Rapid in-vitro propagation method for psychotria serpens leaves
Through the ex vivo rapid breeding method of syllium leaves, disinfection and specific culture medium induce rooting of uncertain buds, the problems of low reproduction rate and genetic instability in syllium planting are solved, and efficient and stable large-scale production is achieved.
Patent Information
- Application Number
- CN202510774253.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-07-11
AI Technical Summary
The existing cypress planting model is difficult to meet the rapidly growing market demand. The traditional reproduction rate is low, the growth cycle is long, and the genetic traits are unstable, so large-scale production cannot be achieved.
The syllium leaves are used as explants, and uncertain buds and rooting are induced by disinfection, cutting and inoculation on specific culture media, the hormone ratio is optimized, the culture cycle is shortened, and the reproduction rate and genetic stability are improved.
It achieves efficient and stable asexual reproduction of pine blue leaves, shortens the seedling cycle, is suitable for large-scale production, meets market demand, and breaks seasonal restrictions.
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Figure CN120283662A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of Isatis indigotica rapid propagation, and particularly relates to an in vitro rapid propagation method for Isatis indigotica leaves. Background Art
[0002] Isatis indigotica Fort. Isatis indigotica , belonging to the genus Isatis of the family Brassicaceae, is a biennial herb. Its dried root (i.e., Isatis root) and leaves (i.e., Folium Isatidis) are both Chinese medicinal materials with a bitter taste and a cold nature, and they belong to the heart and stomach meridians. In traditional medicine, they are widely used for their significant pharmacological activities such as clearing heat and detoxifying, and antiviral effects, and are mainly used to treat influenza, upper respiratory tract infections, viral hepatitis and other diseases. Modern pharmacological research further reveals the multiple pharmacological effects of Isatis indigotica. Its rich bioactive components such as indigo, isoindigo, amino acids and phenolic compounds not only have antiviral, antibacterial and immunomodulatory functions, but also show potential application values in anti-tumor and anti-inflammatory aspects. Therefore, Isatis indigotica shows great application potential in the field of modern drug research and development, especially in the development of antiviral drugs.
[0003] However, due to the large amount of Isatis indigotica used in traditional Chinese medicine and the continuous growth of the demand for Isatis indigotica in the medical field, the traditional planting mode has been difficult to meet its rapidly growing market demand. In the prior art, the tissue culture rapid propagation method of Isatis indigotica uses leaves or the epicotyls of Isatis indigotica seedlings as explants to cultivate tissue culture seedlings. However, these methods have low propagation rates, long growth cycles, unstable genetic traits, and cannot be produced on a large scale. Based on this, it is urgent for those skilled in the art to provide an in vitro rapid propagation method for Isatis indigotica leaves that can achieve efficient proliferation, is not restricted by climate, has a short growth cycle, stable genetic traits, can achieve rapid and stable asexual reproduction, and can be produced on a large scale. Summary of the Invention
[0004] In view of the above technical problems, the present invention provides an in vitro rapid propagation method for Isatis indigotica leaves, which solves the above problems existing in the prior art.
[0005] In order to achieve the above object, the technical solutions adopted by the present invention are as follows: An in vitro rapid propagation method for Isatis indigotica leaves of the present invention comprises the following steps: S1: Selection and disinfection of explants: Cut healthy, pest-free and tender Isatis indigotica leaves, add a small amount of washing powder and rinse under running water. Use forceps to pick up the rinsed Isatis indigotica leaves and place them in a triangular flask, then transfer them into a laminar flow hood. Pour 75% ethanol into the triangular flask, immediately shake the triangular flask to disinfect the Isatis indigotica leaves in 75% ethanol for 30 s, pour out the 75% ethanol, rinse with sterile water for 3 times. Pour 0.1% mercuric chloride into the triangular flask, immediately shake the triangular flask to disinfect the Isatis indigotica leaves in 0.1% mercuric chloride for 8 min, pour out the 0.1% mercuric chloride, and rinse with sterile water for 5 times; S2: Cutting of leaves: Transfer the disinfected leaves in step S1 to a disposable sterile petri dish, remove the leaf edges with sterile scissors, and retain the main vein part; S3: Inoculation of leaves and induction of adventitious buds: Inoculate the small leaf pieces cut in step S2 with the leaf surface facing up onto the culture medium, and induce adventitious buds on the inoculated leaves; S4: Induction of adventitious roots: After cutting the induced adventitious buds into single plants with a sterile scalpel, inoculate them onto the culture medium of 1 / 2MS + 1.0 mg / L NAA for the induction of adventitious roots; S5: Transplanting of regenerated plants: Open the bottle cap of the Isatis indigotica tissue culture seedlings after rooting at room temperature, let them adapt to the room temperature environment for 3 - 4 d, then take out the plants from the tissue culture bottle, wash the culture medium on the roots clean, and transplant them into the planting substrate. Water thoroughly once every 7 - 10 d to survive.
