Tissue culture and rapid propagation method for high-drug-effect golden cypress and culture medium thereof
Through the rapid reproduction method of tissue culture, specific culture media and control conditions are used to solve the problem that traditional reproduction methods are difficult to meet market demand, and efficient and rapid reproduction of corks is achieved, which improves seedling cultivation efficiency and survival rate.
Patent Information
- Application Number
- CN202510281810.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-06-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Traditional breeding methods are difficult to meet the market's demand for high-efficiency cork. The seed breeding cycle is long, the seedling efficiency is low, and the supply of seedlings is seasonal.
The rapid breeding method of tissue culture is adopted to induce the differentiation and rooting of the young buds of corundum by configuring specific culture media and controlling the culture conditions to achieve rapid reproduction.
It improves the reproduction efficiency of cypress, shortens the seedling time, and reduces the cost. The survival rate of the differentiation and induction of young buds is as high as more than 85%, and the rooting rate can reach more than 95%.
Smart Images
Figure BDA0005305948140000091 
Figure BDA0005305948140000101
Abstract
Description
Technical Field
[0001] This application relates to the field of seedling propagation technology, and particularly to a method for rapid tissue culture propagation of Phellodendron amurense with high medicinal efficacy and its culture medium. Background Art
[0002] Phellodendron amurense, a traditional Chinese medicine name, is the dried bark of the plant Phellodendron chinense Schneid. in the Rutaceae family, commonly known as "Sichuan Phellodendron amurense". After stripping the bark, the rough bark is removed and dried in the sun. The outer surface is yellowish-brown or yellowish-brown, flat or with longitudinal grooves, and some may show lenticel scars and remaining grayish-brown rough bark; the inner surface is dark yellow or light brown, with fine longitudinal rib patterns; it is light in weight, hard in texture, fibrous in cross-section, split into layers, dark yellow; it has a faint smell and an extremely bitter taste, and is sticky when chewed. Phellodendron amurense has medicinal effects such as clearing heat and drying dampness, purging fire and removing steaming, detoxifying and treating sores, antibacterial and anti-inflammatory, and has been applied in traditional Chinese medicine.
[0003] Phellodendron amurense is a potential flower border plant with a large demand. It is difficult to meet the market demand solely by traditional propagation methods. Its seedling propagation cycle is long, sowing efficiency is low, and the supply of seedlings is seasonally restricted. Therefore, there is an urgent need to provide a cultivation method that can improve the propagation efficiency and break through limitations such as seasons. Summary of the Invention
[0004] In order to provide a cultivation method for Phellodendron amurense with high medicinal efficacy, this application provides a method for rapid tissue culture propagation of Phellodendron amurense with high medicinal efficacy and its culture medium.
[0005] A method for rapid tissue culture propagation of Phellodendron amurense with high medicinal efficacy provided by this application adopts the following technical solution:
[0006] A method for rapid tissue culture propagation of Phellodendron amurense with high medicinal efficacy includes the following steps:
[0007] S1. Prepare a culture medium for inducing the tender buds of Phellodendron amurense, adjust the pH value of the culture medium to 5.8 - 6.0, and sterilize it for later use;
[0008] S2. Select the tender buds of Phellodendron amurense on a healthy female tree without pests and diseases, wash them clean with distilled water; and disinfect the tender buds;
[0009] S3. Place the disinfected tender buds on a sterilized filter paper to absorb the moisture on the surface of the tender buds, and inoculate them into the culture medium prepared in S1 for cultivation;
[0010] S4. Prepare a culture medium for rooting Phellodendron amurense, adjust the pH value of the culture medium to 5.8 - 6.0, and sterilize it for later use;
[0011] S5. Transfer the induced cluster buds of Phellodendron amurense tender buds into the culture medium prepared in S4 for cultivation.
[0012] Preferably, the method for disinfecting the tender buds in S2 includes the following steps:
[0013] Under aseptic conditions, the tender buds are first disinfected with 75% alcohol for 30 - 40 seconds, rinsed several times with sterile water, then disinfected with 0.1% mercuric chloride for 6 - 8 minutes, and rinsed several times with sterile water.
[0014] Preferably, the culture conditions in S3 are a temperature of 25 ± 2 °C, a humidity of 50 - 70%, and a culture period of 7 - 14 days.
[0015] Preferably, the culture conditions in S5 are a culture room temperature of 25 ± 2 °C, a humidity of 50 - 70%, and a culture period of 21 - 35 days.
