered. Seed pretreatment: The wild Lespedeza seeds after friction treatment are cleaned and disinfected. Warm water soaking: Soak the disinfected seeds in warm water at 35℃~45℃ for 6 h~12 h; Chemical dormancy breaking: After soaking, the seeds are immersed in a chemical dormancy-breaking solution at 18℃~28℃ for 4 h~10 h. The chemical dormancy-breaking solution consists of 0.05% (w / v) gibberellin and 0.5% (w / v) composite osmotic regulator. The composite osmotic regulator includes potassium nitrate, potassium citrate, proline, and disodium EDTA. Based on the total mass of the composite osmotic regulator, potassium nitrate accounts for 40%~60%, potassium citrate accounts for 20%~35%, proline accounts for 5%~15%, and disodium EDTA accounts for 1%~5%. The preparation of the composite osmotic regulator includes: dissolving each component in a phosphate buffer solution with a pH of 6.0~6.8 in proportion, mixing them evenly under light-protected conditions at a temperature below 10℃ to obtain a composite regulator stock solution; then mixing the composite regulator stock solution with the gibberellin stock solution in proportion to prepare the chemical dormancy-breaking solution. Gradient germination: Chemically treated seeds are mixed with a moist germination substrate with a humidity of 60%~80% at a volume ratio of 1:2~1:5 to form a mixed germination bed. The mixture is then cultured at a constant temperature of 18℃~25℃ for 48 h~96 h. During the germination period, maintain light-protected conditions, and keep the substrate moisture of the mixed germination bed at 60%~80%. The moisture-retaining germination substrate is composed of sterilized humus, river sand, and vermiculite in a volume ratio of 3:2:

1. Composite particles are added to the substrate at a concentration of 0.4%~0.7% (w / w) of the total substrate mass, with a particle size of 0.1~0.5 mm. The composite particles have a core-shell structure: the core layer is porous starch loaded with carbendazim, accounting for 15%~25% of the total mass of the composite particles; the middle layer is a polymer film formed by crosslinking sodium alginate and chitosan, accounting for 10%~20%; and the shell layer is an acrylamide-acrylic acid copolymer grafted and crosslinked onto a carboxymethyl cellulose backbone, accounting for 55%~75%. First batch of sowing: Select the seeds that have germinated in the gradient germination process along with some substrate, spread them evenly on the seedbed, with a thickness of 2 cm to 5 cm, cover with moisture-retaining material and manage in the dark after sowing to complete the first batch of sowing. Secondary germination and second batch sowing: The ungerminated seeds remaining from the first batch of sowing are mixed with the original substrate to form a secondary mixed germination bed. Germination is continued for 36 h to 60 h in a dark environment with a temperature of 20℃~28℃ and a substrate humidity of 65%~85%, before the second batch of sowing is carried out. Reseeding treatment: Soak the secondary germinated seeds and the remaining seeds that have not germinated after the second batch of sowing in a 0.01%~0.03% (w / v) gibberellin solution for 3 h~9 h, and then sow them in the reseeding area of ​​the seedbed. Seedling management: After all batches of sowing are completed, cover the sowing surface with a 1 cm to 3 cm thick layer of sterilized and moisturizing covering material, and implement drip irrigation and micro-spraying in coordination based on soil moisture sensor data to maintain substrate moisture at 40% to 80%, and manage the temperature and light environment until seedling emergence is completed.

2. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, The surface cleaning and disinfection treatment includes the following steps: screening to remove light impurities, shriveled seeds, and seeds with a particle size of less than 1.2 mm; rinsing with clean water 2-3 times; immersing the seeds in a 0.5%-1.0% (w / v) sodium hypochlorite solution at 18℃-25℃ in the dark for 8-12 minutes; rinsing with sterile water until the outflow is neutral; and placing the seeds in a ventilated environment at 25℃-30℃ to drain the surface moisture for 30-60 minutes.

3. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, The warm water soaking is carried out in a constant temperature water bath device, and the temperature fluctuation is controlled within ±1℃. During the soaking process, the seeds are stirred at a speed of 30 r / min to 50 r / min for 15 min to 20 min every 2 hours, and isothermal sterile water is added every 3 hours, with the amount of water added being 5% to 8% of the initial soaking water volume. After the soaking is completed, chaff and inferior seeds floating on the liquid surface are removed.

4. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, The physical friction medium is coarse river sand with a particle size of 2 mm to 5 mm, which is sterilized by dry heat at 120℃ to 130℃ for 20 min to 30 min before use; the rolling friction treatment is carried out in a horizontal drum with a rotation speed of 15 r / min to 25 r / min, and the seed breakage rate is monitored in real time to ensure that it does not exceed 5%; after the treatment, a double-layer vibrating screen with an upper sieve aperture of 4 mm to 6 mm and a lower sieve aperture of 1.5 mm is used to separate the seeds from the friction medium.

5. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, During the constant temperature germination stage, the relative humidity of the environment should be maintained at 75%~80%, and ventilation should be carried out 3~4 times a day for 30~50 minutes each time. The entire germination process should be kept away from light.

6. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, After the secondary mixed germination bed is formed and before germination continues, add 10%–15% of the total mass of the secondary mixed germination bed with fresh moisturizing germination substrate and adjust the substrate humidity to 65%–85%. During the germination process, turn the seeds over every 12 hours to a depth of 5–8 cm. For the ungerminated seeds remaining after the second batch of sowing, before soaking in gibberellin solution, perform a second physical friction treatment for 8–12 minutes. The second physical friction treatment uses the same friction medium and operating conditions as the physical skin-breaking step. After soaking, mix the seeds with fresh moisturizing germination substrate at a volume ratio of 1:3 and sow them in the replanting area of ​​the seedbed.

7. The method for breaking dormancy and promoting germination of wild Lespedeza seeds according to claim 1, characterized in that, In the seedling management process, the substrate moisture is dynamically maintained at 40% to 80% based on real-time monitoring data; the moisture-retaining covering material is crushed straw or rice husks that have been sterilized by dry heat at 120℃ to 130℃, and the covering thickness is 1 cm to 3 cm; the seedling environment temperature is controlled at 18℃ to 28℃, the day-night temperature difference is maintained at 5℃ to 8℃, and ventilation is carried out 2 to 3 times a day for 20 to 40 minutes each time.

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