A seed pretreatment method and seedling raising method for improving the breeding efficiency of caragana jubata

Abstract

The application belongs to the technical field of plant breeding, and particularly relates to a seed pretreatment method and seedling raising method for improving the breeding efficiency of Carpinus putolekii, which comprises the following steps: disinfecting Carpinus putolekii seeds, and performing temperature-variable stratification treatment on the disinfected seeds to obtain pretreated Carpinus putolekii seeds; the temperature-variable stratification treatment comprises: 23-27 DEG C warm treatment for 30 days, and 3-5 DEG C cold treatment for 60 days. The pretreatment method can significantly shorten the germination time of Carpinus putolekii, improve the germination efficiency, and provide technical support for the protection and large-scale seedling raising of the species. The results of the examples show that, by using the method provided in the application, the germination time of Carpinus putolekii seeds is reduced to 14 days, the germination rate is 93.1%, and the seedling rate is 89.7%, so the germination is rapid, and the uniformity is high.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation technology, specifically relating to a seed pretreatment method and seedling raising method for improving the propagation efficiency of *Carpinus baohuaensis*. Background Technology

[0002] *Carpinus baohuaensis*, belonging to the genus *Carpinus* in the family Betulaceae, is mainly distributed in Yixing and Jurong cities, Jiangsu Province, and possesses extremely high ecological, landscape, and scientific research value. However, due to its inherent biological characteristics, such as habitat fragmentation, low natural fruit production, deep seed dormancy, and a hard, dense seed coat, its natural population regeneration capacity is extremely weak, and it has been listed as Critically Endangered (CR) in the *China Red List of Biodiversity—Higher Plants*. Currently, the number of wild saplings and seedlings is scarce, and the natural population's self-replenishment mechanism is severely lacking. Under current techniques, *Carpinus baohuaensis* typically requires 20-30 days after sowing to begin germination. Therefore, how to overcome the existing reproductive bottlenecks and further improve the reproductive efficiency of *Carpinus baohuaensis* has become an urgent technical problem to be solved. Summary of the Invention

[0003] The purpose of this invention is to provide a seed pretreatment method and a seedling raising method to improve the breeding efficiency of *Carpinus baohuaensis*. The pretreatment method can significantly shorten the germination time of *Carpinus baohuaensis* and improve the germination efficiency, providing technical support for the protection and large-scale seedling raising of this species.

[0004] This invention provides a seed pretreatment method to improve the breeding efficiency of Carpinus baohuaensis, comprising the following steps: disinfecting Carpinus baohuaensis seeds, subjecting the disinfected seeds to variable temperature stratification treatment, and obtaining pretreated Carpinus baohuaensis seeds. The variable temperature lamination process includes: warm treatment at 23~27℃ for 30 days, followed by cold treatment at 3~5℃ for 60 days.

[0005] As a preferred embodiment, the matrix of the variable temperature stratification treatment comprises: peat moss, sand and perlite; the volume ratio of the peat moss, sand and perlite is 1~3:1~2:1~2.

[0006] As a preferred embodiment, the moisture content of the matrix is ​​≥60%.

[0007] As a preferred embodiment, the disinfection process includes: disinfecting with ethanol (65%~75% by volume) for 30~60 seconds, followed by disinfecting with NaClO solution (1%~2% by mass) for 5~10 minutes.

[0008] As a preferred embodiment, the disinfection process further includes rinsing with sterile water, with the number of rinsing cycles being ≥3.

[0009] As a preferred embodiment, the variable temperature stratification treatment further includes spraying a bactericide every 8 to 12 days.

[0010] As a preferred embodiment, the fungicide includes at least one of the following: carbendazim, pyraclostrobin, and difenoconazole.

[0011] This invention provides a seedling raising method to improve the propagation efficiency of Carpinus baohuaensis, comprising the following steps: treating Carpinus baohuaensis seeds using the above-mentioned seed pretreatment method, and then sowing, germinating, hardening off, transplanting, and allowing the treated Carpinus baohuaensis seeds to recover.

[0012] As a preferred option, the seedling recovery period is 7 to 10 days.

[0013] As a preferred embodiment, the light intensity for germination is ≤400 μmol·m⁻¹. -2 ·s -1 The light intensity for seedling hardening is 80~400 μmol·m⁻¹. -2 ·s -1 .

[0014] Beneficial Effects: This invention provides a seed pretreatment method to improve the breeding efficiency of *Carpinus baohuaensis*, comprising the following steps: disinfecting *Carpinus baohuaensis* seeds, and subjecting the disinfected seeds to temperature-dependent stratification treatment to obtain pretreated *Carpinus baohuaensis* seeds; the temperature-dependent stratification treatment includes: warm treatment at 23-27℃ for 30 days, followed by cold treatment at 3-5℃ for 60 days. This invention, through disinfection and temperature-dependent stratification of *Carpinus baohuaensis* seeds, and by artificially setting alternating temperatures, directionally regulates the degradation of endogenous inhibitors and the synthesis of germination-promoting substances in the seeds, breaking deep physiological dormancy, thereby significantly improving the rate of seed dormancy breaking, shortening the average germination cycle to about 14 days, and basically completing germination within two weeks. This speed is significantly improved compared to existing methods, physiologically solving the technical problem of slow germination of *Carpinus baohuaensis* seeds and significantly improving breeding efficiency. The seed pretreatment method of this invention can simultaneously activate seed physiological activity and inhibit the synthesis of dormancy substances, thus significantly improving the seedling rate of *Carpinus baohuaensis*. The results of the embodiments show that, using the method provided by the present invention, the germination time of seeds is reduced to 14 days, the germination rate is 93.1%, the seedling rate is 89.7%, the germination is rapid, and the uniformity is high.

