Tissue culture rapid propagation method of brettunia and application thereof

Abstract

The application relates to a tissue culture and rapid propagation method of Ben Duo and application thereof, and belongs to the technical field of plant tissue culture. A tissue culture medium combination of the Ben Duo comprises an explant differentiation culture medium, a cluster shoot induction culture medium and a rooting culture medium. The high-efficiency tissue culture and rapid propagation method of the Ben Duo and the culture medium for cultivating Ben Duo regenerated plants prepared by the method can be used for rapidly breeding excellent plants, shortens a seedling raising period, reduces the cost of fry, significantly improves the propagation efficiency of the Ben Duo, realizes high-efficiency and low-cost breeding of the Ben Duo, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a novel rapid tissue culture propagation method and its application. Background Technology

[0002] Bendu is a plant native to Africa, with large, bright green leaves and swollen tubers underground, making it a typical dual-purpose crop of leafy greens and root vegetables. It has a short growth cycle, frequent harvests, and high yields. Under suitable conditions, leaves can be harvested every 15 days, with a single plant yielding approximately 1.5 kilograms of leaves per harvest, exhibiting both rapid growth and high yield. In addition, bendu is rich in dietary fiber, vitamins, and minerals, promoting intestinal peristalsis and aiding digestion. Local Africans boil its leaves or tubers in water to drink, using it to treat constipation and relieve gastrointestinal discomfort; it also helps quickly restore energy, making it suitable for consumption after work. Long-term, moderate consumption can help control blood sugar, relieve fatigue, and enhance physical strength. Besides direct consumption, bendu can also be processed, such as into tea, further increasing product added value and expanding industrial development space. As a niche African crop with both edible and potential health benefits, bendu has considerable development potential in the specialty agricultural product market and the healthy food sector.

[0003] Due to the short seed lifespan and low germination rate of this plant, asexual reproduction is the primary method, mainly through tuber division and cuttings. Tuber division relies heavily on mother plant resources and incurs high initial costs; cuttings have a relatively low survival rate, require meticulous management, and have a low propagation coefficient, making them unsuitable for large-scale seedling production. Furthermore, long-term asexual reproduction can lead to severe seed degeneration, such as smaller tubers, reduced yield, thinner leaves, decreased stress resistance, and slowed growth cycles. Currently, there is little research on tissue culture technology for this plant, and no industrially viable rapid propagation system based on tissue culture has been established. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid tissue culture propagation method for native plants and its application, in order to solve the problem that the existing technology lacks a tissue culture method that can produce native plants with high stress resistance, high quality and high yield.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a novel tissue culture medium combination, including an explant differentiation medium, a clump seedling induction medium, and a rooting medium; The explant differentiation medium is MS-based and also includes the following components at the following concentrations: 6-benzylaminopurine 0.1–0.5 mg / L, naphthaleneacetic acid 0.02–0.2 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L; The clump-forming induction medium is MS-based and also includes the following components at the following concentrations: 6-benzylaminopurine 0.1–0.5 mg / L, naphthaleneacetic acid 0.02–0.1 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L; The rooting medium is MS-based and also includes the following components at the following concentrations: indolebutyric acid 0.2–0.8 mg / L, naphthaleneacetic acid 0.02–0.06 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L.

[0006] The present invention also provides the application of the aforementioned tissue culture medium combination in the culture of this tissue and / or plant.

[0007] This invention also provides a rapid tissue culture propagation method, comprising the following steps: (1) Take stem segments of this degree, disinfect them with 75% alcohol, soak them in mercuric chloride solution, and then obtain virus-free explants; (2) The virus-free explants were cultured in the explant differentiation medium for 5-10 days to obtain germinating seedlings; (3) Place the germinating seedlings in a clump seedling induction medium and culture them for 10-20 days to obtain tissue culture seedlings; (4) Place the tissue culture seedlings in the rooting medium and culture for 15-20 days to obtain the plantlets.

[0008] Preferably, the disinfection time in step (1) is 20-60 seconds, and the soaking time is 5-10 minutes; The mass concentration of the mercuric chloride solution is 0.05% to 0.2%.

[0009] Preferably, the culture temperature in step (2) is 20-28℃, the humidity is 50%-70%, and the light exposure time is 12-18h / d.

[0010] Preferably, the culture temperature in step (3) is 20-28℃, the humidity is 50%-70%, and the light exposure time is 12-18h / d.

[0011] Preferably, the culture temperature in step (4) is 20-28°C, the humidity is 50%-70%, and the light exposure time is 12-18 h / d.

[0012] This invention also provides the application of the aforementioned tissue culture rapid propagation method in local tissue and / or plant culture.

