Method for cultivating aseptic seedlings by using cercis chinensis seeds

Abstract

The invention discloses a method for cultivating aseptic seedlings by using cercis chinensis seeds. In order to overcome the defect of lack of a technical system for cultivating aseptic seedlings by using the cercis chinensis seeds, a strategy of double sterilization and middle physical peeling is adopted, namely, the cercis chinensis seeds coated with complete seed coats are sterilized, the cercis chinensis seeds not coated with the seed coats are sterilized after the seed coats are peeled off, and the aseptic seedlings are cultivated by using the cercis chinensis seeds. The method for cultivating the aseptic seedlings of the cercis chinensis by utilizing the cercis chinensis seeds is simple and convenient to operate, good in repeatability and capable of adapting to different seed size differences. By means of the method, bacteria carried by seed coats can be effectively removed, meanwhile, excessive damage of a powerful disinfectant to internal embryos is avoided, the pollution rate of the cultured cercis chinensis aseptic seedlings is low, the germination rate is high, and protection and sustainable utilization of cercis chinensis are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of seed and seedling cultivation technology. More specifically, it relates to a method for cultivating sterile seedlings using Bauhinia seeds. Background Technology

[0002] Redbud ( Madhuca pasquieri (Dubard) HJLam) is an evergreen, tall tree belonging to the genus *Cercis* in the family Sapotaceae. It is a Class II protected wild plant in China, as well as a wild plant with extremely small populations, and a species included in the national reserve forest. As a tree species with extremely high protection level and economic value, conducting research on artificial propagation and germplasm resource conservation of *Cercis chinensis* is of practical significance for the rescue protection and sustainable utilization of this species.

[0003] Plant tissue culture technology is now widely used for the rapid propagation of various crops. However, due to species differences, plant tissue culture methods for one plant are not applicable to other plants. Tissue culture of *Cercis chinensis* is particularly challenging, and there is currently no technical system for cultivating aseptic seedlings from *Cercis chinensis* seeds. Therefore, providing a method for cultivating aseptic seedlings from *Cercis chinensis* seeds with low contamination rates and high germination or survival rates is a primary problem that urgently needs to be solved for research such as *Cercis chinensis* tissue culture. Summary of the Invention

[0004] This invention addresses the lack of a technical system for cultivating sterile seedlings using Bauhinia seeds by providing a method for cultivating sterile seedlings using Bauhinia seeds, which has a low contamination rate and a high germination rate.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] This invention provides a method for cultivating sterile Bauhinia seedlings using Bauhinia seeds through a strategy of double sterilization plus intermediate physical peeling.

[0007] This invention provides a method for cultivating sterile seedlings using Bauhinia seeds, comprising the following steps: S1. Seed pretreatment: Select fresh and mature Bauhinia fruits and peel off the pericarp under sterile conditions to obtain Bauhinia seeds with intact seed coats. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, and then sterilized with 0.08%–0.12% mercuric chloride or 4%–6% sodium hypochlorite for 5–9 minutes. The mercuric chloride or sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1%–3% sodium hypochlorite for 3–10 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds. S3. Inoculation: Under aseptic conditions, inoculate the sterile seeds obtained in S2 into the culture medium; S4. Cultivation: The seeds obtained in S3 and inoculated into the culture medium are placed under light at 25 ℃~28 ℃ for 20~25 days.

[0008] Specifically, the Bauhinia seeds include large seeds and small seeds; based on the weight of a single seed, the large seeds weigh 2.1 g to 3.5 g, and the small seeds weigh 1.0 g to 2.0 g.

[0009] Specifically, the Bauhinia fruit mentioned are fresh, ripe Bauhinia fruits. The Bauhinia fruits need to be washed and dried before peeling.

[0010] Optionally, the Bauhinia seeds with intact seed coats in S1 are washed with detergent and then dried.

[0011] Preferably, in S2, sterilization is performed using 0.08%–0.12% mercuric chloride or 4.8%–5.2% sodium hypochlorite for 5–9 minutes.

[0012] Specifically, the culture medium in S3 consists of MS medium, sucrose, plant gel, inositol, and water; in the culture medium, the concentration of sucrose is 28–32 g / L, the concentration of plant gel is 3.0–4.0 g / L, and the concentration of inositol is 0.05–0.15 g / L.

[0013] More specifically, in the culture medium, the concentration of sucrose is 30 g / L, the concentration of plant gel is 3.5 g / L, and the concentration of inositol is 0.1 g / L.

