A method for breaking the dormancy of cyperus iria seeds
By combining physical scoring, ultrasonic-assisted soaking, and low-temperature variable-temperature stratification, and treating centella asiatica seeds with low concentrations of gibberellin and naphthaleneacetic acid, the problem of deep dormancy in centella asiatica seeds was solved, resulting in efficient germination and improved seedling survival rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEXI UNIV
- Filing Date
- 2026-07-02
- Publication Date
- 2026-07-31
AI Technical Summary
Existing technologies are insufficient to efficiently break the deep dormancy of Centella asiatica seeds, resulting in low germination rates and long germination cycles. Furthermore, existing methods are prone to operational risks and have low seedling survival rates.
The method combines physical scoring, ultrasonic-assisted soaking, and low-temperature variable-temperature stratification. The seeds are treated with a combination of low-concentration gibberellin and naphthaleneacetic acid, including ethanol disinfection, sanding, ultrasonic treatment, low-temperature variable-temperature stratification, and germination treatment.
It significantly improves the germination rate and seedling survival rate of Centella asiatica seeds, with a germination rate of over 90%. The seedlings grow vigorously and are suitable for both new and old seeds, thus shortening the treatment cycle.
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Figure CN122477818A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant seed treatment technology, and in particular to a method for breaking the dormancy of Centella asiatica seeds. Background Technology
[0002] Centella asiatica, a perennial semi-parasitic herb belonging to the genus Centella of the Santalaceae family, is used medicinally as a whole plant. It possesses properties such as clearing heat and detoxifying, tonifying the kidneys and astringing essence, and is a commonly used traditional Chinese medicine for treating acute mastitis, pneumonia, tonsillitis, and upper respiratory tract infections. Modern pharmacological research shows that Centella asiatica contains various active ingredients such as centellarin, flavonoids, and alkaloids, exhibiting significant antibacterial, antiviral, anti-inflammatory, and immunomodulatory effects, earning it the reputation of "plant antibiotic." In recent years, with the continuous development of its medicinal value and the sustained growth in market demand, wild Centella asiatica resources have been over-harvested, and its habitat has been destroyed, leading to the near depletion of wild resources. Artificial cultivation has become an inevitable way to solve the shortage of Centella asiatica resources. However, Centella asiatica seeds have a deep dormancy characteristic, with a germination rate of less than 10% under natural conditions, and a germination cycle of 6-8 months, resulting in extremely uneven germination, which severely restricts the process of large-scale artificial cultivation of Centella asiatica.
[0003] Currently, existing technologies for breaking the dormancy of *Gnaphalium affine* seeds mainly include low-temperature stratification, gibberellin treatment, and concentrated sulfuric acid etching. Low-temperature stratification is the most commonly used method, but it typically takes 3-4 months to break dormancy, and the germination rate is only 30%-40%, resulting in a long treatment cycle and low efficiency. While gibberellin treatment can improve the germination rate to some extent, using high concentrations of gibberellin alone can easily lead to excessive seedling growth, poor root development, and low transplant survival rates. Concentrated sulfuric acid etching, although it can quickly destroy the seed coat, is highly dangerous to operate, easily damages seeds, and has an unstable germination rate. Furthermore, there are few reports of combining multiple methods synergistically to efficiently break the dormancy of *Gnaphalium affine* seeds, which cannot meet the needs of industrial production and therefore urgently requires improvement. Summary of the Invention
[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for breaking the dormancy of Centella asiatica seeds, thereby solving the above-mentioned technical problems.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for breaking the dormancy of Centella asiatica seeds, comprising: Seed selection and disinfection: Select plump, disease-free seeds of Centella asiatica. After rinsing with clean water to remove impurities, disinfect them in sequence with 75% ethanol solution for 30-60 seconds and 0.1% mercuric chloride solution for 8-12 minutes. Rinse with sterile water and drain. Physical scratch treatment: Place the sterilized seeds in a sandpaper polisher and polish them at 150-200 r / min for 3-8 minutes until fine scratches appear on the seed coat surface without damaging the endosperm. Ultrasonic-assisted seed soaking involves immersing the polished seeds in a mixed soaking solution containing 50-200 mg / L gibberellin and 10-50 mg / L naphthaleneacetic acid, ultrasonically treating them for 15-45 minutes at 150-250 W and 40 kHz, and then soaking them at a constant temperature of 25-30℃ for 12-24 hours. Low-temperature variable-temperature stratification treatment involves mixing soaked seeds with sterilized river sand at a volume ratio of 1:3-5, controlling the moisture content of the sterilized river sand at 60%-70%, and placing the mixture in a stratification box for variable-temperature stratification. First, stratify at 5-10℃ for 10-20 days, then at 15-20℃ for 5-10 days, alternating the cycle for a total stratification time of 30-60 days. During this period, the mixture is turned over and water is added every 5-7 days. For germination treatment, remove the stratified seeds, rinse them with clean water, and place them on moist filter paper. Germinate them until they show white sprouts under the conditions of 22-28℃, relative humidity 80%-90%, light intensity 1000-2000lx, and light exposure 12 hours / day.
