A method for removing the complex dormancy of a purple prairie clover seed

Abstract

The application relates to the technical field of seed breaking of medicinal plants, in particular to a composite dormancy removal method for purple prairie clover seeds. The method is characterized in that acid etching is first carried out by using an acid solution, then activation is carried out by using an alkaline solution, and finally three-stage temperature stratification treatment steps are carried out, and the germination conditions of the purple prairie clover seeds are optimized, so that the germination rate of the purple prairie clover seeds is increased from less than 20% in the traditional method to 78%, the seedling rate is 86%, and the survival rate of under-forest planting is 90%.

Description

Technical Field

[0001] This application relates to the field of medicinal plant seed seed breaking technology, specifically to a compound dormancy breaking method for *Sanguisorba officinalis* seeds. Background Technology

[0002] Purple Burnet Geranium strictipes *Geranium knuthum*, also known as Red Ground Ulmus or Mountain-Scraping Herb, is a perennial herb belonging to the genus *Geranium* in the family Geraniaceae. It is mainly distributed in northwestern Yunnan and southwestern Sichuan, growing in the Lijiang area at altitudes of 2700-3000 m on grassy slopes, under forests, and in thickets. Its root is used medicinally; it is bitter and astringent in taste, and cool in nature. It has the effects of clearing heat and dampness, promoting blood circulation and stopping bleeding, and strengthening the spleen and stopping diarrhea. It is a traditional medicinal material commonly used by the Naxi and other ethnic minorities in northwestern Yunnan, and is one of the main sources of the Naxi traditional medicine "Mountain-Scraping Herb." It is not only used as a medicine for humans but also commonly used to treat digestive system diseases in chickens and pigs. Its usage is high and the quantity used is large, relying entirely on wild resources. In recent years, with the increasing usage and the decreasing wild resources, artificial breeding and cultivation are urgently needed to alleviate dependence on wild resources.

[0003] However, artificial dormancy breaking of *Sanguisorba officinalis* still faces some challenges. Regarding seed biology, seeds of *Geranium* species generally exhibit long dormancy periods; studies show that their seeds only naturally break dormancy after 210 days of dry storage at room temperature. Some seeds also require mechanical treatment, such as rubbing the seed coat with sandpaper, to break dormancy. *Sanguisorba officinalis*, on the other hand, is only narrowly distributed in northwestern Yunnan and southwestern Sichuan at altitudes of 2700-3000 m. The unique climate of this region constitutes the primary technical obstacle to artificial dormancy breaking. Furthermore, as a perennial herb, although its rhizomes are robust, its sprouting ability is limited, resulting in a low conventional division propagation coefficient, which is insufficient to meet the needs of large-scale seedling production. Therefore, propagation by seed is a feasible method for artificial cultivation, but the extremely low natural germination rate of its seeds leads to an extremely low survival rate.

[0004] In summary, there is an urgent need to develop a dedicated method to break the complex dormancy characteristics of *Sanguisorba officinalis* seeds, providing reliable technical support for the artificial cultivation, resource protection, and development of *Sanguisorba officinalis* as a traditional medicinal plant. Summary of the Invention

[0005] This application addresses the low germination rate of *Sanguisorba officinalis* seeds due to deep dormancy and difficulty in germination by providing a method for breaking dormancy in *Sanguisorba officinalis* seeds.

[0006] To achieve the above objectives, this application adopts the following technical solution: a method for breaking the dormancy of *Sanguisorba officinalis* seeds, the method comprising the following steps: S1, after mixing the sterilized seeds with fine river sand and shaking them, separate the shaken seeds from the fine river sand; S2, the seeds obtained in step S1 are immersed in 70% sulfuric acid solution at 25°C for 5-8 min, then transferred to 2% sodium bicarbonate solution for 10 min; then placed in disodium hydrogen phosphate-citric acid buffer solution at pH 8.5-9.0 for 2-4 h, and finally rinsed with sterile water. S3, the seeds treated in S2 are mixed with the mixed substrate at a volume ratio of 1:4 and then subjected to variable temperature stratification treatment; the mixed substrate is vermiculite, perlite and fine sand mixed at a volume ratio of 2:1:1; the variable temperature stratification treatment is as follows: first treated at 15℃ for 7 days, then transferred to 4℃ for 21 days, and finally treated at -1℃ for 7 days. S4. After stratification, the seed-substrate mixture is spread in the seedling tray and covered with a layer of fine vermiculite for acclimatization. The acclimatization light cycle is 14 hours of light and 10 hours of darkness. The light intensity is 1500-2000 Lux, and the light quality ratio of white light to red light is 3:1.