[0014] In a specific embodiment of the present invention, the culture medium is prepared as follows: accurately weigh 4.74 g MS culture medium, 30 g sucrose, 3.5 g plant gel and 0.1 g inositol, add distilled water to a final volume of 1 L, and sterilize at high temperature.

[0015] Specifically, the photocycle for light culture described in S4 is 12 h to 14 h of light and 10 h to 12 h of darkness.

[0016] More specifically, the photocycle for the light culture is 14 hours of light and 10 hours of darkness.

[0017] Specifically, when the Bauhinia seeds are small seeds, in step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, sterilized with 0.08%–0.12% mercuric chloride for 5–9 minutes, the mercuric chloride is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1%–1.2% sodium hypochlorite for 3–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

[0018] Specifically, when the Bauhinia seeds are small seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 70%–75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, sterilized with 0.08%–0.12% mercuric chloride for 6–8 minutes, the mercuric chloride is washed away with sterile water, the seed coat is peeled off to obtain Bauhinia seeds without seed coat, and then sterilized with 1%–1.2% sodium hypochlorite for 4–6 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

[0019] More specifically, when the Bauhinia seeds are small seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 75% alcohol for 25-35 seconds, the alcohol is washed away with sterile water, sterilized with 0.1% mercuric chloride for 6-8 minutes, the mercuric chloride is washed away with sterile water, the seed coat is peeled off to obtain Bauhinia seeds without seed coat, then sterilized with 1% sodium hypochlorite for 4-6 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

[0020] Preferably, when the Bauhinia seeds are small seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 75% alcohol for 30 seconds, the alcohol is washed away with sterile water, sterilized with 0.1% mercuric chloride for 7 minutes, the mercuric chloride is washed away with sterile water, the seed coat is peeled off to obtain Bauhinia seeds without seed coat, then sterilized with 1% sodium hypochlorite for 5 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

[0021] Specifically, when the Bauhinia seeds are large seeds, in step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, sterilized with 4%–6% sodium hypochlorite for 5–9 minutes, the sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 2.8%–3% sodium hypochlorite for 3–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

[0022] Specifically, when the Bauhinia seeds are large seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 70%–75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, sterilized with 4%–6% sodium hypochlorite for 7–9 minutes, the sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 2.8%–3% sodium hypochlorite for 5–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

[0023] More specifically, when the Bauhinia seeds are large seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 75% alcohol for 25-35 seconds, the alcohol is washed away with sterile water, sterilized with 5% sodium hypochlorite for 7-9 minutes, the sodium hypochlorite is washed away with sterile water, the seed coat is peeled off to obtain Bauhinia seeds without seed coat, then sterilized with 3% sodium hypochlorite for 5-8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

[0024] Preferably, when the Bauhinia seeds are large seeds, the Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 75% alcohol for 30 seconds, the alcohol is washed off with sterile water, sterilized with 5% sodium hypochlorite for 8 minutes, the sodium hypochlorite is washed off with sterile water, the seed coat is peeled off to obtain Bauhinia seeds without seed coat, then sterilized with 3% sodium hypochlorite for 5 minutes, the sodium hypochlorite is washed off with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

[0025] The present invention has the following beneficial effects: This invention addresses the current lack of a technical system for cultivating sterile seedlings from Bauhinia seeds. It employs a double sterilization + intermediate physical peeling strategy: first, sterilizing Bauhinia seeds with intact seed coats; then, removing the seed coats and sterilizing the seeds without seed coats. This results in a simple, reproducible method for cultivating sterile Bauhinia seedlings from different seed sizes. The method effectively removes bacteria carried by the seed coat while avoiding excessive damage to the internal embryo from strong disinfectants. This results in sterile Bauhinia seedlings with low contamination rates and high germination rates, which is beneficial for the protection and sustainable use of Bauhinia. Attached Figure Description

[0026] Figure 1 This is a comparison image showing the size of large and small seeds.

[0027] Figure 2 Images of the fruit and seeds of the Bauhinia tree.

[0028] Figure 3 These are seeds of the Bauhinia tree after peeling and sterilization.

[0029] Figure 4 These are seeds of the Bauhinia tree after the radicle has developed.

[0030] Figure 5 This is a picture of the radicle penetrating the culture medium during the cultivation process.

[0031] Figure 6 These are seeds of the Bauhinia tree after the embryo has sprouted.

[0032] Figure 7 These are sterile seedlings of Bauhinia purpurea, which have sprouted from small seeds.

