A method for regulating the growth of Sophora japonica seeds into seedlings

Abstract

The present invention relates to a method for regulating seedling formation of sandy locust seeds, belonging to the technical field of seed treatment and seedling cultivation, and the method comprises: step 1, disinfection and cleaning; step 2, seed soaking treatment: soaking sandy locust seeds in a mixed solution of cellulase and hemicellulase for 2-6 hours, and then soaking the seeds in 0.1%-0.3% KNO3 for 6-10 hours; step 3, placing on a bed for cultivation. The enzyme treatment is combined with KNO3 to treat sandy locust seeds, and the treatment can improve the emergence rate of sandy locust seeds and the number of branches, plant height and total biomass of sandy locust seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of seed treatment and seedling raising, and particularly relates to a method for regulating seedling formation of Sophora japonica seeds with high germination rate, high number of branches, high plant height and total biomass. Background Art

[0002] Sophora moorcroftiana is a perennial shrub of the Fabaceae family, Sophora L., endemic to the Tibet Autonomous Region. It is mainly distributed in the “one river and two rivers” basin at an altitude of 2800-4400 m. According to incomplete statistics, the natural population area in the region is about 20.7×10 4 hm 2 . Its community habitat ranges from hillside shrubs, sandy river floodplains, rocky hillsides, and alluvial sands. It is drought-resistant, barren-resistant, and wind-resistant, and is an important windbreak and sand-fixing plant. According to Tibetan medical literature, its roots, stems, and leaves can be used as medicine, with the effects of clearing heat and detoxifying, anti-inflammatory and antibacterial, insecticidal, cardiotonic, and anti-arrhythmic. It is an important medicinal plant with great development prospects. Sand Sophora japonica is also a high-quality protein feed. Its seeds have a crude protein content of up to 29.4-31.3%, and the crude protein content of young branches and leaves is also above 20%. Tender branches and leaves and fruit pods are important spring supplementary feed for cattle and sheep in the Tibetan valley agricultural areas. In addition, its stems are also used as fuel for life, and it is also widely used as a nectar plant. Over the years, due to the slow renewal of the sand Sophora japonica population and the unreasonable use of firewood felling and grazing, the resources have been severely damaged, the shrub area has shrunk and has shown varying degrees of degradation, which has also caused a series of ecological problems such as soil desertification.

[0003] The hardness rate of Sophora japonica seeds is as high as 92.5%, which is a super hard seed. In recent years, researchers have adopted many methods to carry out experimental research on breaking seed dormancy. An Yipeng et al. found that the storage period reduces the hardness rate of seeds. Under the temperature condition of 15℃~25℃, the germination rate can reach more than 70% by piercing the seed coat with a knife or soaking the seeds in 96% concentrated sulfuric acid, but the treatment with too high concentration of acid has a negative effect on the length of the plumule and radicle. Wang Wenjuan et al. used "moisture absorption-drying back" treatment once and soaked the seeds for 6 hours to increase the germination rate to 91.33%, and the germination rate was increased by 50mg / L gibberellin treatment. CN102487625A, using temperature-changing soaking and concentrated sulfuric acid treatment. CN111492886A, the seed coat of the lower part of the Sophora japonica seeds was cut and placed in a berberine aqueous solution for soaking treatment.

[0004] The prior art uses concentrated sulfuric acid for treatment, and concentrated sulfuric acid is a strong acid with strong corrosiveness and irritation. It is necessary to be particularly careful when handling seeds, and improper operation may cause harm to the human body. In addition, if the concentration is not well controlled, the seeds may be easily burned, affecting the germination rate and emergence rate of the seeds. Although artificial skin breaking has a high germination rate, it requires high skills and experience, and improper operation may damage the internal structure of the seeds. It is difficult to ensure that the degree of breaking the seed coat of each seed is consistent, which may lead to uneven emergence. Manual operation is relatively cumbersome and consumes a lot of time and manpower. In addition, external pathogens may be brought into the seeds during the operation, increasing the risk of infection.

[0005] How to improve the emergence rate and strong seedlings is the key to further improve the survival rate of sand locust trees. The technical solution of this application is dedicated to solving the emergence and strong seedling problems of sand locust trees, hoping to provide a new solution for sand locust tree seed propagation and seedling regulation. Summary of the invention

[0006] The invention aims to provide a method for regulating seedling formation of Sophora japonica seed reproduction, which improves the emergence rate of Sophora japonica seeds and at the same time increases the number of branches, plant height and total biomass of Sophora japonica seedlings.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] 1. Clean and disinfect.

