Method for promoting centella asiatica seed germination

Abstract

The invention relates to a method for promoting centella asiatica seed germination, and belongs to the technical field of medicinal plant cultivation. Aiming at the technical problems of low germination rate and long period of centella asiatica seeds caused by seed coat obstacles and endogenous inhibitors, the method comprises the following steps: firstly, sequentially carrying out mechanical coat breaking, ultrasonic treatment and high-pressure permeation treatment on the seeds to break a physical barrier; mixing the seeds with a wet substrate, carrying out warm lamination at 18-25 DEG C for 25-35 days, then carrying out cold lamination at 2-6 DEG C for 50-70 days, and spraying a KNO3 solution in the cold lamination stage to activate physiological metabolism; after lamination, washing with running water, sequentially soaking with a thiourea solution, carrying out H2O2 treatment for chemical degradation, and carrying out biological adsorption by adopting an adsorbent containing bacillus subtilis and activated carbon; soaking the seeds into a mixed solution containing brassinolide, kinetin and fluridone, and carrying out gradient heating and drying; and finally carrying out germination culture. The method can significantly improve the germination efficiency and uniformity, and is suitable for centella asiatica large-scale seedling raising and medicinal resource development.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant propagation technology, specifically relating to a method for promoting the germination of Centella asiatica seeds. Background Technology

[0002] Centella asiatica ( Centella asiatica (L.) Urban., a traditional Chinese medicine of the Apiaceae family, is widely used in the pharmaceutical, health, and cosmetic fields. Its artificial cultivation mainly relies on seed propagation to maintain genetic diversity, but seed propagation faces the following technical bottlenecks: 1. The interplay of physical and physiological dormancy leads to low germination rates. Centella asiatica seeds have a hard, dense seed coat structure that hinders water and gas exchange, while the embryo exhibits a physiological dormancy mechanism. Under natural conditions, the germination rate is typically less than 30%. Conventional physical treatments (such as warm water soaking) or single hormone treatments (such as gibberellin GA3) fail to simultaneously break down the seed coat barrier and physiological inhibition, resulting in limited improvement in germination rates, with most still remaining below 50%. Improving seed coat permeability requires balancing the degree of mechanical damage; excessive damage to the seed coat can harm the embryo, while insufficient permeability leads to inadequate water absorption during the stratification stage, becoming the primary obstacle to improving germination rates.

[0003] 2. Long stratification cycle and poor germination uniformity. Breaking physiological dormancy requires low-temperature stratification, but current stratification techniques have a cycle of 90-120 days, with germination times scattered across 30-60 days. The root cause lies in the fact that temperature fluctuations during the cold stratification stage easily lead to incomplete dormancy breaking; and the lack of effective signaling molecules to activate seed metabolic synchronization. Although the applicant has attempted to add germination-promoting substances such as nitrates, the application concentration, frequency, and poor matching with stratification temperature and humidity have failed to significantly shorten the cycle or improve uniformity. Long-cycle stratification also increases the risk of mold, further restricting large-scale production.

[0004] 3. Accumulation of endogenous inhibitors interferes with seedling emergence. Inhibitors such as phenolic acids and flavonoids contained in seeds remain after stratification and cannot be completely removed by conventional water rinsing. These substances inhibit radicle elongation, leading to an increased rate of deformed seedlings after germination. Existing chemical degradation methods (such as acid treatment) risk damaging embryo activity, while biosorption technology suffers from inconsistent inhibitor removal due to improper adsorbent selection or low loading efficiency. Inhibitor residues become a key challenge in ensuring seedling quality.

[0005] In summary, the propagation of Centella asiatica seeds faces three major technical challenges: low germination rate, long stratification period, and teratogenic effects of inhibitors. The complexity lies in the fact that the coordinated breaking of physical and physiological dormancy requires precise control of damage and metabolic activation, while inhibitor removal demands a highly efficient and embryo-safe degradation and adsorption combined method. These factors are interrelated, and improvements to a single technology are insufficient to systematically enhance germination efficiency. Therefore, developing a composite method integrating physical treatment, stratification optimization, and inhibitor removal is essential for achieving efficient Centella asiatica seedling cultivation. SUMMARY

[0006] For the hard and dense seed coat and the accumulation of endogenous inhibitors of Centella asiatica, the physical and physiological dormancy is intertwined, and the germination rate is low. The conventional single treatment such as warm water soaking or gibberellin cannot simultaneously break the seed coat barrier and physiological inhibition, and the germination rate is usually less than 50%. At the same time, the low-temperature stratification period is as long as 90-120 days, and the germination uniformity is poor, with a span of 30-60 days, and the lack of effective signal molecules to activate the metabolic synchronicity, in addition, the residual phenolic acid and flavonoid inhibitors after stratification cause abnormal seedlings, and a composite method for simultaneously breaking the seed coat barrier, optimizing the stratification metabolism and efficiently removing the inhibitors is urgently needed to solve the core problems of low germination rate, long period and high abnormal seedling rate.