[0015] This invention discloses a seedling cultivation method for improving the propagation efficiency of *Carpinus baohuaensis*, comprising the following steps: treating *Carpinus baohuaensis* seeds using the aforementioned seed pretreatment method; and then sowing, germinating, hardening off, transplanting, and allowing the treated seeds to recover. This seedling cultivation method can complete seed germination and seedling cultivation within 74-104 days, significantly improving the propagation efficiency of *Carpinus baohuaensis*. It also solves the technical bottlenecks of deep seed dormancy, slow germination, and low seedling survival rate, significantly shortening the seed germination cycle and achieving a high seedling survival rate. The technical solution of this invention is simple to operate, low in cost, requires no complex equipment or large amounts of hormones, and can effectively improve the propagation speed of *Carpinus baohuaensis*. It is suitable for germplasm preservation, population reconstruction, ex-situ conservation, and large-scale seedling cultivation for landscaping, providing technical support for the protection and industrial application of this critically endangered species. Attached Figure Description

[0016] Figure 1 These are photos of carefully selected Carpinus baohuaensis seeds. Figure 2 This is a photograph of the seed germination process; the steps from A to D are, in order, seed germination failure, cotyledon unfolding, true leaf emergence, and hardening off. Figure 3 Images of samples processed at different temperatures; Figure 4 The results of seed germination rates after two weeks under different stratification treatments are shown in the figure. Figure 5 The results of seed germination rates over four weeks under different stratification treatments are shown in the figure. Figure 6 The graph shows the seedling survival rate under different stratification treatments. Detailed Implementation

[0017] This invention provides a seed pretreatment method to improve the breeding efficiency of Carpinus baohuaensis, comprising the following steps: disinfecting Carpinus baohuaensis seeds, subjecting the disinfected seeds to variable temperature stratification treatment to obtain pretreated Carpinus baohuaensis seeds; the variable temperature stratification treatment includes: warm treatment at 23~27℃ for 30 days, followed by cold treatment at 3~5℃ for 60 days.

[0018] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0019] The seeds of *Carpinus baohuaensis* described in this invention are preferably plump and healthy seeds. As a preferred embodiment, this invention first collects, removes impurities from, and selects *Carpinus baohuaensis* seeds to obtain plump and healthy seeds. During collection, it is preferred to select robust *Carpinus baohuaensis* mother trees free from pests and diseases, and harvest fully mature seeds; the optimal harvesting time is when the fruit is yellowish-brown and the buds are split open. During impurity removal, it is preferred to manually remove impurities such as buds, branches, leaves, and stones to obtain pure seeds. This invention preferably uses a buoyancy-based water-floating method for selection. As a specific embodiment, the pure seeds are placed in clean water and gently stirred with a glass rod to gradually separate the seeds into layers. Empty, shriveled, underdeveloped, moldy, and insect-infested seeds are removed using a filter or sieve, and the plump and healthy seeds that sink are collected; these are the selected seeds. Mature, plump, and healthy seeds have a density greater than water; using the water-floating method can efficiently remove inferior seed sources. The present invention preferably involves placing the selected seeds in a cool, ventilated place to dry (avoiding direct sunlight) until the seed surface is dry and feels loose to the touch. Appropriate drying balances the seed moisture content, preventing subsequent mold growth and maintaining the seed's original activity. During the drying process, some paper towels can be placed under the seeds to accelerate water absorption.

[0020] In the pretreatment described in this invention, it is preferable to first disinfect the seeds of *Carpinus baohuaensis*. As a preferred embodiment, the seeds are first disinfected with 65%–75% ethanol (volume percentage) for 30–60 seconds, followed by disinfection with a 1%–2% NaClO solution (mass concentration) for 5–10 minutes. These two solutions have good safety and effectiveness, are readily available, and the ethanol can wash away oils and esters adhering to the outer shell, while the NaClO solution provides disinfection and sterilization. The volume percentage of ethanol used in this invention can be any value within the range of 65%–75%, such as 65%, 70%, or 75%; the disinfection time can be any value within the range of 30–60 seconds, such as 30, 40, 50, or 60 seconds; and the preferred disinfection method is immersion. The concentration of the NaClO solution described in this invention can be any value within the range of 1% to 2%, for example, 1%, 1.5%, or 2%; the disinfection time of the NaClO solution can be any value within the range of 5 to 10 minutes, for example, 5, 6, 7, 8, 9, or 10 minutes; the preferred disinfection method is immersion. After the disinfection treatment described in this invention, it is preferable to further include rinsing with sterile water, rinsing ≥3 times. After rinsing, it is also preferable to drain the water in a sterile environment; the draining time can be any value within the range of 5 to 10 minutes, for example, 5, 6, 7, 8, 9, or 10 minutes.