[0009] 2. Seed soaking treatment:

[0010] Soak the seeds of Sophora japonica in water in a mixture of cellulase and hemicellulase at a temperature of 40°C to ensure enzyme activity and seed safety. Stir gently every hour during the treatment to ensure that the seeds are evenly exposed to the enzyme solution.

[0011] 3. Cultivation.

[0012] Further, the mixed solution of cellulase and hemicellulase is used to soak the Sophora japonica seeds for 1-8 hours, preferably 2-7 hours, preferably 3-6 hours, preferably 4-5 hours, and most preferably 4 hours. The soaking temperature is controlled at 30-50°C, preferably 40°C.

[0013] Furthermore, the disinfection step is: soaking the Sophora japonica seeds in a disinfectant for 5-30 minutes, and washing them after disinfection.

[0014] Furthermore, the disinfectant includes potassium permanganate and other disinfectants commonly used in the art.

[0015] Furthermore, the disinfectant preferably contains 1-10% potassium permanganate, more preferably 1-8%; more preferably 2-7%; more preferably 3-6%; most preferably 5%.

[0016] Furthermore, the immersion time for disinfection is 5-20 minutes, preferably 5-15 minutes; preferably 10 minutes.

[0017] Furthermore, in the seed soaking treatment, the concentrations of cellulase and hemicellulase are 0.1%-0.3% (w / v), preferably 0.2%-0.3% (w / v), and most preferably 0.3% (w / v).

[0018] Furthermore, the soaking time is 1-10 hours, preferably 2-8 hours; more preferably 3-7 hours; more preferably 3-6 hours; more preferably 3-5 hours and most preferably 4 hours.

[0019] After further enzyme treatment, the seeds are then soaked in 0.1%-0.5% KNO3, preferably 0.1%-0.4%; 0.1%-0.3%; most preferably 0.1%-0.2%; the soaking time is 4-12h; preferably 6-10h; most preferably 8h.

[0020] Further, the seed soaking temperature is 30-50°C, preferably 35-45°C, preferably 40°C.

[0021] Further, the sand was sieved through a 1 mm sieve and then sterilized and dried at high temperature, and distilled water was added to make the water content of the sand 15%.

[0022] Further, the seeds are placed on the surface of the sand substrate, and finally the surface of the seeds is covered with sand to a depth of 1-5 cm, preferably 2-4 cm, and most preferably 3 cm.

[0023] Furthermore, the culture temperature is variable temperature culture, the temperature range is 5°C-25°C, 12h5°C, 12h25°C.

[0024] Beneficial Effects

[0025] The seeds of Sophora japonica are super hard seeds, and the high hardness rate means that the shell of the seeds is difficult to be penetrated by water, which will limit the water absorption and germination of the seeds. The existing technology often uses concentrated sulfuric acid treatment, and concentrated sulfuric acid is a strong acid with strong corrosiveness and irritation. It is necessary to be particularly careful when handling seeds, and improper operation may cause harm to the human body. And if the concentration is not well controlled, it is easy to burn the seeds, affecting the germination rate and emergence rate of the seeds. Although the germination rate of artificial skin breaking is high, it requires high skills and experience, and improper operation may damage the internal structure of the seeds. It is difficult to ensure that the degree of breaking the seed coat of each seed is consistent, which may cause uneven emergence. Manual operation is relatively cumbersome, consuming a lot of time and manpower. In addition, external pathogens may be brought into the seeds during the operation, increasing the risk of infection.

[0026] Enzyme treatment is relatively mild, causes less damage to seeds, can better maintain the vitality and integrity of seeds, and is simple to operate and can be controlled more accurately. Sand Sophora seeds contain polysaccharide components such as cellulose and hemicellulose. Cellulase mainly acts on the cellulose components in the plant cell wall, breaking it down into smaller sugar molecules. In the case of hard seeds, cellulase can help destroy the cellulose structure of the seed shell, loosen the fiber structure of the hard shell, thereby promoting the penetration and absorption of water and creating conditions for seed germination. Hemicellulose is another important component of plant cell walls. Hemicellulase can further decompose hemicellulose, further weaken the barrier effect and strength of the sand Sophora seed shell, and increase the water permeability and air permeability of the seeds. Researchers found that the synergistic effect of the two can more thoroughly destroy the hard structure of sand Sophora seeds. This synergistic effect not only accelerates the degradation of cell wall components, but also improves the permeability of the seed coat by improving the physical structure of the seed shell. And it may also help to remove substances that may inhibit germination in the hard shell of sand Sophora seeds. Further, the applicant also screened the optimal concentration ratio of cellulase and hemicellulase. The germination rate was the highest with 0.3% cellulase + 0.2% hemicellulase, and increasing the concentration of both did not further increase the germination rate.