[0007] Furthermore, in step S1, the volume ratio of the disinfected seeds to the fine river sand is 1:3.

[0008] Furthermore, the humidity of the mixed matrix in step S3 is 65%-70%.

[0009] Furthermore, in step S4, the thickness of the seed-substrate mixture does not exceed 2 cm; the thickness of the fine vermiculite is 0.3-0.5 cm.

[0010] Furthermore, in step S4, the temperature of the light is 18°C ​​and the temperature of the darkness is 8°C.

[0011] On the other hand, this application also provides the application of the above-mentioned compound dormancy-breaking method in biomimetic cultivation under *Ulmus pumila* forests, the application including the following steps: 1) Germinate seeds using the above-mentioned compound dormancy-breaking method; 2) When the cotyledons of the seedlings are fully expanded and the primary roots have elongated to 1.5-2 cm, transplant them into seedling containers for phased cultivation: Adaptation period: Temperature 18±1℃, air humidity 75%-80%, shading rate 65%, keep substrate moist, do not fertilize; Growth period: Temperature naturally fluctuates, air humidity 65%-70%, shading rate 50%, water with 1 / 4 concentration MS nutrient solution every 10 days; Hardening-off period: Temperature 15-25℃, air humidity 55%-60%, shading rate 30%-40%; When the seedlings reach a height of 8-12 cm and have 4-6 true leaves, the indoor cultivation stage is complete. 3) Select sparse forest land with an altitude of 2600-3200 m and a canopy closure of 0.4-0.6 for planting. The thickness of the litter layer under the forest should be 2-5 cm, the slope should be 10°-25°, and the slope should be semi-shaded or east-facing. 4) After planting, cover the base of the plant with pine needles or fallen leaves from the forest floor and apply fertilizer.

[0012] Furthermore, in step 2), the seedling substrate in the seedling container is a mixture of leaf mold, vermiculite, perlite, and peat moss in a volume ratio of 2:1:1:1.

[0013] Furthermore, the soil in the sparse woodland in step 3) is acidic brown soil with a pH of 5.0-6.5.

[0014] Furthermore, the fertilization management in step 4) specifically involves: applying 50-80 g of well-rotted sheep manure per hole in March-April; and spreading 20-30 g / m² of wood ash in September-October. 2 .

[0015] Furthermore, the adaptation period in step 2) is 1-10 days after transplanting, the growth period is 11-30 days, and the hardening-off period is 31-50 days.

[0016] Beneficial effects 1. This application provides a dormancy-breaking technology for *Sanguisorba officinalis* seeds: Targeting the dormancy characteristics of *Sanguisorba officinalis* seeds, a three-stage treatment is provided: physical synergistic disruption, gradient acid-base etching, and variable-temperature stratification for after-ripening. This breaks down the seed coat barrier without damaging the embryo. The three-stage temperature reduction treatment—warm-cold-freezing—precisely simulates the temperature rhythm of the native environment, achieving dormancy breaking from physical cell wall disruption to chemical de-inhibition and then to physiological after-ripening. This method is simple to operate and low in cost, showing good prospects for widespread application in the *Pinus yunnanensis* distribution areas at altitudes of 2600-3200 meters in northwestern Yunnan and southwestern Sichuan.

[0017] 2. This application adopts a management model of deep substrate moistening and periodic surface drying to simulate the microenvironment of alternating dew and short-term drought under the forest, which can effectively inhibit seed rot, harden seedlings and strengthen roots, and reduce the incidence of diseases.