[0007] To achieve the purpose of the application, a method for promoting the germination of Centella asiatica seeds is provided, comprising: Step 1, sequentially performing mechanical peeling, ultrasonic treatment and high-pressure infiltration treatment on the seeds; Step 2, mixing the seeds treated in step 1 with a wet substrate, first performing warm stratification and then cold stratification, and periodically applying a nitrate salt solution during the cold stratification stage; wherein the warm stratification temperature is 18-25℃, the time is 25-35 days, the cold stratification temperature is 2-6℃, and the time is 50-70 days; the nitrate salt solution is 0.1%-0.3% KNO3 solution, and is sprayed once every 3-7 days; Step 3, washing the seeds after stratification with running water, sequentially performing chemical degradation treatment with thiourea solution and hydrogen peroxide solution, and then performing biological adsorption treatment; wherein the chemical degradation treatment is first soaked in 0.05%-0.2% thiourea solution for 4-8 hours, and then treated with 3%-7% H2O2 solution for 20-40 minutes; the biological adsorption treatment uses a mixture of Bacillus subtilis with a bacterial amount of ≥1×10 8 CFU / g and activated carbon as the adsorbent; Step 4, immersing the seeds in a mixed solution containing 0.05-0.2mg / L brassinolide, 0.3-0.8mg / L kinetin and 5-20μM fluridone, and then drying with gradient temperature rise, the final temperature of the gradient temperature rise is not more than 50℃, and the final water content of the seeds is ≤10%; Step 5, performing germination culture.

[0008] The present embodiment significantly improves the water and gas exchange efficiency by physical treatment of skin breaking / ultrasonic / high-pressure infiltration, breaks the seed coat barrier, and synchronously completes embryo maturation and dormancy release by combining warm and cold stratification with periodic KNO3 spraying, significantly shortens the stratification period, and efficiently removes inhibitors by chemical degradation of thiourea / H2O2 and microbial-activated carbon adsorption, and guarantees embryo safety. The combination of brassinolide, kinetin and fluridone hormones enhances absorption by gradient drying and precisely regulates the germination signal pathway. The core problems of low germination rate, long period and high rate of abnormal seedlings are comprehensively solved.

[0009] Preferably, in step 1, the mechanical skin breaking uses 400-800 mesh sandpaper to rub the seed coat to the surface wear, then immerse in 0.1%-0.3% non-ionic surfactant solution Tween-20 or Triton X-100, maintain at 25℃ for 10-30 minutes, then ultrasonic treatment in the solution, ultrasonic frequency 30-50 kHz, time 5-15 minutes, the seed treated by ultrasonic is placed in a high-pressure container together with the liquid medium for high-pressure infiltration treatment, high-pressure infiltration pressure 50-110 MPa, temperature 15-25℃, maintain for 5-15 minutes.

[0010] The present embodiment limits the mesh number of sandpaper and the concentration of non-ionic surfactant to ensure uniform generation of micro-cracks in the seed coat, matches the ultrasonic frequency and time with the solution infiltration to expand the crack depth, and precisely controls the high-pressure infiltration pressure and temperature to realize forced water introduction and maintain cell membrane integrity. The three synergistically significantly improve the metabolic efficiency during stratification, laying a foundation for subsequent dormancy release.

[0011] Preferably, the wet substrate in step 2 is wet sand with sand-water mass ratio of 3:1, the treated seed is mixed with the wet sand during warm stratification, and stratified at 20±1℃ for 30 days; then transferred to a cold environment at 4±1℃ for cold stratification for 60 days, and turned over and sprayed with 0.2% KNO3 solution every 5 days, with spraying amount of 10% of the sand weight.