[0027] The applicant's further research found that gibberellin treatment can improve the emergence rate of sand-growing Sophora japonica seeds (this is the same as some research results of the prior art), but the study found that the number of branches, plant height and total biomass of the seedlings after gibberellin treatment were reduced, and the inhibitory effect of gibberellin after enzyme treatment was more significant, indicating that gibberellin had a more significant negative impact on the number of branches, plant height and total biomass of sand-growing Sophora japonica seedlings. In response to this problem, the applicant sought other ingredients to replace gibberellin treatment. The study found that KNO3 can not only improve the emergence of sand-growing Sophora japonica seeds, but also significantly improve the number of branches, plant height and total biomass of sand-growing Sophora japonica seedlings. The reason for the analysis may be that gibberellin mainly promotes germination by regulating the hormone balance in the seeds. As a plant hormone, gibberellin, excessive or inappropriate gibberellin levels may break the balance of hormones in the plant body. This will affect the synthesis, metabolism and signal transduction of other hormones, leading to growth regulation disorders, thereby inhibiting the growth of seedlings. It may also interfere with the metabolic process of the seedlings, such as affecting photosynthesis, respiration or nutrient absorption and distribution. This will lead to insufficient supply of energy and materials, thereby limiting the growth of seedlings. Cellulase and hemicellulase treatment increased the permeability of the seed coat and significantly increased the amount of gibberellins entering the seeds, thereby further inhibiting the growth of Sophora japonica seedlings.

[0028] The nitrogen and potassium in potassium nitrate are essential nutrients for plant growth, which can meet the needs of seed germination and initial growth of seedlings and promote the development of seedlings. In addition, potassium can participate in the activation of various enzymes, which is beneficial to intracellular metabolism and energy conversion, and improves seed vitality. Potassium nitrate also helps to maintain the osmotic balance of cells, allowing seeds to absorb water and swell better under suitable moisture conditions. It can also enhance the stress resistance of plants and make seedlings more tolerant under adverse environmental conditions, thereby promoting the emergence of sand-growing Sophora japonica seeds. Cellulase and hemicellulase treatment break the protective structure of the seeds, making it easier for KNO3 to penetrate into the seeds. Its potassium element may also participate in enzyme activation, and combined with various nutrients after enzyme decomposition, it can better improve the emergence rate of sand-growing Sophora japonica seeds, and significantly increase the number of branches, plant height and total biomass. DETAILED DESCRIPTION

[0029] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0030] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0031] Example 1

[0032] Sources:

[0033] Sophora japonica seeds were collected in Qushui County, Lhasa;

[0034] Cellulase, Shanghai Yuanye Biotechnology Co., Ltd., product number S10041;

[0035] Pectinase, Shanghai Yuanye Biotechnology Co., Ltd., product number S10007;

[0036] Hemicellulase, Shanghai Lianmai Bioengineering Co., Ltd., catalog number LM9025-56-3;

[0037] Xylanase, Beijing Biolab Technology Co., Ltd., product number Y12968.

[0038] Prepare enzyme treatment solution: dissolve enzyme in deionized water and adjust pH to 5.0.

[0039] The specific steps of the experiment are:

[0040] 1. Select mature, plump, uniform and consistent Sophora japonica seeds from the same batch.

[0041] 2. Disinfection: Soak the seeds of Sophora japonica in 5% potassium permanganate solution for 10 minutes. Finally, rinse the disinfected seeds with sterile water 4-5 times.

[0042] 3. Seed soaking treatment:

[0043] The experiment set up multiple treatment groups (see Table 1 below), each group had 3 replications, and each replication had 100 seeds.

[0044] A. Soak Sophora japonica seeds in 0.2% (w / v) of cellulase, pectinase, hemicellulase, xylanase, and a mixture of different enzymes (0.2% (w / v) + 0.2% (w / v)) for 4 hours at a temperature of 40°C to ensure enzyme activity and seed safety. Gently stir every hour during the treatment to ensure that the seeds are evenly exposed to the enzyme solution.

[0045] B. Use 96% concentrated sulfuric acid alone to soak the Sophora japonica seeds for 1 hour. After soaking, rinse the sulfuric acid with purified water immediately for later use.

[0046] C. Use a knife to artificially break the skin. The upper part of the Sophora japonica seed is the location of the embryo and radicle, and the lower part of the seed away from the radicle and embryo is the artificial breaking point.

[0047] The control group (CK) was untreated.

[0048] 4. Sand bed culture. After the sand is sieved through a 1mm sieve, it is sterilized and dried at high temperature. Distilled water is added to make the water content of the sand 15%. The seeds are placed on the surface of the sand matrix, and finally covered with sand to make the depth of the sand burial reach 3cm.