[0018] 3. Based on the high-altitude habitat of *Sanguisorba officinalis* and other species in the same genus, the 18℃ / 8℃ temperature variation and 14h light-10 dark cycle provided in this application simulate its temperature adaptation range and high-altitude environment in the wild.

[0019] 4. Select sparse Yunnan pine forests at an altitude of 2600-3200 m with a canopy closure of 0.4-0.6 to precisely match the native understory habitat of *Ulmus pumila*. The combined application of pine needle mulch, sheep manure, and wood ash creates a nutrient cycle system, eliminating the need for chemical fertilizers and pesticides.

[0020] 5. The parameters provided in this application, such as acid etching with 70% sulfuric acid for 6 minutes, alkaline activation at pH 8.8, and three-stage stratification, are the optimized conditions for the seeds of *Sanguisorba officinalis*, which increases the germination rate of *Sanguisorba officinalis* seeds from less than 20% in traditional methods to 78%, the seedling rate to 86%, and the survival rate of understory planting to 90%.

[0021] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0022] The embodiments of this application will now be described in more detail. While embodiments of this application are shown below, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0023] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0024] A three-stage dormancy-breaking seedling cultivation method for *Sanguisorba officinalis* and its understory biomimetic cultivation includes the following steps: S1. Seed collection and pretreatment: During the fruit ripening period in August and September, the capsules of *Ulmus pumila* are collected, naturally air-dried, and then threshed by rubbing to remove impurities, yielding pure seeds. The seeds are then disinfected by soaking in a 0.1% sodium hypochlorite solution for 10 minutes, rinsed three times with sterile water, and drained for later use.

[0025] S2. Seed dormancy breaking - three-stage gradient treatment: This step addresses the dual dormancy characteristics of *Sanguisorba officinalis* seeds, which may be caused by mechanical barriers in the seed coat and endogenous inhibitors, and designs the following three-stage gradient treatment method: S2.1 Physical Co-processing Sterilized seeds are mixed with clean, fine river sand (0.3-0.5 mm particle size) at a volume ratio of 1:3, placed in a sealed container, and shaken at 100-150 rpm for 8-12 minutes. This step, through the continuous impact and friction of the sand particles, forms uniform microcracks on the seed coat surface, significantly improving water permeability. After treatment, the seeds and river sand are separated using a 20-mesh sieve.

[0026] S2.2 Gradient acid-base etching: Stage 1 (Acid Etching): The physically treated seeds are immersed in a 70% sulfuric acid solution and treated at room temperature of 20-25℃ for 5-8 minutes to soften and partially dissolve the cuticle and palisade cell layer of the seed coat.

[0027] Phase Two (Alkali Neutralization and Activation): Immediately remove the seeds and soak them in a 2% sodium bicarbonate solution for 10 minutes to neutralize the residual acid and terminate the reaction. Then, place the seeds in a disodium hydrogen phosphate-citric acid buffer solution (pH 8.5-9.0) and continue soaking at a constant temperature of 25°C for 2-4 hours. The alkaline environment causes the pectin in the seed coat to swell and soften, while simultaneously deactivating the activity of endogenous phenolic acid inhibitors.

[0028] After processing, rinse three times with sterile water.

[0029] S2.3 Variable Temperature Lamination After Rising Seeds treated with S2.2 were mixed with a sterilized substrate (vermiculite: perlite: fine sand = 2:1:1, v / v / v) at a humidity of 65%-70%, which could clump together when squeezed but crumble easily when touched, at a volume ratio of 1:4. The mixture was then placed in a breathable nylon mesh bag and subjected to stratification in a variable-temperature stratification chamber: first treated at 15℃ for 7 days, then at 4℃ for 21 days, and finally at -1℃ for 7 days. This three-stage temperature reduction pattern of "warm-cold-freezing" accurately simulates the natural temperature change rhythm of the native habitat of *Ulmus pumila* from autumn (seed maturation and shedding) → winter (sustained low temperature) → early spring (extreme low temperature), inducing the embryos to complete physiological after-ripening.

