A method for establishing a rapid system of motherwort plants

Abstract

This application proposes a method for establishing a rapid propagation system for Leonurus japonicus, comprising: inoculating pretreated and sterilized Leonurus japonicus explants onto a callus induction medium for primary culture; after obtaining stable callus tissue from the primary culture, placing the callus tissue into an adventitious bud culture medium to cultivate adventitious buds; once the adventitious buds have grown, placing them into a rooting medium to root, thereby obtaining Leonurus japonicus seedlings suitable for rapid planting. Using this method, the success rate of callus induction can be improved, achieving a Leonurus japonicus callus induction rate >95%, and Leonurus japonicus callus tissue conducive to adventitious bud proliferation and rooting can be obtained, thus establishing a complete and stable rapid propagation system for Leonurus japonicus. This method can significantly shorten the growth cycle of Leonurus japonicus, enabling rapid propagation to meet market demand and providing a technological foundation for the large-scale production of Leonurus japonicus.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, and in particular to a method for establishing a rapid system of Leonurus japonicus. Background Technology

[0002] Motherwort (Leonurus japonicus Houtt.) is a plant belonging to the Lamiaceae family. It possesses properties that promote blood circulation, regulate menstruation, and reduce swelling. It is used to treat irregular menstruation, dysmenorrhea, amenorrhea, lochia retention, edema with oliguria, and acute carbuncles and boils. It is a commonly used bulk medicinal material in clinical prescriptions and a major raw material for pharmaceutical companies. Due to its remarkable efficacy in gynecological medicines, the annual sales of motherwort-related medicines in China exceed 1 billion RMB. In recent years, with in-depth scientific research and increased public health awareness, the medicinal and cosmetic value of motherwort has gained widespread recognition and attention, gradually expanding from the traditional field of traditional Chinese medicine to multiple fields such as cosmetics and health products. However, while market demand is surging, the natural growth state of motherwort has become a bottleneck restricting its supply.

[0003] In its natural environment, the germination rate of motherwort seeds is relatively low. Besides seed quality issues, unsuitable environmental factors such as soil moisture, temperature, and light conditions can all inhibit germination. Furthermore, motherwort grows relatively slowly. Even under suitable growing conditions, it takes a considerable amount of time for motherwort to reach harvestable size. This leads to a longer production cycle, increases planting costs, and limits the market supply of motherwort. Therefore, overcoming the limitations of motherwort's natural growth and improving its yield and quality to meet urgent market demand has become a pressing issue that needs to be addressed.

[0004] Tissue culture technology allows for the rapid and large-scale acquisition of motherwort seedlings, significantly shortening its long natural growth cycle and overcoming the challenge of low germination rates, thus effectively meeting the market's urgent demand for motherwort raw materials. However, current research on motherwort tissue culture still focuses on the early-stage culture of callus tissue. Callus tissue forms under specific chemical induction, undergoes dedifferentiation, and exhibits stem cell characteristics, possessing high regenerative capacity; each cell has the potential to develop into a complete plant.

[0005] Although callus tissue plays a crucial role in plant tissue culture, the tissue culture technology of Leonurus japonicus still faces many challenges, especially in callus induction, proliferation, and differentiation. Therefore, we need to strengthen research on Leonurus japonicus tissue culture technology and explore more efficient and stable callus culture methods to improve its propagation efficiency and quality. To address this issue, this invention proposes a tissue culture regeneration technology. This technology allows for the acquisition of regenerated plants through tissue culture, requiring a shorter cycle and achieving more stable quality and yield. It enables mass propagation of plants and allows for year-round seedling propagation, unaffected by seasonal factors. This is crucial for promoting the sustainable development of the Leonurus japonicus industry and providing the market with more high-quality and efficient Leonurus japonicus seedlings. Summary of the Invention

[0006] This application provides a method for establishing a rapid system based on Leonurus japonicus to solve the problems existing in related technologies. The technical solution is as follows:

[0007] This application provides a method for establishing a rapid system based on Leonurus japonicus, including:

[0008] Pretreated and sterilized Leonurus japonicus explants were inoculated onto callus induction medium for primary culture.

[0009] After obtaining stable callus tissue through the first generation culture, the callus tissue was placed in an adventitious bud culture medium to cultivate buds.