[0021] This invention preferably involves placing sterilized seeds in a substrate for variable-temperature stratification treatment to obtain pretreated *Carpinus baohuaensis* seeds. The variable-temperature stratification treatment includes: a warm treatment at 23-27°C for 30 days, followed by a cold treatment at 3-5°C for 60 days. *Carpinus baohuaensis* seeds exhibit deep physiological dormancy, primarily regulated by endogenous hormone balance (abscisic acid inhibits germination, gibberellin promotes germination) and enzyme activity. Gradual temperature changes can simulate the alternating warm and cold rhythms of the natural environment, precisely regulating the internal physiological metabolism of the seeds and breaking the seed dormancy barrier. The warm treatment temperature can be any value within the range of 23-27°C, for example, 23, 24, 25, 26, or 27°C; the warm treatment duration is 30 days. Similarly, the cold treatment temperature can be any value within the range of 3-5°C, for example, 3, 4, or 5°C; the cold treatment duration is 60 days. Plump seeds have ample nutrient reserves, providing a solid material foundation for germination and seedling growth. Warm treatment at a constant temperature of around 25℃ enhances seed respiration, accumulating the energy needed for germination. Subsequent cold treatment at a constant temperature of around 4℃ effectively inhibits abscisic acid synthesis, promotes gibberellin accumulation, and orderly breaks deep dormancy. Almost all sown seeds germinate within two weeks, improving germination uniformity. As a preferred embodiment, the substrate for the variable-temperature stratification treatment described in this invention includes peat moss, sand, and perlite. Peat moss retains water and fertilizer, sand provides aeration and aids root growth, and perlite provides drainage, aeration, and insulation. The volume ratio of peat moss, sand, and perlite in this invention is any value within the range of 1~3:1~2:1~2, for example, 1:1:1, 1:2:1, 1:1:2, 1:2:2, 2:1:1, 2:2:1, 2:1:2, 2:2:2, 3:1:1, 3:2:1, 3:1:2, or 3:2:2. The substrates used in this invention (peat moss, sand, and perlite) are commonly used materials in forestry seedling cultivation, readily available, low in cost, and scientifically proportioned, naturally adapting to seed growth needs without the need for additional expensive reagents. The variable-temperature stratification treatment described in this invention is preferably carried out in a breathable container; a 5-6 cm thick layer of substrate can be laid at the bottom of the container to prevent seeds from contacting the bottom and causing water accumulation. In a specific embodiment of this invention, the breathable container is a breathable self-sealing bag. In the variable-temperature stratification treatment, it is preferable to periodically open the container for ventilation to maintain air circulation and prevent seed hypoxia. In the variable-temperature stratification treatment of this invention, the moisture content of the substrate is preferably ≥60%. A stable moisture environment needs to be maintained during the seed dormancy release process. Insufficient moisture will lead to reduced seed enzyme activity and stagnation of physiological metabolism, while excessive moisture will easily promote mold growth. Regular water replenishment can precisely maintain a suitable moisture content in the substrate, ensuring normal seed respiration and metabolic activities. The variable-temperature stratification treatment of this invention preferably also includes: spraying a fungicide every 8-12 days; the fungicide preferably includes at least one of the following: carbendazim, pyraclostrobin, and difenoconazole.The concentration of the stock solution of the bactericide described in this invention is preferably any value within the range of 20% to 40%, such as 20%, 30%, or 40%. When using it, it is preferable to dilute the stock solution before use, and the dilution ratio can be any value within the range of 400 to 600 times, such as 400, 500, or 600 times. Regular sterilization can inhibit the reproduction of harmful microorganisms such as mold, prevent seed coat rot and embryo damage, thereby preserving seed viability. This invention focuses on the dormancy characteristics of *Carpinus baohuaensis* seeds, abandoning complex equipment dependence and exogenous hormone intervention, relying on natural physiological regulation mechanisms to achieve seed dormancy breaking and germination, without requiring professional operating skills or high-end equipment.

[0022] The present invention provides a seedling raising method for improving the propagation efficiency of Carpinus baohuaensis, comprising the following steps: treating Carpinus baohuaensis seeds using the above-mentioned seed pretreatment method, and then sowing, germinating, hardening off, transplanting, and allowing the treated Carpinus baohuaensis seeds to recover.

[0023] The substrate used for sowing in this invention is the substrate subjected to temperature-variable stratification treatment; the composition of the substrate will not be detailed here. Using the same substrate simplifies the process, requiring only one preparation, and substrates with similar physicochemical properties promote stable seed germination. Sowing can be carried out in a seedling container, such as a seedling tray or pot. The sowing density is 1-2 seeds per 4 square centimeters. After sowing, it is preferable to cover the seeds with a 1-2 cm thick layer of sowing substrate; gently compact it to ensure close contact between the seeds and the substrate, and then water thoroughly. This invention preferably pre-germinates the seeds after sowing. The germination temperature can be any value within the range of 23-27℃, for example, 23, 24, 25, 26, or 27℃. Around 25℃ is the optimal temperature for simulating the germination of *Carpinus baohuaensis* seeds, maximizing the activation of key enzymes such as amylase and lipase, while airtight sealing and moisture retention prevent seed dehydration and inactivation, accelerating the germination process. In this invention, during germination, it is preferable to cover the seedling container with a lid, which can be a transparent plastic lid or transparent plastic sheet. Covering with a transparent lid helps retain moisture, maintaining a stable temperature and humidity microenvironment, providing optimal conditions for seed water absorption and swelling, and for the radicle to break through the seed coat, thus promoting uniform germination within a short time. The light intensity for germination in this invention can be ≤400 μmol·m⁻¹. -2 ·s -1 Any value within the range, such as 0 (darkness), 200, or 400 μmol·m -2 ·s -1The relative humidity for germination in this invention can be any value within the range of 80% to 85%, for example, 80%, 81%, 82%, 83%, 84%, or 85%. During germination, it is preferable to open the lid for ventilation for 10-15 minutes daily to prevent substrate mold. After the cotyledons germinate, an appropriate amount of nutrient soil is added to the seedling container; there are no special restrictions on the source of the nutrient soil, and conventional commercially available products are acceptable. The nutrient soil allows for continued root extension after the roots emerge, as long as the soil remains moist but not waterlogged. Before use, this invention preferably undergoes sun exposure or routine disinfection; the nutrient soil promotes root expansion and growth. Transplanting is preferably carried out when the seedlings are robust, have fresh and intact leaves, are free from obvious pests and diseases, have developed 4-6 true leaves, and reach a height of 8-12 cm. The preferred substrate for transplanting in this invention is nutrient soil.