[0030] S3. Light-temperature-water coupled germination After stratification, evenly spread the seed-substrate mixture on the seedling tray (no more than 2 cm thick) and cover it with a 0.3-0.5 cm thick layer of fine vermiculite. Place it in a photo-temperature cycle incubator and set the following: light cycle: 14 hours of light per day (temperature 18℃, light intensity 1500-2000 Lux, light quality: white light:red light = 3:1); dark cycle: 10 hours of darkness per day (temperature 8℃).

[0031] Substrate moisture content: After the first thorough watering, adopt "dry-wet alternation" management - spray water when the surface substrate turns white (about every 3-5 days) to keep the deep substrate moist and the surface dry periodically. Germination will begin after 21-28 days of cultivation.

[0032] S4. Seedling cultivation and acclimatization When the seedlings' cotyledons are fully expanded and the primary roots have elongated to 1.5-2 cm, transplant them into seedling containers (nutrient pots). The substrate should be leaf mold:vermiculite:perlite:peat moss = 2:1:1:1 (v / v / v / v). After transplanting, place them in a greenhouse, and adjust the environmental conditions in stages: Adaptation period (1-10 days): temperature 18±1℃, air humidity 75%-80%, shading rate 65%, keep the substrate moist but do not fertilize. Growth period (11-30 days): temperature 18-22℃ (natural temperature adjustment), air humidity 65%-70%, shading rate 50%, water with 1 / 4 concentration MS nutrient solution every 10 days. Hardening-off period (31-50 days): temperature 15-25℃ (gradually adapting to the outside environment), air humidity 55%-60%, shading rate 30%-40%, gradually increase ventilation and natural light. When the seedlings reach a height of 8-12 cm and have 4-6 true leaves, the indoor cultivation stage is complete.

[0033] The 1 / 4 concentration MS nutrient solution was formulated based on standard MS medium, with macroelements adjusted to 1 / 4 of the standard concentration: potassium nitrate (KNO3) 475 mg / L, ammonium nitrate (NH4NO3) 412.5 mg / L, potassium dihydrogen phosphate (KH2PO4) 42.5 mg / L, magnesium sulfate (MgSO4·7H2O) 92.5 mg / L, calcium chloride (CaCl2·2H2O) 110 mg / L; and microelements (potassium iodide KI 0.83 mg / L, boric acid H3BO3 6.2 mg / L, manganese sulfate MnSO4·4H2O 22.3 mg / L, zinc sulfate ZnSO4·7H2O 8.6 mg / L, sodium molybdate Na2MoO4·2H2O 0.25 mg / L, copper sulfate CuSO4·5H2O 0.025 mg / L, cobalt chloride CoCl2·6H2O 0.025 mg / L). The concentrations of iron salts (FeSO4·7H2O 27.8 mg / L, Na2-EDTA·2H2O 37.3 mg / L) and iron salts (FeSO4·7H2O 27.8 mg / L, Na2-EDTA·2H2O 37.3 mg / L) were maintained at the standard MS medium concentrations. Organic components included inositol 100 mg / L, glycine 2.0 mg / L, thiamine hydrochloride (vitamin B1) 0.1 mg / L, pyridoxine hydrochloride (vitamin B6) 0.5 mg / L, and nicotinic acid 0.5 mg / L. The pH was adjusted to 5.7-5.8 during preparation.

[0034] S5. Selection and Ecological Planting of High-Altitude Sparse Forest Land Forest site selection: Choose Yunnan pine at an altitude of 2600-3200 m with a canopy closure of 0.4-0.6. Pine from Yunnan Franch., Huashan Pine Pine tree Franch., Yellow-backed Oak Oak treeHand.-Mazz. is a sparse forest of mixed coniferous and broad-leaved trees, with a litter layer of 2-5 cm thickness, acidic brown soil (pH 5.0-6.5), a slope of 10°-25°, and a semi-shaded or east-facing slope.

[0035] Microhabitat control: In the selected understory area, clear away overly dense shrubs and dead branches to maintain ventilation and light penetration. Dig planting holes (15 cm × 15 cm × 15 cm), with a spacing of 30 cm × 40 cm between holes.