[0006] Preferably, the sandpaper of the sander has a grit of 180-240, a sanding speed of 180 r / min, and a sanding time of 5 minutes.
[0007] Preferably, the ultrasonic treatment parameters are 200W power, 40kHz frequency, and 30 minutes processing time, and the constant temperature soaking temperature is 28℃ and the soaking time is 18 hours.
[0008] Preferably, the concentration of gibberellin in the mixed soaking solution is 100 mg / L and the concentration of naphthaleneacetic acid is 30 mg / L.
[0009] Preferably, the volume ratio of soaked seeds to sterilized river sand is 1:4, and the water content of the river sand is 65%.
[0010] Preferably, the temperature-variable lamination program is 15 days of lamination at 8°C, 7 days of lamination at 18°C, alternating twice, for a total lamination time of 44 days.
[0011] Preferably, the disinfection method for the river sand is to rinse the river sand with clean water, then sterilize it with high-pressure steam at 121°C for 30 minutes, and then cool it to room temperature for later use.
[0012] Preferably, the germination conditions in the germination treatment are a temperature of 25°C, a relative humidity of 85%, a light intensity of 1500 lx, and a light duration of 12 hours / day.
[0013] Preferably, the seeds of Centella asiatica are newly harvested seeds from the current year or old seeds stored for 1-2 years.
[0014] Preferably, the volume fraction of the ethanol solution is 75%, the disinfection time is 45 seconds, the mass fraction of the mercuric chloride solution is 0.1%, and the disinfection time is 10 minutes.
[0015] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: This invention effectively breaks the deep dormancy of *Centella asiatica* seeds through a synergistic treatment combining physical scoring, ultrasonic-assisted soaking, and low-temperature variable-temperature stratification. The use of a combination of low-concentration gibberellin and naphthaleneacetic acid (NAA) for soaking overcomes the technical defects of single high-concentration gibberellin treatment, which leads to excessive seedling growth and poor root development. Gibberellin breaks physiological dormancy and promotes seed germination, while NAA regulates seedling growth and promotes root development. Their synergistic effect results in well-developed and robust seedlings with a transplant survival rate exceeding 90%. Furthermore, this method is not only applicable to newly harvested *Centella asiatica* seeds but also has a good dormancy-breaking effect on *Centella asiatica* seeds stored for 1-2 years, solving the technical problems of low germination rates and difficulty in utilizing old seeds, thus improving seed utilization. Attached Figure Description
[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 A flowchart of a method for breaking the dormancy of centella asiatica seeds is shown. Detailed Implementation
[0018] Reference Figure 1 The present invention provides a further description of an embodiment of a method for breaking the dormancy of centella asiatica seeds.
[0019] The seeds of Centella asiatica used in the examples and comparative examples were all collected from the standardized Centella asiatica planting base in Langya Mountain, Chuzhou City, Anhui Province. After being air-dried and cleaned, the thousand-seed weight was measured to be 2.35g and the initial moisture content was 10.2%.