[0016] The culture medium for a method of rapid tissue culture and propagation of Phellodendron amurense with high drug efficacy provided by this application adopts the following technical solution:
[0017] A culture medium for a method of rapid tissue culture and propagation of Phellodendron amurense with high drug efficacy includes a culture medium for inducing tender buds of Phellodendron amurense and a culture medium for rooting of Phellodendron amurense.
[0018] Preferably, the culture medium for inducing tender buds of Phellodendron amurense includes a basal medium, 6-benzylaminopurine 6-BA, indolebutyric acid IBA, indoleacetic acid IAA, sucrose, and agar.
[0019] Preferably, the concentration of 6-benzylaminopurine 6-BA is 0.1 - 1 mg / L, the concentration of indolebutyric acid IBA is 0.1 - 1 mg / L, the concentration of indoleacetic acid IAA is 0.1 - 1 mg / L, the concentration of sucrose is 20 - 30 g / L, and the concentration of agar is 6 - 7 g / L.
[0020] Preferably, the culture medium for rooting of Phellodendron amurense includes a basal medium, indoleacetic acid IAA, naphthaleneacetic acid NAA, sucrose, and agar.
[0021] Preferably, the concentration of indoleacetic acid IAA is 0.1 - 1 mg / L, the concentration of naphthaleneacetic acid NAA is 0.01 - 0.1 mg / L, the concentration of sucrose is 20 - 30 g / L, and the concentration of agar is 6 - 7 g / L.
[0022] Preferably, the basal medium is MS medium.
[0023] In summary, this application includes at least one of the following beneficial technical effects:
[0024] 1. The culture medium provided by this application for obtaining regenerated plants of Phellodendron amurense can breed excellent plants with high drug efficacy, quickly obtain excellent female seedlings, shorten the seedling raising time, reduce costs, the survival rate of tender bud differentiation induction is up to over 85%, and the rooting rate can reach over 95%;
[0025] 2. In this application, 0.1 - 1 mg / L of 6-benzylaminopurine (6-BA), 0.1 - 1 mg / L of indolebutyric acid (IBA), and 0.1 - 1 mg / L of indoleacetic acid (IAA) are added to the MS medium, which can effectively promote the differentiation of tissue culture seedlings of Phellodendron amurense with high drug efficacy. Detailed implementation manners
[0026] The following further elaborates on this application in conjunction with examples.
[0027] Example 1
[0028] S1. Prepare the medium for inducing the buds of Phellodendron amurense: Select the MS medium as the basal medium, and add 6-benzylaminopurine (6-BA), indolebutyric acid (IBA), indoleacetic acid (IAA), sucrose, and agar; their concentrations are 0.1 mg / L for 6-benzylaminopurine (6-BA), 0.1 mg / L for indolebutyric acid (IBA), 0.1 mg / L for indoleacetic acid (IAA), 20 g / L for sucrose, and 6 g / L for agar; and adjust the pH value of the medium to 5.8, then place it in a high-pressure sterilizer at 121 °C for sterilization for 20 min and set aside for use.
[0029] S2. Select the buds of Phellodendron amurense on the female trees with strong growth and no pests and diseases, and wash them clean with distilled water; and disinfect the buds in a super-clean workbench. In a sterile environment, first disinfect the buds with 75% alcohol for 30 seconds, and rinse them 3 times with sterile water, then disinfect them with 0.1% mercuric chloride for 6 minutes, and rinse them 3 times with sterile water.
[0030] S3. Place the disinfected buds on a sterilized filter paper to absorb the moisture on the surface of the buds, and inoculate them into the medium prepared in S1. Incubate them in an incubator at a temperature of 25 ± 2 °C and a humidity of 50% for 7 days.
[0031] S4. Prepare the medium for rooting of Phellodendron amurense: Select the MS medium as the basal medium, and add the medium, indoleacetic acid (IAA), naphthaleneacetic acid (NAA), sucrose, and agar, and set aside for use after sterilization; their concentrations are 0.1 mg / L for indoleacetic acid (IAA), 0.01 mg / L for naphthaleneacetic acid (NAA), 20 g / L for sucrose, and 6 g / L for agar; and adjust the pH value of the medium to 5.8, then place it in a high-pressure sterilizer at 121 °C for sterilization for 20 min and set aside for use.
[0032] S5. Transfer the induced clustered buds of Phellodendron amurense into the medium prepared in S4, and incubate them in an incubator at a temperature of 25 ± 2 °C and a humidity of 50% for 21 days.