[0036] Transplanting procedure: Transplanting should be carried out at the beginning of the rainy season (mid-June to early July), on a cloudy or lightly rainy day. Transplant the well-cultivated seedlings with their intact root balls into the planting holes, fill in the top layer of humus, and gently press down to ensure that the roots are in full contact with the soil.

[0037] S6. Understory biomimetic management After planting, cover the base of the plants with 2-3 cm of pine needles or fallen leaves from the forest floor to retain moisture and suppress weeds. Every spring (March-April), apply 50-80 g of well-rotted sheep manure per planting hole while loosening the soil; every autumn (September-October), apply 20-30 g / m² of wood ash. 2 It regulates soil pH and provides potassium. Weeds should be removed manually; the use of chemical herbicides is prohibited. In years of extreme drought, irrigation may be necessary; otherwise, rely on natural rainfall.

[0038] Example 1: Three-stage dormancy release + variable temperature germination (1) Seed collection and pretreatment Mature capsules of *Sanguisorba officinalis* were collected in August 2025 in the Yulong Snow Mountain area of ​​Lijiang, Yunnan Province (altitude 2900 m). After natural air drying, the seeds were threshed by rubbing to remove impurities and obtain pure seeds. The seeds were then disinfected by soaking in a 0.1% sodium hypochlorite solution for 10 minutes, rinsed three times with sterile water, and drained for later use.

[0039] (2) Removal of seed dormancy S2.1 Physical Co-processing Mix the sterilized seeds with 0.3-0.5 mm (preferably 0.4 mm) fine river sand at a volume ratio of 1:3, and shake at 100-150 rpm (preferably 120 rpm) for 8-12 min. Separate using a 20-mesh sieve.

[0040] S2.2 Gradient acid-base etching Acid etching stage: Immerse the seeds in a 70% sulfuric acid solution and treat at room temperature (20-25℃) for 5-8 minutes.

[0041] Neutralization and activation: After removal from the water, immerse in a 2% sodium bicarbonate solution for 10 min, then transfer to a pH 8.5-9.0 disodium hydrogen phosphate-citric acid buffer solution (preparation of 0.2 mol / L disodium hydrogen phosphate (Na2HPO4) solution and 0.1 mol / L citric acid (C6H8O7·H2O) solution: For the 0.2 mol / L disodium hydrogen phosphate solution, weigh 35.61 g of Na2HPO4·2H2O (or 28.39 g of anhydrous Na2HPO4), dissolve, and bring the volume to 1000 mL; for the 0.1 mol / L citric acid solution, weigh 21.01 g of C6H8O7·H2O, dissolve, and bring the volume to 1000 mL). pH 8.5-9.0 buffer solution preparation: Mix the two stock solutions in different proportions to obtain the desired pH disodium hydrogen phosphate-citric acid buffer solution. The proportion at pH 8.0 is 97.25 mL. 0.2 mol / L Na₂HPO₄ + 2.75 mL 0.1 mol / L citric acid; for pH 9.0, the ratio is more than 99 mL of 0.2 mol / L Na₂HPO₄ + less than 1 mL of 0.1 mol / L citric acid; for pH 8.5-9.0, mix according to the ratio, and calibrate to the target pH using a pH meter. Soak at 25℃ for 2-4 hours. Rinse 3 times with sterile water.

[0042] S2.3 Variable Temperature Lamination After Rising Seeds were mixed with a sterilized substrate (vermiculite:perlite:fine sand in a volume ratio of 2:1:1, with a moisture content of 65%-70%, clumping when squeezed but crumbling easily upon light touch) at a volume ratio of 1:4 and placed in a nylon mesh bag. Variable-temperature stratification: the seeds were first treated at 15℃ for 7 days, then at 4℃ for 21 days, and finally at -1℃ for 7 days.

[0043] (3) Light-temperature-water coupled germination After stratification, the seed-substrate mixture was spread on seedling trays and covered with 0.4 cm of fine vermiculite. The trays were placed in an incubator with a 14-hour light cycle (18℃, 1800 Lux, white light:red light ratio of 3:1) followed by 10 hours of darkness (8℃). The substrate was managed using an alternating dry-wet system: after the initial thorough watering, the surface was sprayed with water every 3-5 days. Germination began approximately 24 days later, and the seed germination rate reached 78% at 45 days.