[0020] Example 1: Select 1000 mature, plump, and disease-free *Centella asiatica* seeds from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with a 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with a 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds directly into petri dishes lined with two layers of moist filter paper without any dormancy breaking treatment. Place 100 seeds in each petri dish and set up three replicates. Germinate the seeds under the conditions of 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. Spray the filter paper with clean water once a day to keep it moist and record the germination status.
[0021] The experimental results of Example 1 are as follows: After 60 days of continuous observation, the seeds showed no signs of germination, and the germination rate was 0%. This proves that the seeds of this batch of Centella asiatica were in a deep dormancy state and had significant germination obstacles.
[0022] The experimental data table for Example 1 is as follows:
[0023] Example 2: Select 1000 mature, plump, and disease-free seeds of *Centella asiatica* from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at 200W and 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution contains 100 mg / L gibberellin (GA) and 30 mg / L naphthaleneacetic acid (NAA). A mixed aqueous solution of AA was prepared. The soaked seeds were mixed with sterilized river sand at a volume ratio of 1:4, and the moisture content of the river sand was maintained at 65%. The mixture was then placed in a stratification chamber for variable temperature stratification treatment. The variable temperature stratification treatment procedure was to first stratify at a low temperature of 8℃ for 15 days, and then at 18℃ for 7 days. This cycle was repeated twice, for a total stratification time of 44 days. During the stratification period, the seeds were turned over every 6 days, and water was added to maintain the moisture content of the river sand. After stratification, the seeds were taken out, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. 100 seeds were placed in each petri dish, and 3 replicates were set up. Germination was carried out under the conditions of temperature of 25℃, relative humidity of 85%, light intensity of 1500 lx, and light time of 12 hours / day. The filter paper was sprayed with clean water once a day to keep it moist, and the germination status of the seeds was recorded.
[0024] The experimental results of Example 2 are as follows: Germination began on the 10th day after germination, reached its peak on the 15th day, and basically ended on the 25th day, with a germination rate of 89.2% and a germination vigor of 76.5%. The seedlings grew vigorously, with an average plant height of 4.2 cm, an average root length of 3.8 cm, and a transplant survival rate of 92.3%. The experimental data table for Example 2 is as follows:
[0025] Among them, the germination rate (%) is the percentage of the cumulative number of germinated seeds at the end of the germination period out of the total number of seeds tested; The day with the highest number of germinations is defined as the germination peak period, and the germination potential (%) is the percentage of the cumulative number of germinated seeds up to that day out of the total number of seeds tested. After germination, the natural plant height (from the base of the root collar to the tip of the growing point) and the taproot length (from the base of the root collar to the root tip) of all normal seedlings in each replication were measured with vernier calipers, and the average values were calculated as the average plant height and average root length. One hundred seedlings with uniform growth were randomly selected and transplanted into nutrient soil. After being cultivated under suitable conditions for 30 days, the number of surviving seedlings was counted, and the survival rate was calculated as the percentage of surviving seedlings to the total number of transplanted seedlings.
[0026] This embodiment is the best embodiment of the present invention, verifying the effectiveness of the complete technical solution described in the present invention. By physically scratching to break the seed coat barrier, using ultrasonic-assisted soaking to promote the absorption of plant growth regulators, and using low-temperature variable-temperature stratification to simulate natural environmental conditions to break the physiological dormancy of the seeds, the three work synergistically to achieve efficient breaking of the dormancy of Centella asiatica seeds, resulting in the highest germination rate and the best seedling quality.
[0027] Example 3: Select 1000 mature, plump, and disease-free *Centella asiatica* seeds from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with a 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with a 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at a power of 200W and a frequency of 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution is a mixed aqueous solution containing 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid. Mix the soaked seeds with disinfected river sand at a volume ratio of 1:4. The moisture content of the sand was maintained at 65%, and then the sand was placed in a stratification chamber for variable-temperature stratification treatment. The procedure for variable-temperature stratification treatment was to first stratify at a low temperature of 8℃ for 15 days, and then at 18℃ for 7 days, and then repeat this cycle twice, for a total stratification time of 44 days. During the stratification period, the seeds were turned over every 6 days, and water was added to maintain the moisture content of the sand. After stratification, the seeds were taken out, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. 100 seeds were placed in each petri dish, with 3 replicates. Germination was carried out under the conditions of temperature of 25℃, relative humidity of 85%, light intensity of 1500 lx, and light duration of 12 hours / day. The filter paper was sprayed with clean water once a day to keep it moist, and the germination status of the seeds was recorded.