[0012] The sand-water ratio of the wet sand substrate in the present embodiment is optimized to balance water and gas, the warm stratification temperature and time are strictly controlled to promote embryo morphological maturation, and the cold stratification temperature is stably controlled at the threshold for physiological dormancy release, combined with periodic KNO3 spraying to activate the NO-GA signal pathway. The stratification period is significantly shortened, the germination uniformity is significantly improved, and the risk of mold is effectively reduced.

[0013] Preferably, the activated carbon particle size for biological adsorption treatment in step 3 is ≤300 mesh, and the seed to adsorbent mass ratio is 1:40-1:60; the water temperature for flushing is 20-28℃, and the time is 36-60 hours. The activated carbon with ultra-fine particle size ≤300 mesh increases the specific surface area of adsorption; the seed to adsorbent mass ratio is optimized to efficiently enrich inhibitors; and the water temperature and time for flushing are precisely controlled to completely remove degradation products. The rate of abnormal seedlings is significantly reduced, and the health of seedlings is significantly improved.

[0014] Preferably, the mixed solution of step 4 contains 0.1 mg / L brassinolide, 0.5 mg / L kinetin, and 10 μM fluridone, and the soaking time is 12 hours, followed by drying at 30°C for 24 hours, drying at 35°C for 24 hours, and drying at 40°C for 24 hours or more in a gradient temperature drying process until the water content of the seeds is ≤8%. The low concentration of brassinolide, kinetin, and fluridone in this embodiment synergistically promotes cell wall relaxation, division initiation, and ABA signal blocking. The gradient temperature drying program matches the heat tolerance threshold of the seeds, strengthens the infiltration of hormones, and avoids the loss of activity. The seed metabolic network is synchronously activated, and the germination potential and uniformity are further improved.

[0015] Preferably, during the germination culture of step 5, 640-680 nm red light is supplemented for 3-5 hours per day under a 12-hour white light / 12-hour dark cycle, with a light intensity of 40-60 μmol·m -2 ·s -1 ; at the same time, the temperature is cyclically regulated at 23-27°C during the day and 13-18°C at night for temperature variation stimulation; the CO2 concentration in the culture container is simultaneously maintained at 3%-7%; and the humidity of the sowing substrate in the culture container is regulated to start at 75%-85%, and is reduced by 3%-7% every 24-72 hours until it is reduced to 55%-65%.

[0016] The red light supplementation of this embodiment activates the phytochrome pathway and enhances GA synthesis; the diurnal temperature cycle simulates natural temperature differences and stimulates respiratory metabolism; CO2 enrichment improves carbon assimilation efficiency; and the humidity gradient decline induces the accumulation of osmoprotective substances. Multi-dimensional environmental regulation significantly stimulates the physiological potential of seeds, and the germination speed and seedling rate are simultaneously improved.

[0017] Preferably, for seeds that have not germinated after 7 days of culture in step 5, step 6 is implemented, which includes: cyclically treating the seeds in an independent container at 5°C for 8 hours, 15°C for 8 hours, and 25°C for 8 hours per day for 5-10 days, then immersing the seeds in a 5-15 μM chresin solution for 6 hours, and finally mixing 1-3 mg / L nano-zinc oxide particles into the seeds after 24 hours of dark culture at 25°C, and then re-germinating the treated seeds in the environment of step 5.

[0018] The temperature cycle of this embodiment stimulates membrane permeability remodeling through cold and heat alternation; chresin targets ABA signal transduction; and nano-zinc oxide provides zinc ions to enhance antioxidant enzyme activity. The three work synergistically to break through the physiological inhibition of deep-dormant seeds, achieve near-complete germination rate, and maintain a high seedling level.

[0019] Preferably, after the water flushing in step 3 and before the biosorption treatment, the water-flushed seeds are immersed in a sodium sulfite solution with a concentration of 0.05%-0.3% (w / v) for 10-30 minutes at 20-30℃, and then the seeds are washed with clean water for 1-3 times to remove the residual sulfite before the subsequent biosorption treatment.

[0020] The present embodiment uses sodium sulfite pretreatment to direct the reduction of quinone toxins and block the oxidative damage chain reaction; short-time treatment and secondary flushing avoid the toxic harm of residual sulfite. The insertion of this step before chemical degradation further reduces the incidence of abnormal seedlings and improves the development quality of cotyledons and radicles.