[0006] Furthermore, in step S1, place the cut Isatis indigotica leaves in a beaker, add washing powder and rinse under running water for 30 min. The forceps are forceps disinfected with 75% alcohol, and the Erlenmeyer flask is a sterilized Erlenmeyer flask. The amount of 75% ethanol in the Erlenmeyer flask must cover the Isatis indigotica leaves, and the amount of 0.1% mercuric chloride must cover the Isatis indigotica leaves.
[0007] Furthermore, in step S2, cut the leaves into small pieces approximately 1.2 cm × 0.6 cm (length × width) with sterile scissors.
[0008] Furthermore, in step S3, the culture medium is MS + 0.5 mg / L 6 - BA or MS + 0.50 mg / L KT culture medium.
[0009] Even further, in step S3, the inoculated leaves are induced for adventitious buds under the conditions of a temperature of 25 °C, a light intensity of 1500 - 2000 lx, and a light time of 14 h•d -1 for 15 - 22 d.
[0010] Furthermore, in step S4, adventitious roots are induced under the conditions of a temperature of 25 °C, a light intensity of 1500 - 2000 lx, and a light time of 14 h•d -1 for 15 - 20 d to obtain Isatis indigotica plants with a large number of roots.
[0011] Furthermore, the planting substrate in step S5 is peat soil, vermiculite, and fine sand, and the ratio of the number of parts of peat soil, vermiculite, and fine sand is 3:1:1.
[0012] Compared with the prior art, the beneficial effects of the present invention: An in vitro rapid propagation method for Isatis indigotica Fort. leaves obtains complete plants by directly inducing adventitious buds from the leaves and then inducing root formation, avoiding multiple stages such as dedifferentiation, proliferation, and redifferentiation through the callus pathway in the prior art. Using the method of the present invention, it only takes 30 - 40 d for a single leaf to develop into a complete plant, which can shorten the culture cycle, improve the propagation rate, avoid the risk of genetic instability brought about by the callus pathway, and ensure the genetic stability of the regenerated plants. At the same time, by optimizing the hormone ratio in the culture medium, the present invention significantly improves the adventitious bud differentiation rate and rooting rate of Isatis indigotica Fort. leaf explants, enabling efficient and stable asexual propagation to meet the increasing market demand for Isatis indigotica Fort. medicinal materials. The method of the present invention establishes a standardized and scalable in vitro rapid propagation method for Isatis indigotica Fort. leaves, which can break through the seasonal restrictions, shorten the seedling raising cycle, and provide reliable technical support for the large-scale and intensive development of the Isatis indigotica Fort. industry. The explant selection and disinfection steps of the present invention are simple and easy to implement, and the culture medium components and culture conditions are easy to control, making the method easy to promote and apply and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Appendix Figure 1 is the step diagram of the in vitro rapid propagation method for Isatis indigotica Fort. leaves in this example; Appendix Figure 2 is the growth diagram of Isatis indigotica Fort. leaf adventitious bud induction on the 12th d in Example 1; Appendix Figure 3 is the growth diagram of Isatis indigotica Fort. leaf adventitious bud induction on the 22nd d in Example 2; Appendix Figure 4 is the growth diagram of Isatis indigotica Fort. leaf adventitious bud induction on the 22nd d in Example 3; Appendix Figure 5 is the growth state diagram of the complete tissue culture seedlings formed by adventitious root induction of Isatis indigotica Fort. leaves in Example 1; Appendix Figure 6 is the growth state diagram of the complete tissue culture seedlings formed by adventitious root induction of Isatis indigotica Fort. leaves in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0014] In order to make the objectives and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0015] Example 1