[0028] The experimental results of Example 3 are as follows: germination began on the 15th day after germination, reached its peak on the 22nd day, and basically ended on the 35th day, with a germination rate of 52.7% and a germination vigor of 38.4%; the average seedling height was 4.0cm, the average root length was 3.6cm, and the transplant survival rate was 89.7%.
[0029] The experimental data table for Example 3 is as follows:
[0030] This example serves as a control, verifying the necessity of physical scoring treatment. The hard, dense seed coat of *Centella asiatica* seeds presents mechanical barriers, hindering the entry of water and oxygen and limiting embryo growth. Without physical scoring treatment, seed germination rate and germination potential were significantly reduced, and the germination period was prolonged, indicating that physical scoring is one of the key steps in breaking the dormancy of *Centella asiatica* seeds.
[0031] Example 4: Select 1000 mature, plump, and disease-free seeds of *Centella asiatica* from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution (a mixed aqueous solution containing 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid) at a constant temperature of 28°C for 18 hours. After soaking, place the seeds in a container with disinfected water... The sand was mixed evenly at a volume ratio of 1:4, with the moisture content of the river sand maintained at 65%. The mixture was then placed in a stratification chamber for variable-temperature stratification. The procedure involved stratifying at 8°C for 15 days, followed by stratification at 18°C for 7 days, repeating this cycle twice for a total stratification time of 44 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the moisture content of the river sand. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. Each petri dish contained 100 seeds, with three replicates. Germination was carried out at 25°C, 85% relative humidity, 1500 lx light intensity, and 12 hours of light per day. The filter paper was sprayed with clean water daily to keep it moist, and the germination status was recorded.
[0032] The experimental results of Example 4 are as follows: germination began on the 12th day after germination, reached its peak on the 18th day, and basically ended on the 30th day, with a germination rate of 68.5% and a germination potential of 51.2%; the average seedling height was 4.1cm, the average root length was 3.7cm, and the transplant survival rate was 90.5%.
[0033] The experimental data table for Example 4 is as follows:
[0034] This embodiment verifies the effect of ultrasonic-assisted seed soaking. Ultrasonic treatment can generate cavitation effect and mechanical vibration, which can further destroy the integrity of the seed coat and increase its permeability. On the other hand, it can promote the faster entry of plant growth regulator molecules into the seed, thus improving the treatment effect. Without ultrasonic treatment, the seed germination rate and germination potential decreased, indicating that ultrasonic-assisted seed soaking can significantly improve the efficiency of dormancy breaking.
[0035] Example 5: Select 1000 mature, plump, and disease-free *Centella asiatica* seeds from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at 200W and 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution is only water and does not contain any plant growth regulators. After soaking, the seeds were mixed evenly with sterilized river sand at a volume ratio of 1:4, maintaining the moisture content of the river sand at 65%. The mixture was then placed in a stratification chamber for variable-temperature stratification treatment. The procedure for variable-temperature stratification treatment was to first stratify at a low temperature of 8℃ for 15 days, and then at 18℃ for 7 days, alternating this cycle twice, for a total stratification time of 44 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the moisture content of the river sand. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper, with 100 seeds in each petri dish and 3 replicates. Germination was carried out under the conditions of 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. The filter paper was sprayed with clean water once a day to keep it moist, and the germination status of the seeds was recorded.
[0036] The experimental results of Example 5 are as follows: germination began on the 18th day after germination, reached its peak on the 26th day, and basically ended on the 40th day, with a germination rate of 41.3% and a germination potential of 27.6%; the average seedling height was 3.5cm, the average root length was 3.2cm, and the transplant survival rate was 85.2%.