[0021] The beneficial effects of the present application are as follows: 1. The present application solves the germination barrier of Centella asiatica seeds by a four-step synergistic scheme of physical treatment, stratification optimization, inhibitor removal, and hormone activation. Mechanical peeling combined with ultrasonic and non-ionic surfactant treatment forms uniform micro-cracks in the seed coat; high-pressure penetration forcibly improves water / gas permeability, clearing physical barriers for subsequent metabolism. Warm stratification promotes embryo maturation, and cold stratification combined with periodic KNO3 spraying activates the NO-GA signaling pathway, simultaneously relieving physiological dormancy. Thiourea / H2O2 chemical degradation of phenolic acids and flavonoids, and Bacillus subtilis-activated carbon adsorbent efficient enrichment of residual toxins significantly reduce the risk of abnormal seedlings. The combination of brassinolide, kinetin, and fluridone hormones enhances absorption through gradient drying, synergistically promoting cell division, radicle elongation, and ABA signal blocking. In the basic culture conditions of Example 1, the germination rate is significantly improved, the germination start time is greatly shortened, and the seedling health is significantly better than that of the conventional method, laying a technical foundation for large-scale seedling cultivation.

[0022] 2. The present application further stimulates the physiological potential of seeds by multi-dimensional environmental synergistic regulation, achieving a leap in germination efficiency and uniformity. Among them, daily supplementation of 660 nm red light activates the PhyB pathway, enhancing GA synthesis efficiency. Day / night temperature cycling simulates natural temperature differences, significantly improving respiratory metabolism intensity and energy conversion efficiency. The substrate humidity is gradually reduced from 80% to 60%, gradually inducing the accumulation of osmotic protectants, and simultaneously avoiding root tip hypoxia. Compared with Example 1, the germination rate is further improved, the germination uniformity is significantly improved, the seedling rate is improved, and the abnormal seedling rate is further reduced, proving that synergistic regulation of light, temperature, gas, and humidity can maximize the release of seed physiological potential.

[0023] 3. The present application uses stepwise stress activation to break through the deep dormancy limit for ungerminated stubborn seeds. 5℃ / 15℃ / 25℃ daily cycling, through cold and hot alternating stimulation of cell membrane fluidity reconstruction, breaking the dormancy steady state. Fungichromin (FC) treatment directly activates H +-ATPase enzymes, which relieve the inhibition of germination by ABA. The incorporation of nano-ZnO, which continuously releases zinc ions, enhances the activity of SOD / POD enzymes, resisting oxidative damage during the recovery process. In Example 3, the germination rate of the seeds treated twice approaches 100%, and the seedling vigor is maintained at a high level. This scheme significantly expands the germinable seed bank, providing key technical support for resource-efficient utilization.

[0024] Other advantages, objects, and features of the application will be apparent from the following specification and appended claims. DETAILED DESCRIPTION

[0025] The application will be further described in the following examples, which enable any person skilled in the art to practice the application.

[0026] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained commercially unless otherwise specified.

[0027] It should be understood that the terms such as "have", "contain", and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0028] Example 1 A method for promoting the germination of Centella asiatica seeds, comprising: 1. Seed pretreatment Healthy and plump Centella asiatica seeds are selected, and the seed coat is rubbed with 600-mesh sandpaper until the surface is uniformly abraded. The broken seed coat is immersed in a 0.2% (w / v) Tween-20 solution at 25°C for 20 minutes. In the soaking solution, the seeds are treated with 40 kHz ultrasonic waves for 10 minutes at a temperature of 25°C. The seeds are transferred to a high-pressure container along with the solution, and treated at 80 MPa and 20°C for 10 minutes. After slow pressure release, the seeds are rinsed with sterile water.

[0029] 2. Stratification treatment Prepare wet sand with a sand-water mass ratio of 3:1, sterilize at a temperature above 100°C, and cool to room temperature. Mix the treated seeds with the wet sand and place them in a warm stratification environment at 20±1°C for 30 days. After warm stratification, transfer the seed and sand mixture to a cold stratification environment at 4±1°C for 60 days. During cold stratification, turn the mixture every 5 days and evenly spray 0.2% (w / v) KNO3 solution equivalent to 10% of the weight of the wet sand.