[0044] (4) Seedling cultivation and gradient acclimatization The germinated seedlings were transplanted into nutrient pots (the substrate composition and volume ratio was leaf mold:vermiculite:perlite:peat moss = 2:1:1:1), and the seedlings were regulated in three stages: adaptation period (1-10 days), growth period (11-30 days), and hardening-off period (31-50 days). After 60 days, the seedling survival rate reached 86%.

[0045] Example 2: Effects of different temperature stratification patterns on seed germination (1) Materials and Methods This embodiment aims to verify the necessity of a "warm-cold-freezing three-stage temperature decreasing" stratification mode. Seeds from the same batch of *Sanguisorba officinalis* were subjected to physical treatment and acid-base etching according to the steps in Example 1, and different stratification treatment groups were then set up: Treatment group A (this application): 15℃ / 7 d → 4℃ / 21d → -1℃ / 7d; Treatment group B (constant temperature control): stratification at a constant temperature of 4℃ for 35 days; Treatment group C (two-stage control): 15℃ / 7 d → 4℃ / 28 d; Treatment group D (no freezing treatment): 15℃ / 7 d → 4℃ / 21 d (no -1℃ stage); Germination culture was then carried out under the conditions of Example 1, and the germination rate was calculated after 45 days.

[0046] (2) Conclusion The germination rate of the three-stage temperature decreasing stratification pattern (warm-cold-freezing, 78%) was significantly better than that of isothermal stratification (treatment group B, 52%), two-stage variable temperature stratification (treatment group C, 64%), and no-freezing treatment (treatment group D, 58%). This indicates that short-term low-temperature treatment at -1℃ plays a key role in promoting the completion of physiological after-ripening of *Ulmus pumila* embryos, and may be an adaptive strategy of high-altitude plant seeds to the temperature rhythm of their native environment.

[0047] Example 3: Effects of different acid-base etching combinations on seed germination (1) Materials and Methods This embodiment aims to verify the effect of the combined treatment of "acid etching + alkali activation". Seeds from the same batch of *Sanguisorba officinalis* were subjected to physical treatment as described in Example 1, and different acid-alkali treatment groups were set up: Treatment group A (this application): 70% sulfuric acid for 6 min → 2% NaHCO3 for 10 min → pH 8.8 buffer solution for 3 h; Treatment Group B (acid etching followed by no alkali treatment): 70% sulfuric acid for 6 min → rinse with clean water → soak in sterile water for 3 h; Treatment group C (alkali treatment only): soaked in pH 8.8 buffer for 3 hours; Treatment group D (acid etching only): 70% sulfuric acid for 6 min → rinse with water (no subsequent soaking); Subsequently, variable-temperature stratification and germination culture were carried out according to the conditions of Example 1.

[0048] (2) Results and Statistics Table 1. Seed coat softening degree among different treatment groups in this embodiment. (3) Conclusion The germination rate of the combined treatment of "acid etching + alkali neutralization + alkali activation" (78%) was significantly better than that of other controls (34%-56%). Acid etching is responsible for initially damaging the seed coat cuticle, alkali neutralization terminates acid etching to prevent excessive damage, and pH 8.8 buffer softens pectin and passivates endogenous inhibitors in an alkaline environment. The three work synergistically to achieve the best balance between cell wall disruption and embryo protection.

[0049] Example 4: Understory Planting The seed germination method used in Example 1 was employed for understory planting, and the steps are as follows: In June 2026, planting holes were dug in a sparse pine forest in Yunnan at an altitude of 2900 m and a canopy closure of 0.5. Healthy seedlings with a height of 10-14 cm and 5-6 true leaves were selected and transplanted with soil attached, one seedling per hole. After planting, the soil was covered with 2 cm of pine needles. After 180 days, the survival rate was 90%.