[0037] The experimental data table for Example 5 is as follows:
[0038] This embodiment verifies the effect of combined treatment with plant growth regulators. Gibberellin can induce the synthesis of hydrolytic enzymes in seeds, promote the decomposition of stored substances, and break the physiological dormancy of seeds. Naphthaleneacetic acid can promote root growth and improve the stress resistance of seedlings. Without the addition of plant growth regulators, the seed germination rate and germination potential are significantly reduced, and the seedling growth is also weak. This indicates that the combined treatment of gibberellin and naphthaleneacetic acid plays an important role in effectively breaking the dormancy of *Centella asiatica* seeds and improving seedling quality.
[0039] Example 6: Select 1000 mature, plump, and disease-free seeds of *Gnaphalium affine* from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at 200W and 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution contains 100 mg / L gibberellin. After sowing, the seeds were mixed evenly with sterilized river sand at a volume ratio of 1:4, maintaining the moisture content of the river sand at 65%. The mixture was then placed in a stratification chamber for variable-temperature stratification treatment. The procedure for variable-temperature stratification treatment was to first stratify at a low temperature of 8℃ for 15 days, and then at 18℃ for 7 days, alternating this cycle twice, for a total stratification time of 44 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the moisture content of the river sand. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper, with 100 seeds in each petri dish and 3 replicates. Germination was carried out under the conditions of 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. The filter paper was sprayed with clean water once a day to keep it moist, and the germination status of the seeds was recorded.
[0040] The experimental results of Example 6 are as follows: Germination began on the 11th day of germination, reached its peak on the 16th day, and basically ended on the 27th day, with a germination rate of 78.4% and a germination vigor of 65.3%; however, the seedlings showed obvious etiolation, with an average plant height of 6.8cm, an average root length of 2.5cm, and a transplant survival rate of 76.8%.
[0041] The experimental data table for Example 6 is as follows:
[0042] This embodiment verifies the synergistic effect of naphthaleneacetic acid (NAA). Although gibberellin treatment alone can improve seed germination rate, it can easily lead to excessive growth of the above-ground parts of seedlings, poor root development, and low transplant survival rate. Adding an appropriate amount of NAA can effectively inhibit excessive seedling growth and promote root growth, thereby improving seedling quality and transplant survival rate.
[0043] Example 7: Select 1000 mature, plump, and disease-free seeds of *Centella asiatica* from the current year. Rinse with clean water to remove shriveled seeds and impurities. Then disinfect with a 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with a 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at 200W and 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution contains... A mixed aqueous solution of 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid was prepared. The soaked seeds were mixed with sterilized river sand at a volume ratio of 1:4, maintaining the sand moisture content at 65%. The mixture was then placed in a stratification chamber for constant-temperature stratification treatment at 8℃ for 44 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the sand moisture content. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. 100 seeds were placed in each petri dish, with 3 replicates. Germination was carried out at 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. The filter paper was sprayed with clean water once daily to keep it moist, and the germination status was recorded.
[0044] The experimental results of Example 7 are as follows: germination began on the 14th day after germination, reached its peak on the 20th day, and basically ended on the 32nd day, with a germination rate of 62.1% and a germination vigor of 45.7%; the average seedling height was 4.0 cm, the average root length was 3.6 cm, and the transplant survival rate was 89.2%.
[0045] The experimental data table for Example 7 is as follows:
[0046] This embodiment verifies the superiority of low-temperature variable-temperature stratification treatment. The dormancy of Centella asiatica seeds is a comprehensive dormancy, which includes not only seed coat barriers but also physiological dormancy. Constant-temperature stratification treatment can only break physiological dormancy to a certain extent, while variable-temperature stratification treatment can simulate temperature changes under natural conditions, more effectively activate metabolic activities within the seeds, promote the decomposition of dormant substances (such as abscisic acid) and the synthesis of germination substances (such as gibberellins and cytokinins), thereby significantly improving the germination rate and germination potential of the seeds.