[0010] Once the adventitious buds have emerged, they are placed in a rooting medium to grow roots, resulting in motherwort seedlings that can be planted quickly.

[0011] In one embodiment of the above technical solution, the motherwort explant includes one of the following: motherwort leaf, motherwort petiole, and motherwort stem segment with bud.

[0012] In one embodiment of the above technical solution, the method for pretreatment and disinfection of the motherwort explants includes: disinfecting the motherwort explants with 75% ethanol for 30-60 seconds, rinsing them three times with sterile water, then disinfecting them with 2% NaClO for 10-15 minutes. After disinfection, the explants are rinsed in sterile water 5-6 times, each time for 3-4 minutes, placed on filter paper, and air-dried in a sterile environment.

[0013] In one embodiment of the above technical solution, the callus induction culture medium is MS medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA + 0.1 mg / L NAA.

[0014] In one embodiment of the above technical solution, the initial culture conditions include: dark conditions, relative humidity of 50% to 60%, temperature of 25±1℃, and culture time of 14 to 16 days.

[0015] In one embodiment of the above technical solution, the adventitious bud culture medium is MS medium + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA.

[0016] In one embodiment of the above technical solution, the cultivation conditions for adventitious buds include: light intensity of 5000 lx, relative humidity of 50% to 60%, temperature of 25 ± 1℃, and cultivation time of 14 to 16 days.

[0017] In one embodiment of the above technical solution, the rooting medium is 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L NAA.

[0018] In one embodiment of the above technical solution, the rooting culture conditions include: light intensity of 5000 lx, relative humidity of 50% to 60%, temperature of 25±1℃, and culture time of 14 to 16 days.

[0019] In one embodiment of the above technical solution, after the adventitious buds have grown to 2-3 cm, they are then placed in a rooting culture medium to root.

[0020] The advantages or beneficial effects of the above technical solutions include at least the following:

[0021] 1. This application first induces callus tissue from *Leonurus japonicus*. Based on screening suitable conditions for callus induction, superior callus tissue is selected for further propagation and rooting culture of adventitious buds from the callus tissue. Using the method of this application, the success rate of callus induction can be improved, achieving a callus induction rate of >95%, and producing callus tissue conducive to adventitious bud proliferation and rooting, thereby establishing a complete and stable rapid propagation system for *Leonurus japonicus*. The method of this application can significantly shorten the growth cycle of *Leonurus japonicus*, enabling rapid propagation to meet market demand and providing a technical foundation for the large-scale production of *Leonurus japonicus*.

[0022] 2. The callus induction medium designed in this application exhibits extremely high efficiency in inducing callus formation in Leonurus japonicus. Compared with traditional culture methods, its induction rate is significantly improved, not only increasing the yield of Leonurus japonicus callus but also greatly enhancing its quality. This technology provides a reliable guarantee for the seedling cultivation and rapid propagation of Leonurus japonicus. By applying the callus induction medium of this application, the propagation of Leonurus japonicus can be carried out more effectively, meeting the market demand for Leonurus japonicus raw materials and further promoting its application in the fields of medicine, health care, and cosmetics.

[0023] 3. Compared with existing tissue culture techniques, this application significantly improves the induction rate, germination rate, and rooting rate of Leonurus japonicus tissue culture by innovatively adjusting the composition of the culture medium and optimizing environmental factors such as light conditions. This not only improves the rapid propagation efficiency of Leonurus japonicus, accelerating its growth cycle and shortening the breeding time, but also greatly reduces seedling costs. Furthermore, it may provide new solutions for the rapid propagation of other plants, making a greater contribution to the protection and development of plant resources.

[0024] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0025] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0026] Figure 1 This is a picture of a motherwort leaf.

[0027] Figure 2 This is a diagram of callus induced from motherwort leaves.

[0028] Figure 3 This is a diagram of callus induced from shoot explants. Detailed Implementation

[0029] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0030] This application provides a method for establishing a rapid system based on Leonurus japonicus, including:

[0031] S1, Pretreated and sterilized Leonurus japonicus explants were inoculated onto callus induction medium for primary culture;

[0032] S2, after obtaining stable callus tissue in the first generation culture, the callus tissue is placed in adventitious bud culture medium to grow buds;

[0033] S3. After the adventitious buds grow, place them in a rooting medium to root, and obtain motherwort seedlings that can be planted quickly.