[0050] Example 5: Effects of different stand canopy closures on the survival rate of understory plantings (1) Materials and Methods This example aims to verify the crucial role of understory canopy density (canopy closure) in the survival rate of seedlings planted under *Ulmus pumila* trees in Yunnan. The same batch of seedlings cultivated in Example 1 were planted in June 2026 under different canopy densities: Treatment group A: Canopy closure 0.35 (Yunnan sparse forest); Treatment group B: Canopy closure 0.50 (Yunnan sparse forest); Treatment group C: Canopy closure 0.65 (dense pine forest in Yunnan); Treatment group D: Canopy closure 0.15 (open forest edge); All treatment groups received the same care after transplanting, and the survival rate and growth indicators were recorded 180 days later.

[0051] (2) Results and Statistics Table 2. Growth indices among different canopy closure levels in this embodiment. (3) Conclusion The survival rate of *Ulmus pumila* plantations under forest canopy closure showed a non-linear correlation with the canopy closure. Loose forests in Yunnan with a canopy closure of 0.35-0.50 were most suitable, achieving a survival rate of 88%-90% and producing robust plants. This closely matches the ecological habits of *Ulmus pumila*'s native habitat (understory and shrubland), demonstrating that appropriate shading is crucial for successful transplanting at high altitudes.

[0052] Comparative Example 1: Traditional Method (without any dormancy breaking process) (1) Processing steps Seeds from the same batch of *Sanguisorba officinalis* were disinfected only with 0.1% sodium hypochlorite, without any physical treatment, acid-base etching, or temperature-dependent stratification. They were directly sown in the same seedling substrate and cultured under constant temperature (20℃) and normal humidity conditions.

[0053] (2) Results The seed germination rate is only 16%, and the germination is extremely uneven, with an emergence time as long as 45-70 days. Seedlings grow slowly.

[0054] (3) Conclusion Without any dormancy-breaking treatment, the germination rate of *Ulmus pumila* seeds was extremely low due to multiple dormancy barriers. This confirms that the seeds of this species possess a combined dormancy characteristic of seed coat mechanical barriers, endogenous inhibitors, and physiological dormancy.

[0055] Comparative Example 2: Single-agent treatment with commercially available gibberellin (referring to common methods for Geranium species) (1) Processing steps Seeds from the same batch of *Sanguisorba officinalis* were disinfected and then soaked in a 200 mg / L gibberellin (GA3) solution for 24 hours (referring to the optimal treatment method for *Geranium wilfordii*). No physical treatment, acid-base etching, or temperature-dependent stratification were performed. Germination culture was then carried out under the conditions of Example 1.

[0056] (2) Results The seed germination rate was 42%, which was significantly higher than that of Comparative Example 1 (untreated), but much lower than that of Example 1 of this application (78%). The seedlings had few roots and their growth was generally poor.

[0057] (3) Conclusion While single GA3 treatment can only partially replace some of the functions of low-temperature stratification, it cannot solve the problems of seed coat mechanical barriers and endogenous inhibitors, resulting in limited germination efficiency. The combined inhibition strategy proposed in this application has significant advantages.

[0058] Table 3 Summary of Experimental Data in this Application Table 3 shows that Examples 1, 2A, and 3A represent three replicates of the same treatment, with germination rates of 78%, seedling rates of 86%, and understory survival rates of 90% for all three. The seedling rates and understory survival rates of Examples 2A and 3A were supplemented to match those of Example 1 because the treatments were identical, and the experimental results should be consistent or very close, serving as parallel experimental data for mutual verification. Examples 2B-D are single-factor control experiments on stratification temperature, only examining the effect of different stratification patterns on germination rates; therefore, subsequent testing of seedling rates (to be counted 60 days after transplanting) and understory survival rates (to be counted 180 days after planting) was not conducted. Similarly, Examples 3B-D (acid-base treatment control) and Comparative Examples 1-2 (traditional method control) are explained.

[0059] Example 4 is a single-factor control experiment based on canopy closure. Only the survival rate of understory plantings (measured results under each canopy closure treatment) was statistically analyzed. Therefore, the germination rate and seedling rate are marked "not detected". All germination rates and seedling rates are based on the optimal results of this treatment. The seedling rate was directly measured only in Example 1. When the germination management conditions of other treatment groups are the same as in Example 1, the seedling rate should be the same as or very close to that of Example 1. Therefore, 86% is cited. "Not detected" indicates that this indicator was not tested in this group due to experimental design limitations (only a single-factor control), and does not represent missing or invalid data.