[0024] In a preferred embodiment, after the seedlings emerge and develop 2-3 true leaves, the transparent plastic cover is gradually removed for ventilation and hardening-off, with the hardening-off period lasting 15-20 days. The light intensity for hardening-off described in this invention can be 80-400 μmol·m⁻². -2 ·s -1 Any value within the range, such as 80, 200, or 400 μmol·m -2 ·s -1When seedlings exhibit abnormal phenomena such as leaf wilting, yellowing, or stunted growth, they can be temporarily moved back to a greenhouse or a relatively stable environment. The constant environmental conditions allow the seedlings to recover, and then the hardening-off process can be repeated 2-3 times to gradually improve their resistance (cold resistance, drought resistance, and resistance to pests and diseases). In this invention, nutrient solution is preferably sprayed every 7 days during the hardening-off process; the amount of nutrient solution applied can be 1-3 mL / plant, for example, 1, 2, or 3 mL / plant. The nutrient solution used in this invention is preferably diluted 800-1000 times before use, for example, 800, 900, or 1000 times. The preferred effective components of the nutrient stock solution of this invention include: 1.5%~2.5% nitrogen (N), 2.0%~3.0% phosphorus (P2O5), and 2.5%~3.5% potassium (K2O). In the nutrient stock solution, the effective nitrogen content can be 1.5%, 2.0%, or 2.5% (N); the effective phosphorus content can be 2.0%, 2.5%, or 3.0% (P2O5); and the effective potassium content can be 2.0%, 2.5%, or 3.0% (K2O). Gradual hardening of seedlings reduces their dependence on a high-humidity, constant-temperature environment. The specialized substrate balances water retention and aeration, and the nitrogen, phosphorus, and potassium ratio matches the growth needs of the seedling stage, synergistically promoting robust seedlings and environmental adaptability. The preferred application method of this invention is spraying; the preferred spraying tool is a trigger-type sprayer. Evenly spraying the nutrient solution onto the roots and surrounding substrate of seedlings can promote rapid development of the root system and stem, improve their stress resistance and health, and thus increase the seedling survival rate. This invention gradually hardens the seedlings, reducing their dependence on a high-humidity, constant-temperature environment. The specialized substrate balances water retention and aeration, and the nitrogen, phosphorus, and potassium ratio matches the growth needs of the seedlings, synergistically promoting robust seedlings and the formation of environmental adaptability.

[0025] This invention allows for transplanting of seedlings when they are robust, with fresh and intact leaves, no obvious pests or diseases, and have developed 4-6 true leaves and reached a height of 8-12 cm. The preferred substrate for transplanting is the nutrient soil added during seedling cultivation, which is preferably subjected to sun exposure or conventional sterilization before use. The composition and ratio of the nutrient soil are not detailed here. As a preferred embodiment, during transplanting, the seedlings are carefully removed along with the root zone substrate to minimize root damage. They are then transplanted into a nutrient container or nursery, with the planting depth essentially the same as the seedling's original growth depth in the original container. Immediately after transplanting, the seedlings are thoroughly watered and placed in a cool, well-ventilated environment under normal light (natural light) to allow them to recover. The recovery period can be any value within the range of 7-10 days, for example, 7, 8, 9, or 10 days. This optimized step ensures the adaptability of the seedlings after transplanting, reduces the decrease in survival rate caused by transplanting, and provides a reliable guarantee for large-scale production.

[0026] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a seed pretreatment method and seedling raising method for improving the breeding efficiency of *Carpinus baohuaensis* provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 The experiment was conducted in the Baohua Mountain Carpinus Nature Reserve in Jurong City, Jiangsu Province. Seeds from the mother trees of Baohua Carpinus were used, and the seedling cultivation steps are as follows: 1. Seed collection and selection From late October to early November, select healthy, disease-free *Carpinus baohuaensis* mother trees and harvest fully mature seeds (the optimal harvesting time is when the fruit is yellowish-brown and the buds are splitting open). Immediately after harvesting, clean and remove impurities such as buds, branches, leaves, and stones manually to obtain pure seeds. Use a buoyancy-based water-floating method for further selection: place the pure seeds in clean water and gently stir with a glass rod to gradually separate them into layers. Use a filter or sieve to remove floating, empty, underdeveloped, moldy, or insect-damaged seeds, and collect the plump, healthy seeds that sink (see...). Figure 1 Place the selected seeds in a cool, well-ventilated place to dry (avoid direct sunlight; you can place some paper towels underneath to accelerate water absorption). Dry until the surface of the seeds is dry and they feel loose to the touch. Once dried, they are ready for use.