[0034] In one embodiment, the motherwort explant includes one of the following: motherwort leaf, motherwort petiole, and motherwort stem segment with bud.

[0035] In one embodiment, the method for pretreatment and disinfection of the motherwort explants includes: disinfecting the motherwort explants with 75% ethanol for 30-60 seconds, rinsing them three times with sterile water, then disinfecting them with 2% NaClO for 10-15 minutes. After disinfection, the explants are rinsed in sterile water 5-6 times, each time for 3-4 minutes, placed on filter paper, and air-dried in a sterile environment.

[0036] In one embodiment, the callus induction medium is MS medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA + 0.1 mg / L NAA. This application optimizes the types and proportions of growth hormones in the callus induction medium. Compared with conventional media, this medium successfully improves the induction rate of Leonurus japonicus callus, achieving an induction rate of over 95%, thus laying the foundation for the establishment of a rapid propagation system for Leonurus japonicus.

[0037] Preferably, the optimal pH value for the callus induction medium is 5.8 to 6.0.

[0038] In one embodiment, the initial culture conditions included: darkness, relative humidity of 50%–60%, temperature of 25±1℃, and a culture time of 14–16 days. Experiments have verified that the callus induction rate of Leonurus japonicus under darkness conditions was the highest.

[0039] In one embodiment, the adventitious bud culture medium is MS medium + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA. This application optimizes the auxin ratio in the adventitious bud culture medium, which induces the differentiation of numerous clustered buds. After root induction culture, normal plants can be formed, significantly improving the induction rate of Leonurus japonicus tissue culture.

[0040] Preferably, the optimal pH value for the adventitious bud culture medium is 5.8 to 6.0.

[0041] In one embodiment, the cultivation conditions for adventitious buds include: light intensity of 5000 lx, relative humidity of 50% to 60%, temperature of 25 ± 1 °C, and cultivation time of 14 to 16 days.

[0042] In one embodiment, the rooting medium is 1 / 2 MS basal medium + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L NAA. Because motherwort has difficulty rooting, the 1 / 2 MS medium is used as the rooting medium. By reducing the concentration of inorganic salts in the rooting medium, the plant's nutrient and sugar content is decreased, reducing cell stress, promoting chlorophyll synthesis and cell division, and further promoting the rooting of adventitious buds of motherwort. The rooting rate can reach 100%, and the root system grows vigorously, improving plant growth quality and yield.

[0043] Preferably, the optimal pH value for the rooting medium is 5.8 to 6.0.

[0044] In one embodiment, the rooting culture conditions include: light intensity of 5000 lx, relative humidity of 50% to 60%, temperature of 25 ± 1℃, and culture time of 14 to 16 days.

[0045] In one implementation, once the adventitious buds have grown to 2-3 cm, they are placed in a rooting culture medium to root.

[0046] Example 1: Effects of different culture media on callus induction in Leonurus japonicus

[0047] 1. Methods and Steps

[0048] S1: Select leaves that are free from rot, pests, and damage. Disinfect with 75% ethanol for 30-60 seconds, rinse three times with sterile water, and then disinfect with 2% NaClO (sodium hypochlorite) for 15 minutes. After disinfection, rinse in sterile water 5-6 times, 3-4 minutes each time. After rinsing, place on filter paper and dry with sterile air in a clean bench.

[0049] S2: Cut the leaves disinfected in S1 into small cubes of appropriate size and inoculate them into callus induction medium. The culture temperature was 25±1℃, under dark conditions, and the relative humidity was 50%~60%. After 16 days of culture, the induction rate of Leonurus japonicus callus in different induction mediums was counted.

[0050] In step S1 of Example 1, MS medium was used as the basic medium for callus induction. Different exogenous additives were added according to experimental requirements. The pH of the medium was adjusted to 5.8-6.0 with 1 mol / L NaOH solution and sterilized in an autoclave at 121°C for 20 minutes. After sterilization, the medium was dispensed into culture bottles for later use.