[0016] Referring to Appendix Figure 1 , Appendix Figure 2 and Appendix Figure 5 , an in vitro rapid propagation method for Isatis indigotica Fort. leaves in this example includes the following steps: S1: Selection and disinfection of explants: Cut healthy, pest- and disease-free and tender Isatis indigotica leaves and place them in a beaker. Add a small amount of washing powder and rinse under running water for 30 min. Use forceps sterilized with 75% alcohol to pick up the rinsed Isatis indigotica leaves and place them in a sterilized Erlenmeyer flask, then transfer it to a laminar flow hood. Pour 75% ethanol into the Erlenmeyer flask. The amount of 75% ethanol should cover the Isatis indigotica leaves. Immediately shake the Erlenmeyer flask to disinfect the Isatis indigotica leaves in 75% ethanol for 30 s. Pour out the 75% ethanol and rinse 3 times with sterile water. Pour 0.1% mercuric chloride into the Erlenmeyer flask. The amount of 0.1% mercuric chloride should cover the Isatis indigotica leaves. Immediately shake the Erlenmeyer flask to disinfect the Isatis indigotica leaves in 0.1% mercuric chloride for 8 min. Pour out the 0.1% mercuric chloride and rinse 5 times with sterile water; S2: Cutting of leaves: Transfer the disinfected leaves in step S1 to a disposable sterile Petri dish. Use sterile scissors to remove the leaf edges and retain the midrib part. Cut the leaves into small pieces about 1.2 cm × 0.6 cm (length × width); S3: Inoculation of leaves and induction of adventitious buds: Inoculate the small leaf pieces cut in step S2 with the leaf surface facing up onto the MS + 0.5 mg / L 6-BA medium. Incubate the inoculated leaves under the conditions of a temperature of 25 °C, a light intensity of 1500 lx, and a light duration of 14 h•d -1 for adventitious bud induction for 15 d. Through observation, about 7 adventitious buds can grow from each Isatis indigotica leaf; S4: Induction of adventitious roots: After cutting the induced adventitious buds into single plants with a sterile scalpel, inoculate them onto the 1 / 2MS + 1.0 mg / L NAA medium for adventitious root induction. The culture conditions for adventitious root induction are: a temperature of 25 °C, a light intensity of 2000 lx, and a light duration of 14 h•d -1 , and the culture time is 15 d, then Isatis indigotica plants with a large number of roots can be obtained. After statistics, the rooting rate of adventitious buds reaches 82%; S5: Transplanting of regenerated plants: Open the cap of the tissue culture seedlings of Isatis indigotica rooted at room temperature and let them adapt to the room temperature environment for 3 - 4 d. Then take out the plants from the tissue culture flask, wash the medium on the roots clean, and transplant them into a planting substrate with a ratio of peat soil, vermiculite and fine sand of 3:1:1. Water thoroughly once every 7 d and they can survive. After statistics, the survival rate of transplanting regenerated plants reaches 88%.
[0017] Example 2
[0018] Refer to Appendix Figure 1 , Appendix Figure 3 and Appendix Figure 6, An in vitro rapid propagation method for Isatis indigotica leaves in this embodiment. In step S3, the leaf pieces cut in step S2 are inoculated face up onto the MS + 0.50 mg / L KT medium. The inoculated leaves are induced to form adventitious buds under the conditions of a temperature of 25 °C, a light intensity of 2000 lx, and a light duration of 14 h•d -1 . The culture time is 22 days; in step S4, the light intensity for adventitious root induction is 1500 lx, and the culture time is 20 days, then Isatis indigotica plants with a large number of roots can be obtained. In step S5, watering thoroughly once every 8 days can ensure survival, and the remaining steps are the same as in Example 1. After statistics, the rooting rate of adventitious buds reaches 89%; the survival rate of transplanted regenerated plants reaches 92%.
[0019] Example 3
[0020] Referring to Appendix Figure 1 and Appendix Figure 4 , An in vitro rapid propagation method for Isatis indigotica leaves in this embodiment. In step S3, the inoculated leaves are induced to form adventitious buds under the conditions of a temperature of 25 °C, a light intensity of 1750 lx, and a light duration of 14 h•d -1 . The culture time is 22 days; in step S4, the light intensity for adventitious root induction is 1750 lx, and the culture time is 17 days, then Isatis indigotica plants with a large number of roots can be obtained. In step S5, watering thoroughly once every 10 days can ensure survival, and the remaining steps are the same as in Example 1. After statistics, the rooting rate of adventitious buds reaches 93%; the survival rate of transplanted regenerated plants reaches 80%.
[0021] In other embodiments, in step S3, the inoculated leaves can be induced to form adventitious buds under any light intensity condition within the range of 1500 - 2000 lx, and the culture time can be any time within the range of 15 - 22 days; in step S4, the light intensity for adventitious root induction can be any light intensity within the range of 1500 - 2000 lx, and the culture time can be any time within the range of 15 - 20 days, then Isatis indigotica plants with a large number of roots can be obtained. In step S5, watering thoroughly once every 7 - 10 days can ensure survival.