[0033] Figure 8 These are sterile seedlings of Bauhinia purpurea with large seeds. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Comparison of the appearance and size of large and small seeds Figure 1 As shown. After multiple weighings and grading screenings of individual seeds, the weight range of a single seed of large Bauhinia purpurea was determined to be 2.1 g to 3.5 g, and the weight range of a single seed of small Bauhinia purpurea was determined to be 1.0 g to 2.0 g.

[0037] In the following examples, the MS culture medium is from Hangzhou Best Biotechnology Co., Ltd., and its product number is BS1004.

[0038] Example 1 This embodiment provides a method for cultivating sterile seedlings using small seeds of Bauhinia purpurea, including the following steps: S1. Seed pretreatment: Select plump, disease-free, mature, fresh Bauhinia fruits (such as mature, fresh Bauhinia fruits). Figure 2 (As shown), rinse thoroughly with clean water and air dry. In a clean bench, peel off the pericarp to obtain small seeds of *Cercis chinensis* with intact seed coats (as shown). Figure 2 As shown in the image, use a soft-bristled brush to gently scrub the surface of the obtained Bauhinia seeds in a container containing dish soap, and then let them air dry. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed once with sterile water, sterilized with 0.1% mercuric chloride for 7 minutes, rinsed 4-5 times with sterile water to remove excess mercuric chloride, and the seed coat is removed to obtain Bauhinia seeds without seed coat (e.g., ...). Figure 2 (as shown), then sterilize with 1% sodium hypochlorite for 5 minutes, rinse 4-5 times with sterile water to remove excess sodium hypochlorite, and use sterile filter paper to absorb the surface moisture for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into a culture flask containing culture medium (the culture medium is prepared by accurately weighing 4.74 g MS medium, 30 g sucrose, 3.5 g plant gel and 0.1 g inositol, adding distilled water to a final volume of 1 L, and sterilizing at 121 °C for 20 min). S4. Culture: Place the inoculated culture bottles under light at 25 ℃ for 20-25 days until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0039] As an example, after peeling and sterilization, the seeds of the Bauhinia tree are like... Figure 3 As shown; the seeds of the Bauhinia tree after the radicle has grown are as follows Figure 4 As shown; during the cultivation process, the radicles penetrate the culture medium as shown in the image. Figure 5 As shown; the seeds of the Bauhinia tree after sprouting embryos are as follows Figure 6 As shown; the sterile seedlings of small Bauhinia seeds that have grown out are like Figure 7 As shown.

[0040] Example 2 This embodiment provides a method for cultivating sterile seedlings using small seeds of Bauhinia purpurea. The main difference between this method and the method described in Example 1 is that in S2, after sterilization with 75% alcohol for 30 seconds, sterilization with 0.1% mercuric chloride is not used.

[0041] The method described in this embodiment specifically includes the following steps: S1. Seed pretreatment: Select plump, disease-free, mature and fresh Bauhinia fruits, rinse them with clean water and dry them. Peel off the pericarp in a clean bench to obtain small Bauhinia seeds with intact seed coats. Use a soft brush to gently scrub the surface of the Bauhinia seeds in a container with detergent and dry them for later use. S2. Seed sterilization: Sterilize the Bauhinia seeds obtained in S1 with 75% alcohol for 30 seconds in a clean bench, rinse once with sterile water, peel off the seed coat to obtain Bauhinia seeds without seed coat, sterilize with 1% sodium hypochlorite for 5 minutes, rinse 4-5 times with sterile water to wash away excess sodium hypochlorite, and dry the surface moisture with sterile filter paper for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into culture flasks containing culture medium; S4. Culture: Place the inoculated culture bottles under light at 25 ℃ until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0042] Example 3 This embodiment provides a method for cultivating sterile seedlings using small seeds of Bauhinia purpurea. The main difference between this method and the method described in Embodiment 1 is that, in S2, after sterilization with 75% alcohol for 30 seconds, sterilization with 0.1% mercuric chloride is not used, and the sterilization time with 1% sodium hypochlorite after peeling off the seed coat is different, changing from 5 minutes to 8 minutes.