[0047] Example 8: Select 1000 mature, plump, and disease-free seeds of *Centella asiatica* from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water, and drain. Place the disinfected seeds in a sander equipped with 200-grit sandpaper and sand at 180 rpm for 5 minutes, until fine scratches appear on the seed coat surface without damaging the endosperm. Place the sanded seeds in a container containing a soaking solution and ultrasonically treat them for 30 minutes at 200W and 40kHz. Then, continue soaking at a constant temperature of 28℃ for 18 hours. The soaking solution is a mixture of water containing 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid. Solution: Soaked seeds were mixed with sterilized river sand at a volume ratio of 1:4, maintaining the sand moisture content at 65%. The mixture was then placed in a stratification chamber for variable-temperature stratification treatment. The procedure was as follows: stratification at 8℃ for 15 days, then at 18℃ for 7 days, and then at 8℃ for 8 days, for a total stratification time of 30 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the sand moisture content. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. Each petri dish contained 100 seeds, with 3 replicates. Germination was carried out at 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day light exposure. The filter paper was sprayed with clean water once a day to keep it moist, and the germination status was recorded.
[0048] The experimental results of Example 8 are as follows: germination began on the 13th day of germination, reached its peak on the 19th day, and basically ended on the 30th day, with a germination rate of 75.6% and a germination vigor of 60.3%; the average seedling height was 4.1 cm, the average root length was 3.7 cm, and the transplant survival rate was 90.1%.
[0049] The experimental data table for Example 8 is as follows:
[0050] This embodiment verifies the effect of stratification time on the treatment effect. If the stratification time is too short, the physiological dormancy of the seeds cannot be completely broken, resulting in a decrease in germination rate and germination potential. When the total stratification time is 44 days, the physiological dormancy of the seeds of Centella asiatica can be fully broken, and the best germination effect can be obtained.
[0051] Example 9: 1000 seeds of *Centella asiatica* stored for one year were selected, rinsed with clean water to remove shriveled seeds and impurities, then disinfected with 75% ethanol solution for 45 seconds, rinsed 4 times with sterile water, then disinfected with 0.1% mercuric chloride solution for 10 minutes, rinsed 6 times with sterile water, and drained. The disinfected seeds were then placed in a sander equipped with 200-grit sandpaper and sanded at 180 rpm for 5 minutes, until fine scratches appeared on the seed coat surface without damaging the endosperm. The sanded seeds were then placed in a container containing a soaking solution and ultrasonically treated for 30 minutes at 200W and 40kHz. Finally, they were soaked at a constant temperature of 28℃ for 18 hours. The soaking solution was a mixed aqueous solution containing 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid. The soaked seeds were mixed with sterilized river sand at a volume ratio of 1:4, with the sand moisture content maintained at 65%. The mixture was then placed in a stratification chamber for variable-temperature stratification. The procedure involved stratification at 8°C for 15 days, followed by stratification at 18°C for 7 days, repeated twice for a total stratification time of 44 days. During stratification, the seeds were turned over every 6 days, and water was added to maintain the sand moisture content. After stratification, the seeds were removed, rinsed with clean water, and placed in petri dishes lined with two layers of moist filter paper. Each petri dish contained 100 seeds, with three replicates. Germination was carried out at 25°C, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. The filter paper was sprayed with clean water daily to keep it moist, and the germination status was recorded.
[0052] The experimental results of Example 9 are as follows: germination began on the 11th day after germination, reached its peak on the 16th day, and basically ended on the 26th day, with a germination rate of 85.7% and a germination vigor of 72.4%; the average seedling height was 4.0 cm, the average root length was 3.7 cm, and the transplant survival rate was 91.5%.
[0053] The experimental data table for Example 9 is as follows:
[0054] This embodiment verifies the applicability of the method of the present invention to aged seeds. The seed coat permeability of centella asiatica seeds stored for 1 year has increased, but physiological dormancy still exists. After treatment with the method of the present invention, a high germination rate and good seedling quality can still be obtained, indicating that the method of the present invention has wide applicability. It is not only suitable for new seeds harvested in the current year, but also for aged seeds stored for 1-2 years.