[0030] 3. Chemical degradation, sulfite treatment, and biosorption After cold stratification, the seeds were washed with running water at 25℃ for 48 hours. The washed seeds were immersed in 0.1% (w / v) sodium sulfite (Na2SO3) solution for 20 minutes at 25℃. The seeds were washed thoroughly with clean water for 3 times to remove the residual sulfite. The seeds were immersed in 0.1% (w / v) thiourea solution for 6 hours at room temperature. After draining, the seeds were transferred to 5% (w / v) H2O2 solution for 30 minutes. After treatment, the seeds were washed thoroughly with sterile water. The washed seeds were mixed with biosorbent at a mass ratio of 1:50, and gently stirred for 3 hours. The excess biosorbent was removed. The biosorbent was Bacillus subtilis with a bacterial load of ≥1.5×10 8 CFU / g loaded on activated carbon with a particle size of ≤300 μm.

[0031] 4. Hormone solution soaking and gradient drying The seeds were immersed in a mixed solution containing 0.1 mg / L brassinolide (BR), 0.5 mg / L kinetin (KT), and 10 μM fluridone (FL) for 12 hours at room temperature. The solution was drained, and the seeds were spread for gradient drying: 30℃ for 24 hours, 35℃ for 24 hours, and 40℃ until the seed moisture content was ≤8%.

[0032] 5. Germination culture The dried seeds were evenly sown in moist sterilized seedling substrate with an initial substrate humidity of about 60-70%. The sowing container was placed in an artificial climate chamber, and the conventional light and temperature conditions were set as follows: 12 hours of white light (light intensity 50 μmol·m -2 ·s -1 ) / 12 hours of darkness, day temperature 25℃ / night temperature 18℃, maintaining an ambient CO2 concentration of 0.04%, and the substrate humidity was maintained at an appropriate level of about 60-70% according to seedling experience, without red light supplementation, CO2 concentration regulation, and humidity gradient change. The seed germination conditions, including the radicle breaking through the seed coat, were observed and recorded regularly.

[0033] Example 2 A method for promoting the germination of Centella asiatica seeds, which is different from Example 1 in that the germination culture stage adopts an optimized scheme.

[0034] The seed pretreatment was the same as in Example 1. The stratification treatment was the same as in Example 1. The chemical degradation, sulfite treatment, and biosorption were the same as in Example 1. The hormone solution soaking and gradient drying were the same as in Example 1.

[0035] The difference is that the germination culture is as follows: the dried seeds are evenly sown in moist sterilized seedling substrate with an initial substrate humidity of 80%. The sowing container is placed in an artificial climate chamber that can be accurately controlled, and the light period and supplemental light are set as follows: 12 hours of white light (light intensity 50 μmol·m-2 ·s -1 ) / 12 hours of darkness. During the white light period, supplemental irradiation with 660 nm red light for 4 hours daily, with a light intensity of 50 μmol·m -2 ·s -1 The temperature cycle was set to 25°C during the day (light period) and 15°C during the night (dark period). The CO2 concentration was adjusted to maintain a level of 5% within the culture vessel by injecting CO2.

[0036] Furthermore, humidity is controlled using a gradient, with substrate humidity decreasing by 5% every 48 hours. Specifically, it starts at 80%, decreases to 75% after 48 hours, then to 70% after another 48 hours, then to 65% after yet another 48 hours, and finally to 60%, with the ultimate target humidity range of 55%-65%, which is maintained until germination is complete. Humidity is precisely controlled through weighing for water replenishment or drying. Seed germination is observed and recorded regularly.

[0037] Example 3 A method for promoting the germination of Centella asiatica seeds, based on Example 2, further employs a secondary treatment of ungerminated, stubborn seeds, including... Seed pretreatment was the same as in Example 1. Stratification was the same as in Example 1. Chemical degradation, sulfite treatment, and biosorption were the same as in Example 1. Hormone solution soaking and gradient drying were the same as in Example 1. Germination culture was the same as in Example 2.

[0038] The difference lies in the secondary treatment of stubborn seeds. After 7 days of germination culture in step 5, seeds that still have not germinated are screened out. These ungerminated seeds are placed in individual containers. A temperature cycling treatment is implemented, with daily cycles set as follows: 5℃ for 8 hours, 15℃ for 8 hours, and 25℃ for 8 hours. This cycle is continued for 7 days. After the temperature cycling is completed, the seeds are immersed in a 10 μM closporin (FC) solution for 6 hours. After soaking and draining, the seeds are cultured in a dark environment at 25℃ for 24 hours. After the dark culture is completed, the seeds are uniformly mixed with 2 mg / L nano zinc oxide (ZnO) particles with a particle size of 1-100 nm. Finally, the seeds that have undergone the second treatment are re-sown in a sterilized and moist substrate and placed back in the environmental conditions of step 5 for a second germination culture. The germination status of this batch of seeds is continuously observed and recorded.