[0051] Table 1. Formula for Motherwort callus induction culture medium

[0052] Grouping Culture medium formulation A MS medium + 30 g / L sucrose + 6 g / L agar + 2 mg / L 6-BA + 1 mg / L NAA B MS medium + 30 g / L sucrose + 6 g / L agar + 2 mg / L 6-BA + 0.5 mg / L NAA C MS medium + 30 g / L sucrose + 6 g / L agar + 2 mg / L 6-BA + 0.1 mg / L NAA D MS medium + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA + 1 mg / L NAA E MS medium + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA + 0.5 mg / L NAA F MS medium + 30 g / L sucrose + 6 g / L agar + 1 mg / L 6-BA + 0.1 mg / L NAA

[0053] 2. Results

[0054] The effects of different culture media on the induction of Leonurus japonicus callus are shown in Table 2. The highest callus induction rate was achieved when the culture medium was MS medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA + 0.1 mg / L NAA.

[0055] Table 2. Statistical table of callus induction in different culture medium formulations of Leonurus japonicus.

[0056]

[0057] Induction rate = (Number of explants forming callus / Number of inoculated explants) × 100%.

[0058] Example 2: Effects of different explants on callus induction in Leonurus japonicus

[0059] 1. Methods and Steps

[0060] S1: Select leaves, petioles, and stem segments with buds that are free from rot, pests, and damage. Disinfect them with 75% ethanol for 30-60 seconds, rinse three times with sterile water, and then disinfect them with 2% NaClO (sodium hypochlorite) for 15 minutes. After disinfection, rinse them in sterile water 5-6 times, 3-4 minutes each time. After rinsing, place them on filter paper and dry them with sterile air in a clean bench.

[0061] S2: The explants sterilized in S1 were cut into small cubes of appropriate size and inoculated into callus induction medium. The medium formula was MS basal medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA + 0.1 mg / L NAA; the relative humidity was 50% to 60%, the temperature was 25 ± 1℃, and the culture was carried out in the dark for 16 days. The induction of different explants was observed.

[0062] 2. Results

[0063] The results are attached. Figure 1 , 2 3; Using leaves as explants, after 11 days of dark culture, milky white callus tissue appeared at the wound site. After 16 days of dark culture, the callus tissue turned pale yellow and was relatively dense. Figure 2 Using petioles as explants, no dedifferentiation occurred after 16 days of dark culture; using stem segments with buds as explants, the cut wound swelled after 16 days of dark culture, but clustered buds and adventitious buds appeared. Figure 3 Therefore, leaves were chosen as the ideal explant material.

[0064] Example 3: Effects of different light conditions on callus induction in Leonurus japonicus

[0065] 1. Methods and Steps

[0066] S1: Select leaves that are free from rot, pests, and damage. Disinfect with 75% ethanol for 30-60 seconds, rinse three times with sterile water, and then disinfect with 2% NaClO (sodium hypochlorite) for 15 minutes. After disinfection, rinse in sterile water 5-6 times, 3-4 minutes each time. After rinsing, place on filter paper and dry with sterile air in a clean bench.

[0067] S2: Cut the leaves disinfected in S1 into small cubes of appropriate size and inoculate them into callus induction medium. The medium formula is MS medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA + 0.1 mg / L NAA; the relative humidity is 50% to 60%, the temperature is 25±1℃, and the culture is carried out for 16 days under different light intensities. The induction rate of motherwort callus is then calculated.

[0068] Table 3. Statistical table of callus induction in Leonurus japonicus under different light conditions.

[0069]

[0070] Induction rate = (Number of explants forming callus / Number of inoculated explants) × 100%.

[0071] 2. Results

[0072] The effects of different light conditions on callus induction of Leonurus japonicus are shown in Table 3. The callus induction rate was highest under the light condition of 0 lx, that is, under dark culture of Leonurus japonicus.

[0073] Example 4: Screening of culture medium for inducing adventitious shoots of Leonurus japonicus

[0074] 1. Methods and Steps

[0075] Select well-grown, healthy embryogenic callus tissue, transfer it to adventitious bud induction medium, and culture it for 14 days at 5000 lx, relative humidity of 50%–60%, and 25±1℃. Calculate the adventitious bud induction rate of Leonurus japonicus under different induction medium conditions.