[0013] The present invention has the following technical effects and advantages: The efficient tissue culture and rapid propagation method for Bendu established in this invention, along with the culture medium prepared for cultivating Bendu regenerated plants, can rapidly propagate superior plants, shorten the seedling cycle, reduce fry costs, achieve a stem segment axillary bud seedling rate of over 80%, a bud proliferation coefficient of 4-5, and a rooting rate of over 95%, significantly improving the propagation efficiency of Bendu and realizing efficient and low-cost propagation of Bendu. This method is suitable for large-scale production and provides high-quality seedlings for the functional exploration and development of Bendu. Attached Figure Description

[0014] Figure 1 This is a flowchart of the tissue culture rapid propagation method of the present invention; Figure 2 These are germinating seedlings obtained using the tissue culture rapid propagation method of the present invention. Figure 3 The tissue culture seedlings of this type are obtained by the rapid tissue culture propagation method of the present invention; Figure 4 The plantlets obtained by the tissue culture rapid propagation method of the present invention are native plants. Detailed Implementation

[0015] This invention provides a novel tissue culture medium combination, including an explant differentiation medium, a clump seedling induction medium, and a rooting medium; The explant differentiation medium is MS-based and further comprises the following components at the following concentrations: 6-benzylaminopurine (6-BA) 0.1–0.5 mg / L, preferably 0.5 mg / L; naphthaleneacetic acid (NAA) 0.02–0.2 mg / L, preferably 0.2 mg / L; sucrose 20–40 g / L, preferably 30 g / L; agar 5–10 g / L, preferably 7 g / L; pH 5.8. The clump-forming induction medium is MS-based and further comprises the following components at the following concentrations: 6-BA 0.1–0.5 mg / L, preferably 0.2 mg / L; NAA 0.02–0.1 mg / L, preferably 0.05 mg / L; sucrose 20–40 g / L, preferably 30 g / L; agar 5–10 g / L, preferably 7 g / L; pH 5.8. The rooting medium is MS-based and also includes the following components at the following concentrations: indolebutyric acid (IBA) 0.2–0.8 mg / L, preferably 0.4 mg / L; NAA 0.02–0.06 mg / L, preferably 0.02 mg / L; sucrose 20–40 g / L, preferably 30 g / L; agar 5–10 g / L, preferably 7 g / L; pH 5.8.

[0016] The present invention also provides the application of the aforementioned tissue culture medium combination in the culture of this tissue and / or plant.

[0017] This invention also provides a rapid tissue culture propagation method, comprising the following steps: (1) Take stem segments of this degree, disinfect them with 75% alcohol, soak them in mercuric chloride solution, and then obtain virus-free explants; (2) Place the virus-free explants in the explant differentiation medium and culture for 5-10 days, preferably 7 days, to obtain germinating seedlings; (3) Place the germinating seedlings in a clump seedling induction medium and culture them for 10-20 days, preferably 10 days, to obtain tissue culture seedlings; (4) Place the tissue culture seedlings in a rooting medium and culture for 15 to 20 days, preferably 15 days, to obtain the plantlets.

[0018] In this invention, the stem segment is preferably a young, non-lignified stem segment from a female plant of this type.

[0019] In this invention, the disinfection time in step (1) is 20-60 seconds, preferably 30 seconds, and the soaking time is 5-10 minutes, preferably 8 minutes; The mass concentration of the mercuric chloride solution is 0.05% to 0.2%, preferably 0.1%.

[0020] In this invention, the culture temperature in step (2) is 20-28°C, preferably 25°C; the culture humidity is 50%-70%, preferably 60%; and the culture light duration is 12-18 h / d, preferably 16 h / d.

[0021] In this invention, the culture temperature in step (3) is 20-28°C, preferably 25°C; the culture humidity is 50%-70%, preferably 60%; and the culture light duration is 12-18 h / d, preferably 16 h / d.

[0022] In this invention, the culture temperature in step (4) is 20-28°C, preferably 25°C; the culture humidity is 50%-70%, preferably 60%; and the culture light duration is 12-18 h / d, preferably 16 h / d.

[0023] This invention also provides the application of the aforementioned tissue culture rapid propagation method in local tissue and / or plant culture.

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1: Preparation of explant differentiation culture medium Take 0.5 mg of 6-BA, 0.2 mg of NAA, 30 g of sucrose, and 7 g of agar and make up to 1 L of MS culture medium. Adjust the pH to 5.8 to obtain the explant differentiation medium.

[0026] Example 2: Preparation of induction medium for clump-forming seedlings Take 0.2 mg of 6-BA, 0.05 mg of NAA, 30 g of sucrose, and 7 g of agar and make up to 1 L of MS culture medium. Adjust the pH to 5.8 to obtain the induction medium for clump seedlings.