[0033] Example 2
[0034] S1. Prepare a medium for inducing young shoots of Phellodendron amurense: Select MS medium as the basal medium and add 6-benzylaminopurine (6-BA), indolebutyric acid (IBA), indoleacetic acid (IAA), sucrose, and agar. Their concentrations are as follows: the concentration of 6-BA is 0.1 mg / L, the concentration of IBA is 0.5 mg / L, the concentration of IAA is 0.5 mg / L, the concentration of sucrose is 25 g / L, and the concentration of agar is 6.5 g / L. Adjust the pH value of the medium to 5.9, and place it in a high-pressure sterilizer at 123 °C for sterilization for 22 minutes and then set aside for use.
[0035] S2. Select young shoots of Phellodendron amurense from healthy female plants without pests and diseases, and wash them clean with distilled water. Disinfect the young shoots in a laminar flow hood. In a sterile environment, first disinfect the young shoots with 75% alcohol for 35 seconds, rinse them 3 times with sterile water, then disinfect them with 0.1% mercuric chloride for 7 minutes, and rinse them 3 times with sterile water.
[0036] S3. Place the disinfected young shoots on sterilized filter paper to absorb the moisture on the surface of the young shoots, and inoculate them into the medium prepared in S1. Incubate them in an incubator at a temperature of 25 ± 2 °C and a humidity of 60% for 7 days.
[0037] S4. Prepare a medium for rooting of Phellodendron amurense: Select MS medium as the basal medium and add the medium, indoleacetic acid (IAA), naphthaleneacetic acid (NAA), sucrose, and agar, and set aside for use after sterilization. Their concentrations are as follows: the concentration of IAA is 0.5 mg / L, the concentration of NAA is 0.01 mg / L, the concentration of sucrose is 25 g / L, and the concentration of agar is 6.5 g / L. Adjust the pH value of the medium to 5.9, and place it in a high-pressure sterilizer at 123 °C for sterilization for 22 minutes and then set aside for use.
[0038] S5. Transfer the induced clustered shoots of Phellodendron amurense to the medium prepared in S4, and incubate them in an incubator at a temperature of 25 ± 2 °C and a humidity of 60% for 21 days.
[0039] Example 3
[0040] S1. Prepare a medium for inducing young shoots of Phellodendron amurense: Select MS medium as the basal medium and add 6-benzylaminopurine (6-BA), indolebutyric acid (IBA), indoleacetic acid (IAA), sucrose, and agar. Their concentrations are as follows: the concentration of 6-BA is 0.1 mg / L, the concentration of IBA is 1 mg / L, the concentration of IAA is 1 mg / L, the concentration of sucrose is 30 g / L, and the concentration of agar is 7 g / L. Adjust the pH value of the medium to 6.0, and place it in a high-pressure sterilizer at 125 °C for sterilization for 25 minutes and then set aside for use.
[0041] S2. Select the fresh and tender buds of Phellodendron amurense Rupr. on healthy female plants without diseases and pests, and wash them clean with distilled water; disinfect the buds in a laminar flow hood. Under sterile conditions, first disinfect the buds with 75% alcohol for 40 seconds, rinse them 3 times with sterile water, then disinfect them with 0.1% mercuric chloride for 8 minutes, and rinse them 3 times with sterile water;
[0042] S3. Place the disinfected buds on sterilized filter paper to absorb the moisture on the surface of the buds, and inoculate them into the medium prepared in S1. Incubate them in an incubator at a temperature of 25±2°C and a humidity of 70% for 7 days;
[0043] S4. Prepare the medium for the rooting of Phellodendron amurense Rupr.: Select the MS medium as the basal medium, and add the medium, indoleacetic acid (IAA), naphthaleneacetic acid (NAA), sucrose, and agar. After sterilization, it is ready for use; the concentrations are as follows: the concentration of indoleacetic acid (IAA) is 1 mg / L, the concentration of naphthaleneacetic acid (NAA) is 0.01 mg / L, the concentration of sucrose is 30 g / L, and the concentration of agar is 7 g / L; adjust the pH value of the medium to 6.0, and place it in a high-pressure sterilizer at 125°C for sterilization for 25 minutes and then it is ready for use;
[0044] S5. Transfer the induced cluster buds of Phellodendron amurense Rupr. into the medium prepared in S4, and incubate them in an incubator at a temperature of 25±2°C and a humidity of 70% for 21 days.