[0028] 2. Gradient Temperature Layering (1) Matrix preparation: Mix peat moss: sand: perlite in a volume ratio of 2:1:1 to prepare a moisturizing matrix. Sterilize the matrix at high temperature in advance to remove impurities and harmful microorganisms, ensuring that the matrix is ​​clean, breathable and has good moisture retention. Let it stand for about 2 hours to cool to room temperature before use.

[0029] (2) Preparation of disinfectant solution and seed disinfection treatment: Preparation of 1% NaClO solution: The bleaching solution has an effective chlorine content of about 5%. Mix the bleaching solution and water at a volume ratio of 1:4 to obtain a NaClO solution of about 1%.

[0030] Seed disinfection procedure (in a sterile / clean work area), wearing gloves throughout: a) The clean bench should be wiped and sterilized with 75% ethanol solution in advance, and then irradiated with ultraviolet light for 6 hours; b) Clean the tweezers with a 75% ethanol solution and let them air dry. c) Rinse with deionized water for 2 minutes to remove surface residue. Place the rinsed items in a laminar flow hood to air dry, with sterile disposable petri dishes underneath. d) Soak in 70% ethanol solution for 60 seconds, then immediately transfer to 1% NaClO solution for 10 minutes.

[0031] e) Rinse three times with sterile water, air dry in a clean bench, and drain on sterile disposable petri dishes or sterile filter paper.

[0032] (3) Variable temperature stratification: After disinfection, the seeds are subjected to corresponding physical and chemical treatments and mixed with a moisture-retaining substrate, and then placed in a self-sealing bag. A 5cm thick layer of substrate is laid at the bottom of the bag to prevent the seeds from contacting the bottom of the bag and causing water accumulation. The self-sealing bags are warm-treated at 25℃ for 30 days and then cold-treated at 4℃ for 60 days, with a total stratification period of 90 days.

[0033] (4) Management during stratification: During stratification, check the substrate moisture every 5 days. When the substrate moisture content is below 60%, i.e., when the surface appears loose and dry, add water to keep the substrate moist but not waterlogged (the substrate should be able to clump together when squeezed by hand and crumble when released). Spray a diluted benzoyl-pyraclostrobin solution (the total effective ingredient content of the stock solution is 30%, and the suspension of pyraclostrobin and difenoconazole is mixed at 19% and 11% respectively, with a dilution concentration of 1 / 500) every 10 days for sterilization to prevent seed mold and rot. Open the self-sealing bag regularly for 5 minutes to maintain air circulation inside the bag and avoid seed hypoxia.

[0034] 3. Sowing and germination management (see the cultivation process of seed germination, cotyledon unfolding, true leaf emergence, and hardening off for details). Figure 2 ) (1) Preparation of seedling substrate: Use the same moisture-retaining substrate (peat moss: sand: perlite = 2:1:1) as in step 2, water it thoroughly with a watering can to ensure that the substrate has sufficient moisture and is evenly distributed before use.

[0035] (2) Sowing operation: In the spring of the following year, the seeds that have been stratified are sown in the substrate of the seedling tray at a density of 1 seed per 4 square centimeters. Then, cover the seeds with a layer of the same substrate about 1 cm thick, gently compact it to make the seeds and substrate in close contact, and then water thoroughly to settle the roots.

[0036] (3) Germination management: After sowing, place the seedling trays in a constant temperature environment, controlling the ambient temperature at 25℃. Cover the seedling trays with a transparent plastic lid (or transparent plastic sheet) to retain moisture, maintaining a relative humidity of 80%~85%. Open the lid for ventilation for 10 minutes every day to prevent substrate mold. The light is provided in darkness for 28 days. After the cotyledons germinate, add nutrient soil (named organic cultivation substrate, purchased from Jiangsu Xingnong Substrate Technology Co., Ltd.) to the seedling trays to promote root expansion and growth.

[0037] 4. Seedling hardening and transplanting (1) Seedling hardening: After 28 days of germination, transplant to a location with 200 μmol·m light. -2 ·s -1Under light conditions, once the seedlings emerge and have developed 2-3 true leaves, gradually remove the transparent plastic cover to allow for ventilation and hardening off, with a hardening-off period of 15 days. If seedlings exhibit abnormal phenomena such as leaf wilting, yellowing, or stunted growth, they can be temporarily moved back to a greenhouse or a relatively stable environment to allow them to recover under constant environmental conditions. This hardening-off process should then be repeated three times to gradually improve the seedlings' resistance (cold resistance, drought resistance, and disease and pest resistance). Nutrient solution should be precisely controlled and sprayed every 7 days with a diluted nutrient solution (the effective components of the nutrient stock solution are: nitrogen (N) 2.0%, phosphorus (P2O5) 2.5%, potassium (K2O) 3.0%, diluted to a concentration of 1 / 1000). Use a trigger-type sprayer to spray approximately 2 mL of nutrient solution per seedling, evenly covering the roots and surrounding substrate.