[0043] The method described in this embodiment specifically includes the following steps: S1. Seed pretreatment: Select plump, disease-free, mature and fresh Bauhinia fruits, rinse them with clean water and dry them. Peel off the pericarp in a clean bench to obtain small Bauhinia seeds with intact seed coats. Use a soft brush to gently scrub the surface of the Bauhinia seeds in a container with detergent and dry them for later use. S2. Seed sterilization: Sterilize the Bauhinia seeds obtained in S1 with 75% alcohol for 30 seconds in a clean bench, rinse once with sterile water, peel off the seed coat to obtain Bauhinia seeds without seed coat, sterilize with 1% sodium hypochlorite for 8 minutes, rinse 4-5 times with sterile water to wash away excess sodium hypochlorite, and dry the surface moisture with sterile filter paper for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into culture flasks containing culture medium; S4. Culture: Place the inoculated culture bottles under light at 25 ℃ until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0044] Example 4 This embodiment provides a method for cultivating sterile seedlings using large seeds of Bauhinia purpurea, including the following steps: S1. Seed pretreatment: Select plump, disease-free, mature and fresh Bauhinia fruits, rinse them with clean water and dry them. Peel off the pericarp in a clean bench to obtain large Bauhinia seeds with intact seed coats. Use a soft brush to gently scrub the surface of the Bauhinia seeds in a container with detergent and dry them for later use. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed once with sterile water, sterilized with 5% sodium hypochlorite for 8 minutes, rinsed with sterile water 4-5 times to remove excess sodium hypochlorite, and the seed coat is peeled off to obtain Bauhinia seeds without seed coat. Then, they are sterilized with 3% sodium hypochlorite for 5 minutes, rinsed with sterile water 4-5 times to remove excess sodium hypochlorite, and the surface moisture is absorbed with sterile filter paper for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into culture flasks containing culture medium; S4. Culture: Place the inoculated culture bottles under light at 25 ℃ until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0045] As an example, the sterile seedlings with large Bauhinia seeds that have grown out are like... Figure 8 As shown.

[0046] Example 5 This embodiment provides a method for cultivating sterile seedlings using large seeds of Bauhinia purpurea. The main difference between this method and the method described in Example 4 is that in S2, after sterilization with 75% alcohol for 30 seconds, sterilization is carried out with 0.1% mercuric chloride for 8 minutes.

[0047] The method specifically includes the following steps: S1. Seed pretreatment: Select plump, disease-free, mature and fresh Bauhinia fruits, rinse them with clean water and dry them. Peel off the pericarp in a clean bench to obtain large Bauhinia seeds with intact seed coats. Use a soft brush to gently scrub the surface of the Bauhinia seeds in a container with detergent and dry them for later use. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed once with sterile water, sterilized with 0.1% mercuric chloride for 8 minutes, rinsed with sterile water 4-5 times to remove excess mercuric chloride, and the seed coat is peeled off to obtain Bauhinia seeds without seed coat. Then, they are sterilized with 3% sodium hypochlorite for 5 minutes, rinsed with sterile water 4-5 times to remove excess sodium hypochlorite, and the surface moisture is absorbed with sterile filter paper for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into culture flasks containing culture medium; S4. Culture: Place the inoculated culture bottles under light at 25 ℃ until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0048] Example 6 This embodiment provides a method for cultivating sterile seedlings using large seeds of Bauhinia purpurea. The main difference between this method and the method described in Example 4 is that in S2, after sterilization with 75% alcohol for 30 seconds, sterilization with 0.1% mercuric chloride for 8 minutes, after peeling off the seed coat, sterilization with 3% sodium hypochlorite for 10 minutes is performed.

[0049] The method specifically includes the following steps: S1. Seed pretreatment: Select plump, disease-free, mature and fresh Bauhinia fruits, rinse them with clean water and dry them. Peel off the pericarp in a clean bench to obtain large Bauhinia seeds with intact seed coats. Use a soft brush to gently scrub the surface of the Bauhinia seeds in a container with detergent and dry them for later use. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized in a clean bench with 75% alcohol for 30 seconds, rinsed once with sterile water, sterilized with 0.1% mercuric chloride for 8 minutes, rinsed with sterile water 4-5 times to remove excess mercuric chloride, and the seed coat is peeled off to obtain Bauhinia seeds without seed coat. Then, they are sterilized with 3% sodium hypochlorite for 10 minutes, rinsed with sterile water 4-5 times to remove excess sodium hypochlorite, and the surface moisture is absorbed with sterile filter paper for later use. S3. Inoculation: Under aseptic conditions, inoculate the sterilized seeds into culture flasks containing culture medium; S4. Culture: Place the inoculated culture bottles under light at 25 ℃ until sterile seedlings grow. The photoperiod is 14 h light and 10 h dark (D:L=10 h:14 h).