[0022] Beneficial effects of this embodiment: In this embodiment, adventitious buds are directly induced from leaves and then induced to root to obtain complete plants, avoiding multiple stages such as dedifferentiation, proliferation, and redifferentiation in the existing technology using the callus pathway. Using the in vitro rapid propagation method of Isatis indigotica Fort. leaves in this embodiment, it only takes 30 - 40 d to develop from one leaf into a complete plant, which can shorten the culture cycle, improve the propagation rate, avoid the risk of genetic instability brought by the callus pathway, and ensure the genetic stability of the regenerated plants. At the same time, in this embodiment, by optimizing the hormone ratio in the medium, the adventitious bud differentiation rate and rooting rate of Isatis indigotica Fort. leaf explants are significantly improved, realizing efficient and stable asexual propagation to meet the growing market demand for Isatis indigotica Fort. medicinal materials. In summary, this embodiment has established a standardized and scalable in vitro rapid propagation method for Isatis indigotica Fort. leaves, which can break through the seasonal limitation, shorten the seedling raising cycle, and provide reliable technical support for the large-scale and intensive development of the Isatis indigotica Fort. industry. The explant selection and disinfection steps in this embodiment are simple and easy to implement, and the medium components and culture conditions are easy to control, making this method easy to promote and apply and suitable for large-scale production.
[0023] The above are only the preferred embodiments of the present application and the description of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, and should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. An in vitro rapid propagation method for Isatis indigotica Fort. leaves, characterized in that: It includes the following steps: S1: Selection and disinfection of explants: Cut healthy, pest- and disease-free and tender Isatis indigotica leaves, add washing powder and rinse under running water. Use forceps to pick up the rinsed Isatis indigotica leaves and place them in a triangular flask, then transfer them to a laminar flow hood. Pour 75% ethanol into the triangular flask, immediately shake the triangular flask, disinfect for 30 s, pour out the 75% ethanol, rinse 3 times with sterile water. Pour 0.1% mercuric chloride into the triangular flask, immediately shake the triangular flask, disinfect for 8 min, pour out the 0.1% mercuric chloride, and rinse 5 times with sterile water; S2: Cutting of leaves: Transfer the disinfected leaves in step S1 to a sterile petri dish, and use sterile scissors to remove the leaf edges, leaving the main vein part; S3: Inoculation of leaves and induction of adventitious buds: Inoculate the leaf pieces cut in step S2 with the leaf surface facing up onto the medium, and induce adventitious buds for the inoculated leaves; S4: Induction of adventitious roots: After cutting the induced adventitious buds into single plants with a sterile scalpel, inoculate them onto the medium of 1 / 2MS + 1.0 mg / L NAA for the induction of adventitious roots; S5: Transplanting of regenerated plants: Open the bottle cap of the Isatis indigotica tissue culture seedlings after rooting at room temperature. After adapting to the room temperature environment for 3 - 4 d, take out the plants from the tissue culture bottle, wash the medium on the roots clean, and then transplant them into the planting substrate. Water thoroughly once every 7 - 10 d.
2. The in vitro rapid propagation method of Isatis indigotica leaves according to claim 1, characterized in that: In step S1, the cut Isatis indigotica leaves are placed in a beaker, added with washing powder and rinsed under running water for 30 min. The forceps are forceps disinfected with 75% alcohol, and the triangular flask is a sterilized triangular flask. The amount of 75% ethanol in the triangular flask must cover the Isatis indigotica leaves, and the amount of 0.1% mercuric chloride must cover the Isatis indigotica leaves.
3. The in vitro rapid propagation method of Isatis indigotica Fort. leaves according to claim 1, characterized in that: In step S2, the leaves are cut into small pieces of 1.2 cm × 0.6 cm with sterile scissors.
4. The in vitro rapid propagation method of Isatis indigotica Fort. leaves according to claim 1, characterized in that: In step S3, the medium is MS + 0.5 mg / L 6 - BA or MS + 0.50 mg / L KT medium.
5. The in vitro rapid propagation method of Isatis indigotica Fort. leaves according to claim 4, characterized in that: In the step S3, the inoculated leaves are induced to form adventitious buds under the conditions of a temperature of 25 °C, a light intensity of 1500 - 2000 lx, and a light duration of 14 h•d -1 , and the culture time is 15 - 22 d.
6. The in vitro rapid propagation method of Isatis indigotica leaves according to claim 1, characterized in that: In the step S4, adventitious root induction is carried out under the conditions of a temperature of 25 °C, a light intensity of 1500 - 2000 lx, and a light duration of 14 h•d -1 and the culture time is 15 - 20 d.
7. The in vitro rapid propagation method of Isatis indigotica Fort. leaves according to claim 1, characterized in that: The planting substrate in step S5 is peat soil, vermiculite and fine sand, and the parts ratio of peat soil, vermiculite and fine sand is 3:1:1.