[0038] (2) Transplanting: Transplant seedlings when they are healthy, with fresh and intact leaves, no obvious pests or diseases, and have 4-6 true leaves and a height of 8-12cm. The preferred transplanting substrate is a mixture of leaf mold, garden soil, and perlite in a volume ratio of 3:2:1. Before use, the substrate should preferably be exposed to sunlight or undergo routine disinfection. When transplanting, carefully remove the seedling along with the root zone substrate to minimize root damage. Then transplant it into a self-sealing nutrient bag or nursery, with the planting depth being basically the same as the seedling's growth depth in the original self-sealing nutrient bag. Immediately after transplanting, water thoroughly to settle the roots and place the seedling in a cool, ventilated environment with normal light (natural light) for 10 days to allow it to recover. After the recovery period, transfer it to normal maintenance and management.

[0039] Example 2 The procedure was carried out in accordance with Example 1, except that in step 3, the germination management process, the light condition was changed from darkness to 200 μmol·m -2 ·s -1 Processing under illumination.

[0040] Example 3 The procedure was carried out in accordance with Example 1, except that in step 3, germination management, the light condition was changed from dark treatment to 400 μmol·m -2 ·s -1 Step 4. Seedling hardening and transplanting (1) Seedling hardening: After 28 days of germination, replace with 200 μmol·m -2 ·s -1 Processing under illumination.

[0041] Comparative Example 1 The procedure is carried out in accordance with Example 1, except that step 2 (3) temperature-controlled stratification and (4) management during stratification are omitted; that is, direct sowing is performed after disinfection.

[0042] Comparative Example 2 The procedure is carried out in accordance with Example 2, except that step (3) temperature-controlled stratification and (4) management during stratification are omitted in step 2; that is, direct sowing is performed after disinfection.

[0043] Comparative Example 3 The procedure is carried out in accordance with Example 3, except that step 2 (3) temperature-controlled stratification and (4) management during stratification are omitted; that is, direct sowing is performed after disinfection.

[0044] Comparative Example 4 The procedure is carried out in accordance with Example 1, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 60 days.

[0045] Comparative Example 5 The procedure is carried out in accordance with Example 2, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 60 days.

[0046] Comparative Example 6 The procedure is carried out in accordance with Example 3, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 60 days.

[0047] Comparative Example 7 The procedure is carried out in accordance with Example 1, except that in step 2 (3) temperature-variable layering, the self-sealing bag is replaced with 90 days instead of being warm-treated at 25°C for 30 days and cold-treated at 4°C for 60 days.

[0048] Comparative Example 8 The procedure is carried out in accordance with Example 2, except that in step 2 (3) temperature-variable layering, the process of warming the self-sealing bag at 25°C for 30 days and then cold-treating it at 4°C for 60 days is replaced with 90 days.

[0049] Comparative Example 9 The procedure is carried out in accordance with Example 3, except that in step 2 (3) temperature-variable layering, the process of warming the self-sealing bag at 25°C for 30 days and then cold-treating it at 4°C for 60 days is replaced with 90 days.

[0050] Comparative Example 10 The procedure is carried out in accordance with Example 1, except that in step 2 (2), the preparation of disinfectant solution and the addition of seed shell polishing treatment before seed disinfection treatment are carried out, and in step 2 (3), the variable temperature layering is omitted, and the self-sealing bag is warm-treated at 25°C for 30 days, that is, after the seed shell is polished and disinfected, it is cold-treated at 4°C for 60 days.

[0051] Comparative Example 11 The procedure is carried out in accordance with Example 2, except that in step 2 (2), the preparation of disinfectant solution and the addition of seed shell polishing treatment before seed disinfection treatment are carried out, and in step 2 (3), the variable temperature layering is omitted, and the self-sealing bag is warm-treated at 25°C for 30 days, that is, after the seed shell is polished and disinfected, it is cold-treated at 4°C for 60 days.

[0052] Comparative Example 12 The procedure is carried out in accordance with Example 3, except that in step 2 (2), the preparation of disinfectant solution and the addition of seed shell polishing treatment before seed disinfection treatment are performed, and in step 2 (3), the variable temperature layering is omitted, and the self-sealing bag is warm-treated at 25°C for 30 days, that is, after the seed shell is polished and disinfected, it is cold-treated at 4°C for 60 days.

[0053] Comparative Example 13 The procedure was carried out in accordance with Example 1, except that in step 2 (2), after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and in step 2 (3), the variable temperature stratification was omitted. The self-sealing bag was warm-treated at 25°C for 30 days and then cold-treated at 4°C for 60 days.

[0054] Comparative Example 14 The procedure was carried out in accordance with Example 1, except that in step 2 (2), after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and in step 2 (3), the variable temperature stratification was omitted. The self-sealing bag was warm-treated at 25°C for 30 days and then cold-treated at 4°C for 60 days.

[0055] Comparative Example 15 The procedure was carried out in accordance with Example 1, except that in step 2 (2), after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and in step 2 (3), the variable temperature stratification was omitted. The self-sealing bag was warm-treated at 25°C for 30 days and then cold-treated at 4°C for 60 days.

[0056] Comparative Example 16 The procedure was carried out in accordance with Example 1, except that in step 2, after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and the self-sealing bags were not subjected to warm treatment at 25℃ for 30 days before direct sowing.

[0057] Comparative Example 17 The procedure was carried out in accordance with Example 2, except that in step 2, after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and the self-sealing bags were not subjected to warm treatment at 25℃ for 30 days before direct sowing.