[0039] Comparative Example 1 (DL1) Conventional methods using basic layering processing include: During stratification, seeds were directly mixed with moist sand at a sand-to-water ratio of 3:1. Warm stratification was performed at 20°C for 30 days, and cold stratification at 4°C for 60 days, without KNO3 spraying.

[0040] During germination culture, seeds were sowed on the substrate with 60% humidity, 25℃ constant temperature, 12h light / 12h dark, light intensity of 50μmol·m -2 ·s -1 Without red light supplement, CO2 regulation, and humidity gradient change.

[0041] Comparative Example 1 differs from Example 1 in that no pretreatment, chemical degradation and biosorption, hormone treatment and optimized culture were performed.

[0042] Comparative Example 2 (DL2) No pretreatment, stratification treatment was the same as Example 1. Chemical degradation and biosorption were the same as Example 1. Hormone solution soaking and gradient drying were the same as Example 1. Germination culture was the same as Example 1.

[0043] Comparative Example 2 differs from Example 1 in that step 1 is skipped, no sandpaper skinning, no ultrasonic, and no high-pressure infiltration.

[0044] Comparative Example 3 (DL3) No cold stratification and KNO3 treatment, including: Pretreatment: the same as Example 1.

[0045] Stratification treatment was only 20℃ warm stratification for 30 days, without cold stratification and KNO3 spraying.

[0046] Subsequent chemical degradation, hormone, drying, and regular germination steps were the same as Example 1.

[0047] Comparative Example 3 differs from Example 1 in that step 2 is only warm stratification, without cold stratification and nitrate stimulation.

[0048] Comparative Example 4 (DL4) No chemical degradation and biosorption, including: Pretreatment and stratification were the same as Example 1.

[0049] Step 3 was skipped, and the seeds were only rinsed with water for 48h after stratification, and directly entered the hormone soaking. No sodium sulfite, thiourea, H2O2, and biosorption.

[0050] Hormone solution soaking and gradient drying and germination culture were the same as Example 1.

[0051] Comparative Example 4 differs from Example 1 in that step 3 is completely missing, without inhibitor removal.

[0052] Comparative Example 5 (DL5) No hormone treatment and gradient drying, including: Pretreatment, stratification treatment, and chemical degradation, sulfite treatment, and biosorption were all the same as Example 1.

[0053] Skip step 4, directly air dry to 8% moisture content after bio-adsorption, and soak in hormone-free solution.

[0054] Germination culture is the same as Example 1.

[0055] Comparative Example 5 differs from Example 1 in that it lacks hormone combination solution soaking and BR / KT / FL treatment.

[0056] Effect comparison and analysis Table 1: Comparison of germination data of examples and comparative examples Wherein, T 50 is the time required for 50% seed germination, used to measure germination uniformity. The seedling rate is the proportion of seedlings that survive and have normal root systems 7 days after germination. The final germination rate of recalcitrant seeds with secondary treatment in Example 3 data.

[0057] As can be seen from Table 1, the baseline control (DL1) uses conventional stratification treatment without pre-treatment, chemical degradation, and hormone regulation, with a germination rate of only 25.3% and a germination start time of 18.2 days, indicating that the seed coat of the Radix Isatidis seed has serious seed coat barriers and inhibitor accumulation, hormone imbalance, and is in a dormant state in both physical and physiological terms.

[0058] Example 1 uses a synergistic treatment of physical seed coat breaking, stratification optimization, inhibitor removal, and hormone regulation, with a germination rate of 75.2% and a germination start time of 8.5 days, which is 197% higher and 113% faster than DL1. This indicates that breaking deep dormancy requires full intervention of physical damage, low temperature signaling, inhibitor degradation, and hormone balance.