[0076] MS medium was used as the basic medium for inducing adventitious shoots of Leonurus japonicus. Different exogenous additives were added according to experimental requirements. The pH of the medium was adjusted to 5.8-6.0 with 1 mol / L NaOH solution and sterilized in an autoclave at 121°C for 20 minutes. After sterilization, the medium was dispensed into culture flasks for later use.

[0077] Table 4. Statistical table of adventitious shoot induction of Leonurus japonicus in different culture medium formulations.

[0078]

[0079] Induction rate = (Number of explants forming adventitious shoots / Number of inoculated explants) × 100%

[0080] 2. Results

[0081] The effects of different culture media on the induction of adventitious shoots of Leonurus japonicus are shown in Table 4. The highest adventitious shoot induction rate was achieved when the culture medium formula was MS medium + 30 g / L sucrose + 6 g / L agar + 1.0 mg / L 6-BA.

[0082] Example 5: Screening of rooting medium for Leonurus japonicus

[0083] 1. Methods and Steps

[0084] Select adventitious shoots of Leonurus japonicus that have grown to 2-3 cm, transfer them to a rooting induction medium, and culture them for 14 days at 5000 lx, relative humidity of 50%-60%, and 25±1℃. Calculate the rooting induction rate of Leonurus japonicus under different induction medium conditions.

[0085] The rooting induction medium for Leonurus japonicus used 1 / 2 MS medium as the basic medium, and different proportions of exogenous additives were added according to experimental requirements. The pH of the medium was adjusted to 5.8-6.0 with 1 mol / L NaOH solution, and the medium was sterilized in an autoclave at 121℃ for 20 minutes. After sterilization, it was dispensed into culture bottles for later use.

[0086] Table 5. Statistical table of adventitious root induction of Leonurus japonicus in different culture medium formulations.

[0087]

[0088] Induction rate = (Number of explants forming adventitious roots / Number of inoculated explants) × 100%

[0089] 2. Results

[0090] The effects of different culture media on the induction of adventitious roots of Leonurus japonicus are shown in the table. The highest induction rate of adventitious roots was achieved when the culture medium formula was 1 / 2 MS medium + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L NAA.

[0091] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0092] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0093] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for establishing a fast system of Leonurus japonicus Houtt. plant, characterized in that, The application relates to a rapid propagation method of motherwort. The pretreated and disinfected motherwort explants are inoculated on a callus induction medium to carry out primary culture; the callus induction medium is MS medium+30g / L sucrose+6g / L agar+1.0mg / L 6-BA+0.1mg / L NAA; the primary culture conditions include dark conditions, air relative humidity of 50%-60%, temperature of 25+ / -1 DEG C and culture time of 14-16 days; After the stable callus is obtained through the primary culture, the callus is placed in an adventitious bud culture medium to grow sprouts; the adventitious bud culture medium is MS medium+30g / L sucrose+6g / L agar+1mg / L 6-BA; the culture conditions of the adventitious bud include light intensity of 5000lx, air relative humidity of 50%-60%, temperature of 25+ / -1 DEG C and culture time of 14-16 days; After the adventitious bud grows, the adventitious bud is placed in a rooting culture medium to root, so that motherwort seedlings which can be used for rapid planting are obtained; the rooting culture medium is 1 / 2 MS basic medium+30g / L sucrose+6g / L agar+0.5mg / L NAA; the culture conditions of the rooting include light intensity of 5000lx, air relative humidity of 50%-60%, temperature of 25+ / -1 DEG C and culture time of 14-16 days; The motherwort explants are motherwort leaves.

2. The method of establishing a fast system of Leonurus japonicus Houtt. plant according to claim 1, characterized in that, The pretreatment and disinfection method of the motherwort explants comprises the following steps: disinfecting the motherwort explants with 75% ethanol for 30-60 seconds, washing the motherwort explants with sterile water for three times, disinfecting the motherwort explants with 2% NaClO for 10-15 minutes, washing the motherwort explants with sterile water for 5-6 times after disinfection, and drying the motherwort explants on filter paper in a sterile environment.

3. The method according to claim 1 or 2, wherein the method is characterized by, After the adventitious bud grows to 2-3cm, the adventitious bud is placed in a rooting culture medium to root.

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