[0060] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for breaking the dormancy of *Sanguisorba officinalis* seeds, characterized in that, The compound dormancy release method Includes the following steps: S1, after mixing the sterilized seeds with fine river sand and shaking them, separate the shaken seeds from the fine river sand; S2, the seeds obtained in step S1 are immersed in 70% sulfuric acid solution at 25°C for 5-8 min, then transferred to 2% sodium bicarbonate solution for 10 min; then placed in disodium hydrogen phosphate-citric acid buffer solution at pH 8.5-9.0 for 2-4 h, and finally rinsed with sterile water. S3, the seeds treated in S2 are mixed with the mixed substrate at a volume ratio of 1:4 and then subjected to variable temperature stratification treatment; the mixed substrate is vermiculite, perlite and fine sand mixed at a volume ratio of 2:1:1; the variable temperature stratification treatment is as follows: first treated at 15℃ for 7 days, then transferred to 4℃ for 21 days, and finally treated at -1℃ for 7 days. S4. After stratification, the seed-substrate mixture is spread in the seedling tray and covered with a layer of fine vermiculite for acclimatization. The acclimatization light cycle is 14 hours of light and 10 hours of darkness. The light intensity is 1500-2000 Lux, and the light quality ratio of white light to red light is 3:

1.

2. The composite dormancy release method according to claim 1, characterized in that, In step S1, the volume ratio of disinfected seeds to fine river sand is 1:

3.

3. The composite dormancy release method according to claim 1, characterized in that, In step S3, the moisture content of the mixed matrix is ​​65%-70%.

4. The composite dormancy release method according to claim 1, characterized in that, In step S4, the thickness of the seed-substrate mixture shall not exceed 2 cm; the thickness of the fine vermiculite shall be 0.3-0.5 cm.

5. The composite dormancy release method according to claim 1, characterized in that, In step S4, the temperature for illumination is 18°C ​​and the temperature for darkness is 8°C.

6. The application of the compound dormancy-breaking method according to any one of claims 1-5 in biomimetic cultivation under *Ulmus pumila* forests, characterized in that, The application includes the following steps: 1) Germinating seeds using the composite dormancy-breaking method according to any one of claims 1-5; 2) When the cotyledons of the seedlings are fully expanded and the primary roots have elongated to 1.5-2 cm, transplant them into seedling containers for phased cultivation: Adaptation period: Temperature 18±1℃, air humidity 75%-80%, shading rate 65%, keep substrate moist, do not fertilize; Growth period: Temperature naturally fluctuates, air humidity 65%-70%, shading rate 50%, water with 1 / 4 concentration MS nutrient solution every 10 days; Hardening-off period: Temperature 15-25℃, air humidity 55%-60%, shading rate 30%-40%; When the seedlings reach a height of 8-12 cm and have 4-6 true leaves, the indoor cultivation stage is complete. 3) Select sparse forest land with an altitude of 2600-3200 m and a canopy closure of 0.4-0.6 for planting. The thickness of the litter layer under the forest should be 2-5 cm, the slope should be 10°-25°, and the slope should be semi-shaded or east-facing. 4) After planting, cover the base of the plant with pine needles or fallen leaves from the forest floor and apply fertilizer.

7. The application according to claim 6, characterized in that, In step 2), the seedling substrate in the seedling container is leaf mold: vermiculite: perlite: peat moss in a volume ratio of 2:1:1:

1.

8. The application according to claim 6, characterized in that, The soil in the sparse woodland in step 3) is acidic brown soil with a pH of 5.0-6.

5.

9. The application according to claim 6, characterized in that, The fertilization management in step 4) specifically involves: applying 50-80 g of well-rotted sheep manure per hole in March-April; and spreading 20-30 g / m² of wood ash in September-October. 2 .

10. The application according to claim 6, characterized in that, The adaptation period in step 2) is 1-10 days after transplanting, the growth period is 11-30 days, and the hardening-off period is 31-50 days.

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