[0059] DL2 lacks physical pre-treatment, but retains subsequent chemical and hormone treatment. However, due to the lack of improvement in seed coat permeability, the germination rate of 52.7% is significantly lower than that of Example 1. Mechanical seed coat breaking such as sandpaper rubbing directly weakens the mechanical resistance of the seed coat, and ultrasonic waves and non-ionic surfactants synergistically promote the formation of micro-cracks, and high pressure infiltration of about 80 MPa forcibly improves the permeability of water and gas. The lack of one of the three leads to insufficient water / gas exchange during stratification, and physiological dormancy is not completely resolved. Therefore, physical pre-treatment is the key to breaking through the seed coat barrier.

[0060] DL3 lacks cold stratification and KNO3, and only warm stratification is performed, with a germination rate of 41.6% and a T 50The extension is up to 21.7 days. 2-6℃ cold stratification is the core to break physiological dormancy, and low temperature accumulation breaks the dormancy state of embryos; periodic spraying of 0.2% KNO3 provides NO signal molecules, activates the GA synthesis pathway and inhibits the accumulation of ABA. Compared with the germination rate of 75.2% in Comparative Example 1 after cold stratification, the germination rate of DL3 is 41.6%, which can significantly accelerate the germination process. Therefore, the cold stratification and nitrate treatment steps cannot be replaced.

[0061] DL4 lacks chemical degradation and adsorption, and the germination rate of 48.2% is lower than that of 75.2% in Example 1, and the rate of abnormal seedlings is as high as 15.7%, which is significantly higher than that of 4.2% in Example 1. Thiourea and H2O2 degrade phenolic acids and flavonoids through redox reactions; sodium sulfite further neutralizes quinone toxins; and bacillus subtilis and activated carbon composite adsorbents efficiently enrich residual inhibitors. The lack of this step leads to the accumulation of toxic substances, inhibiting radicle elongation and inducing cotyledon malformation. Therefore, the inhibitor removal step can ensure the health of seedlings. The high rate of abnormal seedlings in DL4 may be due to the residual inhibitors causing root tip browning and cotyledon malformation.

[0062] DL5 lacks hormone treatment, and the germination rate of 59.4% is 21% lower than that of 75.2% in Example 1. 0.1mg / L brassinolide (BR) promotes cell wall relaxation and radicle elongation; 0.5mg / L kinetin (KT) stimulates cell division; and 10μM fluridone (FL) blocks ABA signal transduction. The synergistic effect of the three through gradient drying enhances absorption, significantly activating seed metabolic networks. Therefore, the hormone combination step can precisely regulate germination metabolism.

[0063] Example 2 is further implemented based on Example 1. Red light supplementation of 660nm, 4h / d can activate phytochrome PhyB, promote GA synthesis; diurnal temperature cycling of 25℃ / 15℃ simulates natural environmental temperature differences, enhancing respiratory metabolic efficiency; 5% CO2 enrichment enhances carbon assimilation capacity of the Calvin cycle; and a humidity gradient of 80% to 60% gradually induces the accumulation of osmotic protectants. This comprehensive environmental regulation significantly improves the germination rate from 75.2% in Example 1 to 92.6% in Example 2, T 50 The germination rate is reduced from 10 days to 7 days, and the seedling rate is improved to 90.3%. The synergistic optimization of surface light, temperature, gas, and humidity can significantly stimulate the physiological potential of seeds.

[0064] Example 3 targets the stubborn seeds that have not germinated after 7 days in Example 2, and implements a temperature cycle of 5℃ / 15℃ / 25℃ to stimulate membrane permeability through cold and hot alternation; and a concentration of about 10μM of chalinophyllogen to directly activate H +- ATPase enzyme to remove ABA inhibition; nano zinc oxide provides zinc ions to enhance antioxidant enzyme activity. The final total germination rate reached 98.1%, and the seedling rate maintained 88.7%. The slight decrease in seedling rate may be due to the loss of vigor of deep-dormant seeds after stress, but it also proves that the scheme of this embodiment can efficiently activate the deep-dormant seed reserve.

[0065] While embodiments of the application have been disclosed in connection with the above description and the drawings, it should be understood that there is no intent to limit the application to the specific form set forth, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the application.