[0058] Comparative Example 18 The procedure was carried out in accordance with Example 3, except that in step 2, after preparing the disinfectant solution and disinfecting the seeds, the seeds were soaked in 500ppm GA3 gibberellin solution for 24 hours, and the self-sealing bags were not subjected to warm treatment at 25℃ for 30 days before direct sowing.

[0059] Comparative Example 19 The procedure is carried out in accordance with Example 1, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 120 days.

[0060] Comparative Example 20 The procedure is carried out in accordance with Example 2, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 120 days.

[0061] Comparative Example 21 The procedure is carried out in accordance with Example 3, except that in step 2 (3) temperature-variable layering, the self-sealing bag is not warm-treated at 25°C for 30 days, that is, it is only cold-treated at 4°C for 120 days.

[0062] Comparative Example 22 The process is carried out in accordance with Example 2, except that in step 2 (3) temperature stratification, the self-sealing bag is placed in an outdoor, dark and ventilated place for 90 days for overwintering treatment, that is, the temperature stratification process is completed according to the natural changes in temperature (daytime temperature 2℃~25℃; nighttime temperature -2℃~15℃).

[0063] Experimental Example 1 The experiment was conducted in the Baohua Mountain Carpinus Nature Reserve in Jurong City, Jiangsu Province, using seeds from the mother trees of the Baohua Carpinus.

[0064] Seeds of *Carpinus baohuaensis* were collected and processed using the seed collection and selection method described in step 1 of Example 1. 411 plump seeds were obtained and sown. Nine differential treatment groups were set up: Comparative Examples 1-3 were denoted as T0, Comparative Examples 4-6 as T1, Comparative Examples 7-9 as T2, Examples 1-3 as T3, Comparative Examples 10-12 as T4, Comparative Examples 13-15 as T5, Comparative Examples 16-18 as T6, Comparative Examples 19-21 as T7, and Comparative Example 22 as T8. Each group was set up with three biological replicates, and all experimental data were taken as the average of the three replicates to ensure the reliability and repeatability of the experimental results. Some group samples are shown below. Figure 3 The T0~T7 treatment groups were further divided into 3 light subunits during germination treatment, with L1 being darkness (dark treatment) and L2 being normal light (≈200 μmol·m⁻¹). -2 ·s -1 L3 is a strong light source with an intensity of approximately 400 μmol·m⁻¹. -2 ·s -1The specific grouping of the experiments is shown in Table 1.

[0065] After sowing, germination was recorded at two and four weeks after sowing in each treatment group, and the germination rate was calculated as follows: Germination rate = (Number of germinated seeds / Initial number of replicate seeds) × 100%. The statistical results for each treatment group are shown in Tables 2 and 3. Figure 4 and Figure 5 The seedling survival rate was defined as the proportion of seedlings that had successfully recovered from transplanting and grown normally for more than one month, out of the initial number of plump seeds in the replicate. The seedling survival rate results for each treatment group are shown in Table 4 and [Table data would be inserted here]. Figure 6 During the experiment, it was found that light conditions had no significant effect on germination, so the statistical results of different light conditions were combined.

[0066] Table 1 Experimental Grouping

[0067] Table 2 Seed germination rates after two weeks under different stratification treatments

[0068] Table 3 Seed germination rates after four weeks under different stratification treatments

[0069] Table 4. Seedling survival rate under different stratification treatments (based on plump seeds)

[0070] From Table 2, Table 3, Figure 4 and Figure 5 It can be seen that there is no significant difference in germination rate between two weeks and four weeks in the T3 treatment group (Examples 1-3), indicating that the average germination period of seeds using the method of the present invention is shortened to about 14 days, and the germination uniformity is high. The germination rates of the T3 treatment group (Examples 1-3) at two weeks and four weeks are both higher than those of other control treatment groups, showing the best effect. Among them, the T8 treatment group (90 days of stratification under natural temperature variations) showed no germination at two weeks, and the seeds only began to germinate at four weeks.

[0071] From Table 4 and Figure 6 It can be seen that the seedling survival rate of the T3 treatment group (warm treatment at constant 25℃ for 30 days followed by cold treatment at constant 4℃ for 60 days) was as high as 89.70%, which was significantly higher than that of other treatment groups.

[0072] By comparing and analyzing the experimental results, the following core conclusions can be drawn: 1. Rapid dormancy breaking and significantly shortened germination cycle: This invention sets up eight continuous temperature and physicochemical treatment gradients, combined with three light treatments (strong light, normal light, and no light) during germination for synergistic verification. Ultimately, the optimal germination stratification conditions suitable for the dormancy characteristics of *Carpinus baohuaensis* seeds were selected (T3 treatment group: constant 25℃ warm treatment for 30 days followed by constant 4℃ cold treatment for 60 days). The core principle is that the constant 25℃ warm treatment activates the activity of germination-related enzymes such as dehydrogenases within the seed, promoting enhanced seed respiration and storing energy for dormancy breaking; the subsequent constant 4℃ cold treatment simulates a natural low-temperature environment, inhibiting the synthesis of dormancy-related substances (such as abscisic acid) within the seed and promoting the accumulation of germination-related hormones (such as gibberellins), thus achieving the orderly breaking of deep seed dormancy. Compared with traditional constant temperature stratification, conventional variable temperature stratification treatment and existing related patented technologies, this invention significantly accelerates the rate of seed dormancy breaking, shortens the average germination cycle to about 14 days, and has a high degree of germination uniformity. Compared with the germination cycle of existing technologies, the improvement effect is obvious, and it effectively solves the technical problem of slow seed germination of *Carpinus baohuaensis*.