[0045] Example 4
[0046] The difference between Example 4 and Example 1 is that the concentration of 6-benzylaminopurine (6-BA) used in Example 4 is 0.5 mg / L.
[0047] Example 5
[0048] The difference between Example 5 and Example 1 is that the concentration of 6-benzylaminopurine (6-BA) used in Example 5 is 1 mg / L.
[0049] Example 6
[0050] The difference between Example 6 and Example 1 is that the concentration of 6-benzylaminopurine (6-BA) used in Example 6 is 0.05 mg / L.
[0051] Example 7
[0052] The difference between Example 7 and Example 1 is that the concentration of 6-benzylaminopurine (6-BA) used in Example 7 is 1.5 mg / L.
[0053] Example 8
[0054] The difference between Example 8 and Example 1 is that the number of days of incubation in S3 in Example 8 is 10 days.
[0055] Example 9
[0056] The difference between Example 9 and Example 1 is that in Example 9, the number of days of cultivation in S3 is 14 days.
[0057] Example 10
[0058] The difference between Example 10 and Example 1 is that in Example 10, the number of days of cultivation in S3 is 3 days.
[0059] Example 11
[0060] The difference between Example 11 and Example 1 is that in Example 11, the number of days of cultivation in S3 is 17 days.
[0061] Example 12
[0062] The difference between Example 12 and Example 1 is that in Example 12, the concentration of naphthalene acetic acid (NAA) used is 0.05 mg / L.
[0063] Example 13
[0064] The difference between Example 13 and Example 1 is that in Example 13, the concentration of naphthalene acetic acid (NAA) used is 0.1 mg / L.
[0065] Example 14
[0066] The difference between Example 14 and Example 1 is that in Example 14, the concentration of naphthalene acetic acid (NAA) used is 0.001 mg / L.
[0067] Example 15
[0068] The difference between Example 15 and Example 1 is that in Example 15, the concentration of naphthalene acetic acid (NAA) used is 0.15 mg / L.
[0069] Example 16
[0070] The difference between Example 16 and Example 1 is that in Example 16, the number of days of cultivation in S5 is 29 days.
[0071] Example 17
[0072] The difference between Example 17 and Example 1 is that in Example 17, the number of days of cultivation in S5 is 35 days.
[0073] Example 18
[0074] The difference between Example 18 and Example 1 is that in Example 18, the number of days of cultivation in S5 is 14 days.
[0075] Example 19
[0076] Example 19 is different from Example 1 in that the number of days of cultivation in S5 in Example 19 is 42 days.
[0077] Comparative Example 1
[0078] S1. Prepare a culture medium for inducing Phellodendron amurense buds: Select MS medium as the basal medium and add 6-benzylaminopurine 6-BA, indolebutyric acid IBA, indoleacetic acid IAA, sucrose, and agar. Their concentrations are as follows: the concentration of 6-benzylaminopurine 6-BA is 0.1 mg / L, the concentration of indolebutyric acid IBA is 0.1 mg / L, the concentration of indoleacetic acid IAA is 0.1 mg / L, the concentration of sucrose is 20 g / L, and the concentration of agar is 6 g / L. Adjust the pH value of the culture medium to 5.8, place it in a high-pressure sterilizer at 121 °C for sterilization for 20 min, and then set aside for use.
[0079] S2. Select the Phellodendron amurense buds on the female trees with strong growth and no pests and diseases, and wash them clean with distilled water. Disinfect the buds in a laminar flow hood. In a sterile environment, first disinfect the buds with 75% alcohol for 30 seconds, rinse them several times with sterile water, then disinfect them with 0.1% mercuric chloride for 6 minutes, and rinse them several times with sterile water.
[0080] S3. Place the disinfected buds on a sterilized filter paper to absorb the moisture on the surface of the buds, inoculate them into the culture medium prepared in S1, and culture them in an incubator at a temperature of 25 ± 2 °C and a humidity of 50% for 30 days.
[0081] Comparative Example 2
[0082] S1. Prepare a culture medium for rooting Phellodendron amurense: Select MS medium as the basal medium and add a culture medium, indoleacetic acid IAA, naphthaleneacetic acid NAA, sucrose, and agar, and set aside for use after sterilization. Their concentrations are as follows: the concentration of indoleacetic acid IAA is 0.1 mg / L, the concentration of naphthaleneacetic acid NAA is 0.01 mg / L, the concentration of sucrose is 20 g / L, and the concentration of agar is 6 g / L. Adjust the pH value of the culture medium to 5.8, place it in a high-pressure sterilizer at 121 °C for sterilization for 20 min, and then set aside for use.