[0055] Example 10: To verify the preferred range of the polishing parameters in this invention, the following polishing experimental group was set up, with all other steps being exactly the same as in Example 2: (1) Grinding grit sets: Grind with 180 grit, 200 grit and 240 grit sandpaper respectively, at a speed of 180 r / min and a time of 5 min.
[0056] The results showed that the seed damage rate in the 180-mesh group was 15%, the mold rate was relatively high, and the germination rate was 75.2%; the seed coat scratches in the 240-mesh group were not obvious, and the germination rate was 81.6%; the germination rate in the 200-mesh group was the highest, at 89.2%. Therefore, 200-mesh sandpaper is the best choice.
[0057] (2) Polishing time group: Polish for 2 min, 5 min and 8 min respectively under the conditions of 200 grit sandpaper and 180 r / min.
[0058] The results showed that the seed coat scratches in the 2-minute group were too shallow, with a germination rate of 80.5%; the 8-minute group showed 5% endosperm damage, with a germination rate of 83.1%, and an increase in deformed seedlings; the 5-minute group had the highest germination rate of 89.2%. Therefore, 5 minutes is the optimal polishing time.
[0059] (3) In the ultrasonic-assisted soaking step, Example 2 was the optimal group, and experiments were conducted with gibberellin concentration gradients (50 mg / L, 100 mg / L, 200 mg / L) and naphthaleneacetic acid concentration gradients (10 mg / L, 30 mg / L, 50 mg / L).
[0060] The results showed that when the gibberellin concentration was 100 mg / L and the naphthaleneacetic acid concentration was 30 mg / L, the seed germination rate of 89.2% and the overall seedling growth were the best. When the gibberellin concentration was 200 mg / L and the naphthaleneacetic acid concentration was 50 mg / L, the concentration was too high and had an inhibitory effect, reducing the germination rate to 70.3% and increasing the seedling deformity rate. When the gibberellin concentration was 50 mg / L and the naphthaleneacetic acid concentration was 10 mg / L, the concentration was too low and the ability to break dormancy was insufficient, reducing the germination rate to 65.4%.
[0061] (4) In the variable temperature layering step, three variable temperature programs were set: 8℃ / 18℃, 6℃ / 15℃, and 10℃ / 20℃.
[0062] The results showed that the 8℃ / 18℃ combination was the most effective in breaking dormancy, with a germination rate of 89.2%. The 6℃ / 15℃ combination had a germination rate of 89.2%, but the overall temperature was too low, resulting in incomplete dormancy breaking. The 0℃ / 20℃ combination had a germination rate of 76.5%, but the low-temperature stage was too high, indicating insufficient cold stimulation.
[0063] The experimental data table for Example 10 is as follows:
[0064] Comparative example: Select 1000 mature, plump, and disease-free *Centella asiatica* seeds from the current year. Rinse with clean water to remove shriveled seeds and impurities, then disinfect with 75% ethanol solution for 45 seconds, rinse four times with sterile water, and then disinfect with 0.1% mercuric chloride solution for 10 minutes, rinse six times with sterile water. Drain and set aside. Mix the disinfected seeds with disinfected river sand at a volume ratio of 1:4, maintaining the sand moisture content at 65%, and stratify in a 4℃ refrigerator for 90 days. Remove the stratified seeds, rinse them clean with clean water, and place 100 seeds in each petri dish lined with two layers of moist filter paper, with three replicates. Germinate the seeds under the conditions of 25℃, 85% relative humidity, 1500 lx light intensity, and 12 hours / day of light. Spray the filter paper with clean water once a day to keep it moist, and record the germination status.
[0065] The results of the comparative experiment are as follows: germination began on the 30th day after germination, reached its peak on the 50th day, and basically ended on the 80th day, with a germination rate of 36.8% and a germination potential of 21.5%; the average seedling height was 3.2cm, the average root length was 2.8cm, and the transplant survival rate was 78.6%.