[0073] 2. High germination and seedling survival rates, and good stability: This invention selects plump seeds using a selective method, combined with scientific gradient temperature stratification and sowing and germination management, effectively ensuring the stability of seed germination rates. The T3 temperature-controlled group (constant 25℃ warm treatment for 30 days followed by constant 4℃ cold treatment for 60 days) achieved a germination rate of 93.1% and a seedling survival rate as high as 89.7% after four weeks, significantly improving both germination and seedling survival rates compared to natural temperature stratification (T8). Simultaneously, the optimized seedling hardening and transplanting scheme further ensures the robustness and uniformity of the seedlings, meeting the practical needs of large-scale seedling production and providing a reliable guarantee for the large-scale propagation of *Carpinus baohuaensis*.

[0074] 3. Controllable process with outstanding pollution prevention and viability preservation effects: This invention incorporates regular water replenishment and sterilization steps throughout the gradient temperature stratification process, effectively maintaining the moisture balance of the seed stratification environment (moist substrate but not waterlogged), inhibiting the growth of harmful microorganisms such as mold, and preventing seed viability decline and rotting during dormancy recovery due to water loss, oxygen deficiency, or contamination. This provides key technical support for achieving high germination rates and high seedling survival rates.

[0075] 4. Scientific treatment method with strong adaptability: Experimental results show that while gibberellin treatment (T5, T6) and sandpaper-polishing of the outer shell (T4) can promote seed germination of *Carpinus baohuaensis* to some extent, their effect on improving the germination rate is not significant. However, combining these treatments with T3 variable temperature treatment (constant 25℃ warm treatment for 30 days followed by constant 4℃ cold treatment for 60 days) can further improve the seed germination rate, indicating that the gradient variable temperature stratification core technology of this invention has a good synergistic effect with other auxiliary treatments. Furthermore, the method of this invention is simple to operate, with clear steps and well-defined treatment parameters. The experimental results have been verified three times with good consistency, making it suitable for large-scale promotion and implementation. It can effectively solve the problem of breeding difficulties for this critically endangered species, *Carpinus baohuaensis*.

[0076] In summary, *Carpinus baohuaensis*, as a critically endangered species, possesses a unique seed dormancy mechanism (deep physiological dormancy and mild physical dormancy), which traditional methods cannot accurately adapt to, leading to breeding difficulties. This invention precisely overcomes its dormancy barrier through a synergistic approach involving gradient temperature control, moisture management, and sterilization, while simultaneously protecting seed viability and fostering robust seedlings, thus meeting the species' physiological growth needs. The technical solution of this invention not only enables the cultivation of healthy, resilient seedlings but also provides effective support for the germplasm preservation, population reconstruction, ex-situ conservation, and landscaping of *Carpinus baohuaensis*, offering practical technical support for the species' protection and sustainable utilization, and making a positive contribution to the conservation and ecological restoration of critically endangered species.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A seed pretreatment method for improving the breeding efficiency of *Carpinus baohuaensis*, characterized in that, Includes the following steps: The seeds of Carpinus baohua were disinfected, and the disinfected seeds were subjected to temperature-dependent stratification to obtain pretreated Carpinus baohua seeds. The variable temperature lamination process includes: warm treatment at 23~27℃ for 30 days, followed by cold treatment at 3~5℃ for 60 days.

2. The seed pretreatment method according to claim 1, characterized in that, The matrix for the variable temperature stratification treatment includes peat moss, sand, and perlite; the volume ratio of peat moss, sand, and perlite is 1~3:1~2:1~2.

3. The seed pretreatment method according to claim 2, characterized in that, The moisture content of the matrix is ​​≥60%.

4. The seed pretreatment method according to claim 1, characterized in that, The disinfection process includes: disinfecting with 65%~75% ethanol by volume for 30~60 seconds, followed by disinfecting with 1%~2% NaClO solution by mass for 5~10 minutes.

5. The seed pretreatment method according to claim 4, characterized in that, The disinfection process also includes rinsing with sterile water, with rinsing times ≥ 3 times.

6. The seed pretreatment method according to claim 1, characterized in that, The variable temperature stratification treatment also includes spraying a bactericide every 8 to 12 days.

7. The seed pretreatment method according to claim 6, characterized in that, The disinfectant includes at least one of the following: carbendazim, pyraclostrobin, and difenoconazole.

8. A seedling raising method for improving the propagation efficiency of *Carpinus baohuaensis*, characterized in that, Includes the following steps: Seeds of *Carpinus baohuaensis* were treated using any one of the seed pretreatment methods described in 1 to 7, and the treated seeds were then sown, germinated, hardened off, transplanted, and allowed to recover.

9. The seedling raising method according to claim 8, characterized in that, The recovery period for seedlings is 7 to 10 days.

10. The seedling raising method according to claim 8, characterized in that, The light intensity for germination is ≤400 μmol·m⁻¹ -2 ·s -1 The light intensity for seedling hardening is 80~400 μmol·m⁻¹. -2 ·s -1 .

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