[0083] S2. Select the Phellodendron amurense buds on the female trees with strong growth and no pests and diseases, and wash them clean with distilled water. Disinfect the buds in a laminar flow hood. In a sterile environment, first disinfect the buds with 75% alcohol for 30 seconds, rinse them several times with sterile water, then disinfect them with 0.1% mercuric chloride for 6 minutes, and rinse them several times with sterile water.
[0084] S3. Place the disinfected buds on a sterilized filter paper to absorb the moisture on the surface of the buds, inoculate them into the culture medium prepared in S1, and culture them in an incubator at a temperature of 25 ± 2 °C and a humidity of 50% for 30 days.
[0085] Performance detection test
[0086] I. The differentiation induction survival rate and rooting rate of the tender buds in Examples 1-19 and Comparative Examples 1-2 were counted, and the results are shown in Table 1.
[0087] The specific detection results are as follows:
[0088] Table 1 Performance detection results
[0089]
[0090]
[0091] It can be seen from the detection method in Table 1 that for a method for rapid tissue culture propagation of Phellodendron amurense with high drug efficacy and its culture medium provided by the present application, the differentiation induction survival rate of tender buds is as high as over 85%, and the rooting rate can reach over 95%. This shows that the method for rapid tissue culture propagation of Phellodendron amurense and its culture medium provided by the present application can effectively promote the success of tissue culture seedlings of Phellodendron amurense with high drug efficacy.
[0092] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the Patent Law.
Claims
1. A method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy, characterized in that: The following steps are involved: S1. Prepare a medium for inducing Phellodendron chinense shoots, adjust the pH value of the medium to 5.8-6.0, and sterilize it for later use; S2. Select the young shoots of Phellodendron chinense from healthy and pest-free female trees and clean them with distilled water; and sterilize the young shoots; S3. Place the sterilized sprouts on sterilized filter paper to absorb moisture from the sprout surface, and inoculate in the medium configured by S1 for cultivation; S4. Prepare a culture medium for Phellodendron chinense rooting, adjust the pH value of the culture medium to 5.8-6.0, and sterilize it for later use; S5. The induced Phellodendron amurense shoots are transferred into the culture medium configured in S4 for cultivation.
2. The method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 1, characterized in that: S2 The method for disinfecting tender shoots comprises the following steps: Under a sterile environment, the young shoots are first disinfected with 75% alcohol for 30-40 seconds and rinsed with sterile water several times, then disinfected with 0.1% mercuric chloride for 6-8 minutes and rinsed with sterile water several times.
3. The method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 1, characterized in that: The culture conditions of S3 are as follows: temperature 25±2° C., humidity 50-70%, and culture for 7-14 days.
4. The method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy and its culture medium according to claim 1, characterized in that: The culture conditions in S5 are as follows: the culture room temperature is 25±2° C., the humidity is 50-70%, and the culture period is 21-35 days.
5. A culture medium for a method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy, characterized in that: The utility model comprises a culture medium for inducing young shoots of Phellodendron amurense and a culture medium for rooting of Phellodendron amurense.
6. The culture medium for the method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 5, characterized in that: The culture medium for inducing young shoots of Phellodendron amurense comprises a basic culture medium, 6-benzylaminopurine 6-BA, indolebutyric acid IBA, indoleacetic acid IAA, sucrose and agar.
7. The culture medium for the method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 6, characterized in that: The concentration of 6-benzylaminopurine 6-BA is 0.1-1 mg / L, the concentration of indolebutyric acid IBA is 0.1-1 mg / L, the concentration of indoleacetic acid IAA is 0.1-1 mg / L, the concentration of sucrose is 20-30 g / L, and the concentration of agar is 6-7 g / L.
8. The culture medium for the method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 5, characterized in that: The culture medium for Phellodendron amurense rooting comprises a basic culture medium, indoleacetic acid IAA, naphthylacetic acid NAA, sucrose and agar.
9. The culture medium for the method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 8, characterized in that: The concentration of the indoleacetic acid IAA is 0.1-1 mg / L, the concentration of the naphthylacetic acid NAA is 0.01-0.1 mg / L, the concentration of sucrose is 20-30 g / L, and the concentration of agar is 6-7 g / L.
10. The culture medium for the method for rapid propagation of Phellodendron chinense tissue culture with high medicinal efficacy according to claim 6 or 8, characterized in that: The basic culture medium is MS culture medium.