[0066] The experimental data for the comparative example are shown in the table below:
[0067] This comparative example, as a reference to existing technologies, further demonstrates the superiority of the method of the present invention. Compared with the traditional low-temperature stratification method, the germination rate of the method of the present invention is increased by 52.4 percentage points, the germination potential is increased by 55 percentage points, the germination cycle is shortened by more than 50 days, and the seedling quality and transplant survival rate are also significantly improved.
[0068] The above description of the embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for breaking the dormancy of seeds of Juncus effusus, characterized in that, include: Seed selection and disinfection: Select plump, disease-free seeds of Centella asiatica. After rinsing with clean water to remove impurities, disinfect them in sequence with 75% ethanol solution for 30-60 seconds and 0.1% mercuric chloride solution for 8-12 minutes. Rinse with sterile water and drain. Physical scratch treatment: Place the sterilized seeds in a sandpaper polisher and polish them at 150-200 r / min for 3-8 minutes until fine scratches appear on the seed coat surface without damaging the endosperm. Ultrasonic-assisted seed soaking involves immersing the polished seeds in a mixed soaking solution containing 50-200 mg / L gibberellin and 10-50 mg / L naphthaleneacetic acid, ultrasonically treating them for 15-45 minutes at 150-250 W and 40 kHz, and then soaking them at a constant temperature of 25-30℃ for 12-24 hours. Low-temperature variable-temperature stratification treatment involves mixing soaked seeds with sterilized river sand at a volume ratio of 1:3-5, controlling the moisture content of the sterilized river sand at 60%-70%, and placing the mixture in a stratification box for variable-temperature stratification. First, stratify at 5-10℃ for 10-20 days, then at 15-20℃ for 5-10 days, alternating the cycle for a total stratification time of 30-60 days. During this period, the mixture is turned over and water is added every 5-7 days. For germination treatment, remove the stratified seeds, rinse them with clean water, and place them on moist filter paper. Germinate them until they show white sprouts under the conditions of 22-28℃, relative humidity 80%-90%, light intensity 1000-2000lx, and light exposure 12 hours / day.
2. The method for breaking the seed dormancy of Juncus setchuanensis according to claim 1, characterized in that, The sandpaper used in the sander has a grit of 180-240, a sanding speed of 180 r / min, and a sanding time of 5 minutes.
3. The method for breaking the seed dormancy of Juncus setchuanensis according to claim 2, characterized in that, The ultrasonic treatment parameters are 200W power, 40kHz frequency, and 30 minutes processing time. The constant temperature soaking temperature is 28℃ and the soaking time is 18 hours.
4. The method for breaking the seed dormancy of Juncus setchuanensis according to claim 3, characterized in that, The mixed soaking solution contains 100 mg / L gibberellin and 30 mg / L naphthaleneacetic acid.
5. A method for breaking the dormancy of Centella asiatica seeds according to claim 4, characterized in that, The volume ratio of soaked seeds to sterilized river sand was 1:4, and the water content of the river sand was 65%.
6. A method for breaking the dormancy of Centella asiatica seeds according to claim 5, characterized in that, The temperature-variable stratification program consists of 15 days of stratification at 8°C and 7 days of stratification at 18°C, alternating twice, for a total stratification time of 44 days.
7. A method for breaking the dormancy of Centella asiatica seeds according to claim 6, characterized in that, The disinfection method for the river sand is to rinse the river sand with clean water, then sterilize it with high-pressure steam at 121℃ for 30 minutes, and then cool it to room temperature for later use.
8. A method for breaking the dormancy of Centella asiatica seeds according to claim 7, characterized in that, The germination conditions during the germination treatment were: temperature 25℃, relative humidity 85%, light intensity 1500 lx, and light duration 12 hours / day.
9. A method for breaking the dormancy of Centella asiatica seeds according to claim 8, characterized in that, The seeds of Centella asiatica are either newly harvested seeds from the current year or old seeds stored for 1-2 years.
10. A method for breaking the dormancy of Centella asiatica seeds according to claim 9, characterized in that, The ethanol solution has a volume fraction of 75% and a disinfection time of 45 seconds, while the mercuric chloride solution has a mass fraction of 0.1% and a disinfection time of 10 minutes.