[0050] Test Example 1: Contamination Rate and Germination Rate Detection In this invention, sterile Bauhinia seedlings of the same number of bottles were cultivated using the methods described in Examples 1 to 6, and the seed contamination rate and germination rate corresponding to different methods were statistically analyzed.

[0051] Contamination rate = (Number of contaminated bottles / Number of inoculated bottles) × 100%; Germination rate = Number of uncontaminated seeds with radicles breaking through the seed coat / Total number of inoculated seeds × 100%.

[0052] The statistical results of seed contamination rate and germination rate using the methods described in Examples 1-6 are shown in Table 1. As can be seen from the results in Table 1, the strategy of double sterilization followed by intermediate physical peeling can significantly reduce the contamination rate of *Cercis chinensis* seeds and improve their germination rate. For small seeds, the sterilization strategy of first sterilizing with mercuric chloride and then with sodium hypochlorite is more effective. For large seeds, sterilization with sodium hypochlorite twice is more effective.

[0053] Table 1. Statistical results of seed contamination rate and germination rate

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for cultivating sterile seedlings using Bauhinia seeds, characterized in that, Includes the following steps: S1. Seed pretreatment: The pericarp of the Bauhinia fruit was removed under sterile conditions to obtain Bauhinia seeds with intact seed coats. S2. Seed sterilization: The Bauhinia seeds obtained in S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, and then sterilized with 0.08%–0.12% mercuric chloride or 4%–6% sodium hypochlorite for 5–9 minutes. The mercuric chloride or sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1%–3% sodium hypochlorite for 3–10 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds. S3. Inoculation: Under aseptic conditions, inoculate the sterile seeds obtained in S2 into the culture medium; S4. Cultivation: The seeds obtained in S3 and inoculated into the culture medium are placed under light at 25 ℃~28 ℃ for 20~25 days.

2. The method according to claim 1, characterized in that, The Bauhinia seeds include large seeds and small seeds; based on the weight of a single seed, the large seeds weigh 2.1 g to 3.5 g, and the small seeds weigh 1.0 g to 2.0 g.

3. The method according to claim 1, characterized in that, The culture medium described in S3 consists of MS medium, sucrose, plant gel, inositol, and water; in the culture medium, the concentration of sucrose is 28–32 g / L, the concentration of plant gel is 3.0–4.0 g / L, and the concentration of inositol is 0.05–0.15 g / L.

4. The method according to claim 1, characterized in that, The photocycle for the light culture described in S4 is 12 h to 14 h of light and 10 h to 12 h of darkness.

5. The method according to claim 2, characterized in that, When the Bauhinia seeds are small seeds, in step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, and then sterilized with 0.08%–0.12% mercuric chloride for 5–9 minutes. The mercuric chloride is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1%–1.2% sodium hypochlorite for 3–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

6. The method according to claim 5, characterized in that, In step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, and then sterilized with 0.08%–0.12% mercuric chloride for 6–8 minutes. The mercuric chloride is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1%–1.2% sodium hypochlorite for 4–6 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

7. The method according to claim 6, characterized in that, In step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, and then sterilized with 0.1% mercuric chloride for 6–8 minutes. The mercuric chloride is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 1% sodium hypochlorite for 4–6 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

8. The method according to claim 2, characterized in that, When the Bauhinia seeds are large seeds, in step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 20–40 seconds, the alcohol is washed away with sterile water, and then sterilized with 4%–6% sodium hypochlorite for 5–9 minutes. The sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 2.8%–3% sodium hypochlorite for 3–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is absorbed to obtain sterilized seeds.

9. The method according to claim 8, characterized in that, In step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 70%–75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, and then sterilized with 4%–6% sodium hypochlorite for 7–9 minutes. The sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 2.8%–3% sodium hypochlorite for 5–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

10. The method according to claim 9, characterized in that, In step S2, the Bauhinia seeds obtained in step S1 are sterilized under aseptic conditions with 75% alcohol for 25–35 seconds, the alcohol is washed away with sterile water, and then sterilized with 5% sodium hypochlorite for 7–9 minutes. The sodium hypochlorite is washed away with sterile water, the seed coat is peeled off, and Bauhinia seeds without seed coat are obtained. Then, they are sterilized with 3% sodium hypochlorite for 5–8 minutes, the sodium hypochlorite is washed away with sterile water, and the surface moisture of the seeds is dried to obtain sterilized seeds.

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