Claims

1. A method for promoting the germination of Centella asiatica seeds, characterized in that, include: Step 1: The seeds are subjected to mechanical cracking, ultrasonic treatment, and high-pressure permeation treatment in sequence; Step 2: Mix the seeds treated in Step 1 with the moist substrate, perform warm stratification followed by cold stratification, and periodically apply a nitrate-containing solution during the cold stratification stage; the warm stratification temperature is 18-25℃, the duration is 25-35 days, and the cold stratification temperature is 2-6℃, the duration is 50-70 days; the nitrate solution is a 0.1%-0.3% KNO3 solution, sprayed every 3-7 days; Step 3: After stratification, the seeds are rinsed with running water and then subjected to chemical degradation treatment with thiourea solution and hydrogen peroxide solution, followed by biosorption treatment. The chemical degradation treatment involves soaking the seeds in a 0.05%-0.2% thiourea solution for 4-8 hours, followed by treatment with a 3%-7% H2O2 solution for 20-40 minutes. The biosorption treatment uses a bacterial count ≥1×10⁻⁶. 8 A mixture of Bacillus subtilis and activated carbon at CFU / g was used as an adsorbent; Step 4: Soak the seeds in a mixed solution containing 0.05-0.2 mg / L brassinolide, 0.3-0.8 mg / L kinetin, and 5-20 μM fluazinam, then dry them by gradient heating, with the final temperature of the gradient heating not exceeding 50℃, and the final moisture content of the seeds ≤10%. Step 5: Germination culture.

2. The method according to claim 1, characterized in that, In step 1, mechanically breaking the seed coat involves rubbing it with 400-800 grit sandpaper until the surface is worn. Then, the seed coat is immersed in a 0.1%-0.3% nonionic surfactant solution, Tween-20 or Triton X-100, and maintained at 25°C for 10-30 minutes. After that, the seed coat is subjected to ultrasonic treatment in the solution at a frequency of 30-50 kHz for 5-15 minutes. The ultrasonically treated seed coat, along with the liquid medium, is then placed in a high-pressure container for high-pressure permeation treatment at a pressure of 50-110 MPa and a temperature of 15-25°C for 5-15 minutes.

3. The method according to claim 2, characterized in that, In step 2, the moist substrate is wet sand with a sand-to-water ratio of 3:

1. During warm stratification, the treated seeds are mixed with wet sand and stratified at 20±1℃ for 30 days. Then, the mixture is transferred to a cold stratification environment at 4±1℃ for 60 days. The mixture is turned over and sprayed with 0.2% KNO3 solution every 5 days, with the spray volume being 10% of the sand weight.

4. The method according to claim 3, characterized in that, Step 3: The activated carbon particle size of the bio-adsorption treatment is ≤300 mesh, and the mass ratio of seeds to adsorbent is 1:40-1:60; the water rinsing temperature is 20-28℃, and the time is 36-60 hours.

5. The method according to claim 4, characterized in that, The mixed solution in step 4 contains 0.1 mg / L brassinolide, 0.5 mg / L kinetin, and 10 μM fluazinon. The soaking time is 12 hours, followed by a gradient drying process of 30°C for 24 hours, 35°C for 24 hours, and 40°C for 24 hours or more until the seed moisture content is ≤8%.

6. The method according to claim 1 or 5, characterized in that, During the germination culture process in step 5, under a 12-hour white light / 12-hour dark cycle, supplemental irradiation with 640-680 nm red light for 3-5 hours daily, with a light intensity of 40-60 μmol·m⁻¹, is provided. -2 ·s -1 Simultaneously, the temperature is cyclically adjusted at 23-27℃ during the day and 13-18℃ at night to provide temperature stimulation; the CO2 concentration in the culture container is maintained at 3%-7%; and the humidity of the sowing substrate in the culture container is adjusted from 75%-85% to 3%-7% every 24-72 hours until it drops to 55%-65%.

7. The method according to claim 6, characterized in that, For seeds that have not germinated after 7 days of culture in step 5, step 6 is performed, which includes: treating the seeds in an independent container at 5℃ for 8 hours, 15℃ for 8 hours, and 25℃ for 8 hours daily for 5-10 days, then immersing them in a 5-15 μM closporin solution for 6 hours, then dark culturing at 25℃ for 24 hours, and finally mixing in 1-3 mg / L nano zinc oxide particles. Finally, the treated seeds are placed back into the environmental conditions of step 5 for a second germination culture.

8. The method according to claim 7, characterized in that, After rinsing with running water in step 3 and before biosorption treatment, immerse the rinsed seeds in a sodium sulfite solution with a concentration of 0.05%-0.3% (w / v) at 20-30℃ for 10-30 minutes. After treatment, rinse the seeds with clean water 1-3 times to remove residual sulfite before proceeding with subsequent biosorption treatment.

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