[0027] Example 3: Preparation of rooting medium Take 0.4 mg of IBA, 0.02 mg of NAA, 30 g of sucrose, and 7 g of agar and make up to 1 L of MS culture medium. Adjust the pH to 5.8 to obtain the rooting medium.

[0028] Example 4: This method of rapid tissue culture propagation Young, non-lignified stem segments from healthy, disease-free female plants were selected. After washing with distilled water, the segments were sequentially disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, soaked in 0.1% mercuric chloride solution for 8 minutes, and rinsed five times with sterile water to obtain virus-free explants. These virus-free explants were inoculated into the explant differentiation medium prepared in Example 1 and cultured for 7 days at 25°C, 60% humidity, and 16 h / d light to obtain germinating seedlings. The survival rate of the germinating seedlings was calculated. The germinating seedlings were then inoculated into the explant differentiation medium prepared in Example 2 and cultured for 10 days at 25°C, 60% humidity, and 16 h / d light to obtain tissue culture seedlings. The proliferation coefficient of the clustered buds in the tissue culture seedlings was calculated. Finally, the tissue culture seedlings were inoculated into the rooting medium prepared in Example 3 and cultured for 15 days at 25°C, 60% humidity, and 16 h / d light to obtain native plants. The rooting rate of the native plants was calculated. The results are as follows Figures 1-4 As shown.

[0029] The results showed that the survival rate of germinating seedlings was 85%, the proliferation coefficient of tissue culture seedlings was 4-5, and the rooting rate of the plants was 96%.

[0030] As can be seen from the above embodiments, the present invention provides a rapid propagation method for basal tissue culture and its application. The efficient rapid propagation method for basal tissue culture established by the present invention and the culture medium prepared for cultivating basal regenerated plants can rapidly propagate superior plants, shorten the seedling cycle, reduce the cost of fry, and significantly improve the reproductive efficiency of basal tissue culture, realizing efficient and low-cost propagation of basal tissue culture, which is suitable for large-scale production.

[0031] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tissue culture medium composition, characterized in that, Including explant differentiation medium, clump seedling induction medium, and rooting medium; The explant differentiation medium is MS-based and also includes the following components at the following concentrations: 6-benzylaminopurine 0.1–0.5 mg / L, naphthaleneacetic acid 0.02–0.2 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L; The clump-forming induction medium is MS-based and also includes the following components at the following concentrations: 6-benzylaminopurine 0.1–0.5 mg / L, naphthaleneacetic acid 0.02–0.1 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L; The rooting medium is MS-based and also includes the following components at the following concentrations: indolebutyric acid 0.2–0.8 mg / L, naphthaleneacetic acid 0.02–0.06 mg / L, sucrose 20–40 g / L, and agar 5–10 g / L.

2. The use of the tissue culture medium combination according to claim 1 in the culture of this tissue and / or plant.

3. A novel tissue culture rapid propagation method, characterized in that, Includes the following steps: (1) Take stem segments of this degree, disinfect them with 75% alcohol, soak them in mercuric chloride solution, and then obtain virus-free explants; (2) The virus-free explants were cultured in the explant differentiation medium for 5-10 days to obtain germinating seedlings; (3) Place the germinating seedlings in a clump seedling induction medium and culture them for 10-20 days to obtain tissue culture seedlings; (4) Place the tissue culture seedlings in rooting medium and culture for 15-20 days to obtain the plantlets. The explant differentiation culture medium, clump seedling induction culture medium, and rooting culture medium mentioned in steps (2) to (4) are the explant differentiation culture medium, clump seedling induction culture medium, and rooting culture medium as described in claim 1.

4. The tissue culture rapid propagation method according to claim 3, characterized in that, The disinfection time in step (1) is 20-60 seconds, and the soaking time is 5-10 minutes; The mass concentration of the mercuric chloride solution is 0.05% to 0.2%.

5. The tissue culture rapid propagation method according to claim 4, characterized in that, The culture temperature in step (2) is 20-28℃, the humidity is 50%-70%, and the light exposure time is 12-18h / d.

6. The tissue culture rapid propagation method according to claim 5, characterized in that, The culture temperature in step (3) is 20-28℃, the humidity is 50%-70%, and the light exposure time is 12-18h / d.

7. The tissue culture rapid propagation method according to claim 6, characterized in that, The culture temperature in step (4) is 20-28℃, the humidity is 50%-70%, and the light exposure time is 12-18h / d.

8. The application of the tissue culture rapid propagation method according to any one of claims 3 to 7 in the culture of this tissue and / or plant.

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