[0049] During the germination test, the relative humidity of the sand (substrate) was maintained by adding distilled water at regular intervals every day, and the evaporated water of each treatment group was weighed and added every day to keep the moisture content of the substrate constant. As for the greenhouse, the average temperature at night was controlled at 5°C, and the average temperature during the day was controlled at 25°C.

[0050] 5. Measurement indicators

[0051] The seed germination rate was counted after 30 days.

[0052] Germination rate (%) = (number of seed germinations / total number of test seeds) × 100%.

[0053] Test results:

[0054] Table 1 Effects of different treatments on the germination rate of Sophora japonica seeds

[0055]

[0056]

[0057] As can be seen from Table 1, cellulase has a better effect than other enzymes in single enzyme treatment, which may be related to the highest cellulose content in the seed coat. The effect is close to that of 96% concentrated sulfuric acid soaking treatment, but it is quite different from the effect of artificial skin breaking treatment. Further treatment of Sophora japonica seeds with cellulase combined with other enzymes has significantly improved the effect compared with single enzyme treatment. Among them, the best effect of cellulase and hemicellulase combined treatment reached 78.33%, exceeding 69.67% of 96% concentrated sulfuric acid soaking and 74% of artificial skin breaking. Analysis of the reasons: The high hardness rate of Sophora japonica seeds means that the seed shell is difficult to be penetrated by water, which will limit the water absorption and germination of the seeds. Sophora japonica seeds contain polysaccharides such as cellulose and hemicellulose. Cellulase mainly acts on the cellulose component in the plant cell wall, breaking it into smaller sugar molecules. In the case of hard seeds, cellulase can help destroy the cellulose structure of the seed shell, making the fiber structure of the hard shell loose, thereby promoting water penetration and absorption, creating conditions for seed germination. Hemicellulose is another important component of the plant cell wall. Hemicellulase can further decompose hemicellulose, further weaken the barrier effect and strength of the seed shell, and increase the water permeability and air permeability of the seeds. The synergistic effect of the two can more thoroughly destroy the hard structure of Sophora japonica seeds. This synergistic effect not only accelerates the degradation of cell wall components, but also improves the permeability of the seed coat by improving the physical structure of the seed shell. It may also help to remove substances that may inhibit germination in the hard shell.

[0058] Example 2 Determination of the effect of different concentrations of cellulase + hemicellulase on the emergence rate of Sophora japonica seeds

[0059] The method of Example 1 was the same as above, only the concentrations of cellulase and hemicellulase in the mixed solution were adjusted. The results are shown in the following table.

[0060] Table 2 Effects of different concentrations of cellulase + hemicellulase on the germination rate of Sophora japonica seeds

[0061]

[0062] As can be seen from Table 2, appropriately increasing the dosage of cellulase can further improve the germination rate of Sophora japonica seeds. The germination rate of 0.3% cellulase + 0.2% hemicellulase is the highest, reaching 82.33%. Further increasing the concentration of both did not increase the germination rate, and reducing the concentration of both decreased the germination rate of Sophora japonica seeds.

[0063] Example 3 Determination of the effects of cellulase + hemicellulase combined with other treatment agents on the emergence rate, number of branches, plant height and total biomass of Sophora japonica seeds

[0064] Same as the method of Example 1 above, wherein the seed soaking treatment is:

[0065] A. Soak Sophora japonica seeds in 0.3% cellulase + 0.2% hemicellulase for 4 hours at 40°C. Then soak the seeds in 1, 10, and 50 mg / L GA3 for 8 hours respectively;

[0066] B. Soak seeds with 1, 10, or 50 mg / L GA3 for 12 h;

[0067] C. Soak the seeds of Sophora japonica in 0.3% cellulase + 0.2% hemicellulase for 4 hours at 40°C, and then soak them in 0.1%, 0.2%, and 0.5% KNO3 for 8 hours respectively;

[0068] D. Soak seeds in 0.1%, 0.2% or 0.5% KNO3 for 12 hours.

[0069] After 30 days, the seed emergence rate was counted and thinning was carried out, and 30 plants were randomly left in each treatment. During the experiment, watering and weeding were carried out according to conventional methods. After 90 days of cultivation, 10 plants were randomly selected from each treatment for harvest, and the number of branches, plant height, and total biomass were measured.

[0070] Table 3 Effects of combined treatments of cellulase + hemicellulase and other treatment agents on seedling emergence rate, branch number, plant height and total biomass of Sophora japonica

[0071]

[0072]

[0073] From Table 3 above, it can be seen that gibberellin treatment alone can increase the emergence rate of sand-growing Sophora japonica seeds, and low concentrations of gibberellin after enzyme treatment can further increase its emergence rate, but the number of branches, plant height and total biomass are reduced, and the inhibitory effect is more significant after enzyme treatment, indicating that gibberellin has a more significant negative impact on the number of branches, plant height and total biomass of sand-growing Sophora japonica seedlings. KNO3 alone or combined with enzyme treatment can not only improve the emergence of sand-growing Sophora japonica seeds, but also significantly improve the number of branches, plant height and total biomass of sand-growing Sophora japonica seedlings. The reason for the analysis may be that gibberellin mainly promotes germination by regulating the hormone balance in seeds. As a plant hormone, excessive or inappropriate gibberellin levels may break the balance of hormones in plants. This will affect the synthesis, metabolism and signal transduction of other hormones, leading to growth regulation disorders, thereby inhibiting the growth of seedlings. It may also interfere with the metabolic process of seedlings, such as affecting photosynthesis, respiration or nutrient absorption and distribution. This will lead to insufficient energy and material supply, thereby limiting the growth of seedlings. The cellulase and hemicellulase treatment increases the permeability of the seed coat, and the amount of gibberellins entering the seed interior increases significantly, thereby further inhibiting the growth of sand-growing Sophora japonica seedlings. Nitrogen and potassium in potassium nitrate are essential nutrients for plant growth, which can meet the needs of seed germination and early growth of seedlings and promote the development of seedlings. And potassium can participate in the activation of multiple enzymes, which is conducive to intracellular material metabolism and energy conversion, and improves seed vitality. Potassium nitrate also helps to maintain the osmotic balance of cells, so that seeds can better absorb water and swell under suitable moisture conditions, and can also enhance the stress resistance of plants, so that seedlings are more tolerant under adverse environmental conditions, thereby promoting the emergence of sand-growing Sophora japonica seeds. After the cellulase and hemicellulase treatment breaks the protective structure of the seed, KNO3 is more easily infiltrated into the seed interior, and its potassium may also participate in enzyme activation, and then cooperate with various nutrients after enzyme decomposition, so that the emergence rate of sand-growing Sophora japonica seeds can be better improved, and the number of branches, plant height and total biomass can be significantly improved.

[0074] In this specification, the same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the embodiments described later, the description is relatively simple, and the relevant parts can be referred to the partial description of the previous embodiments.

[0075] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for regulating the growth of Sophora japonica seeds into seedlings, characterized in that: The method comprises: step 1, disinfection and cleaning; step 2, seed soaking treatment: soaking Sophora japonica seeds in a mixed solution of cellulase and hemicellulase for 2-6 hours at a soaking temperature of 30-50°C, and then soaking the seeds in 0.1%-0.3% KNO3 for 6-10 hours; step 3, bed culture: the concentrations of the cellulase and hemicellulase are 0.2%-0.3% (w / v) respectively.

2. The method for regulating and growing seedlings of Sophora japonica L. seeds according to claim 1, characterized in that: In the step 2, the concentrations of cellulase and hemicellulase are 0.2% (w / v) respectively.

3. The method for regulating and growing seedlings of Sophora japonica L. seeds according to claim 1, characterized in that: In the step 2, the mixed solution of cellulase and hemicellulase is used to soak the Sophora japonica seeds for 3-5 hours.

4. The method for regulating and growing seedlings of Sophora japonica L. seeds according to claim 1, characterized in that: In the step 2, the seeds are soaked in 0.2% KNO3.

5. The method for regulating seedling growth of Sophora japonica L. according to claim 1, characterized in that: In the step 2, the seeds are placed in 0.2% KNO3 and soaked for 7-9 hours.

6. The method for regulating seedling growth of Sophora japonica L. according to claim 1, characterized in that: In the step 1, the disinfectant is 2% potassium permanganate, and the soaking time is 20-40 minutes.

7. The method for regulating the growth of Sophora japonica seeds into seedlings according to claim 1, characterized in that: In step 3, the seeds are planted on the surface of the sand matrix, and finally the surface of the seeds is covered with sand to a depth of 1-5 cm.

8. The method for regulating and growing seedlings of Sophora japonica L. seeds according to claim 7, characterized in that: In step 3, the culture temperature is variable temperature culture, and the temperature range is 5°C-25°C.

9. The method for regulating and growing seedlings of Sophora japonica L. seeds according to claim 1, characterized in that: In the step 1, the seeds of Sophora japonica are soaked in a disinfectant for 5-30 minutes, and then disinfected and washed.

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