A method for regenerating plant tissue of angelica sinensis

Abstract

The application discloses a regeneration culture method of Angelica sinensis plant tissues. The method comprises the following steps: obtaining the disinfected Angelica sinensis seeds, performing germination culture on the disinfected Angelica sinensis seeds on an induction culture medium to obtain tissue culture seedlings, obtaining the hypocotyls of the tissue culture seedlings as explants, then performing callus induction culture on the explants on a callus culture medium to obtain callus tissues, performing adventitious bud induction culture on the callus tissues on a subculture medium to obtain adventitious buds, and finally performing rooting induction culture on the adventitious buds on a rooting culture medium to obtain complete plants. The Angelica sinensis seedlings cultivated by the method have high survival rates, are not limited by seasons and regions, a large number of Angelica sinensis seedlings can be cultivated, the method is easy to operate, the production cost is low, and the environment is not polluted.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a method for regenerating plant tissues of Angelica sinensis. Background Technology

[0002] Angelica sinensis (Oliv.) Diels is a perennial herbaceous plant belonging to the Apiaceae family. It is mainly produced in southeastern Gansu Province, with Minxian County producing the largest quantity and highest quality. Other major producers include Yunnan, Sichuan, and Shaanxi provinces. Its dried root is a commonly used traditional Chinese medicine, possessing properties that nourish blood, regulate menstruation and relieve pain, moisten dryness and promote bowel movement, and boost immunity. Angelica sinensis is not only used for medicinal purposes but also for health maintenance and beauty. It is also one of China's major export medicinal plants, with demand increasing year by year.

[0003] Currently, Angelica sinensis is mainly bred or cultivated using traditional propagation methods. However, traditional propagation methods have a low propagation coefficient and slow seedling growth. Especially in the early growth stage, seedlings are more sensitive to environmental conditions and are susceptible to pests and diseases as well as adverse weather conditions, resulting in a high seedling loss rate during the seedling cultivation process. Consequently, they cannot meet the needs of market development and it is difficult to achieve large-scale production. Summary of the Invention

[0004] The purpose of this invention is to provide a method for regenerating plant tissues of Angelica sinensis. This tissue culture method is not limited by season or region, and the resulting seedlings have a high survival rate. Moreover, it only requires the corresponding laboratory facilities and technical conditions to produce Angelica sinensis seedlings all year round, which meets market demand and realizes large-scale production.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] This invention discloses a method for regenerating plant tissues of Angelica sinensis, the method comprising:

[0007] S1: Obtain sterilized Angelica sinensis seeds, germinate the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, and obtain the hypocotyl of the tissue culture seedlings as explants;

[0008] S2: The explants are cultured on callus culture medium to induce callus formation and obtain callus tissue;

[0009] S3: The callus tissue is subjected to adventitious shoot induction culture on a subculture medium to obtain adventitious shoots;

[0010] S4: The adventitious buds are induced to root on a rooting medium to obtain complete plants.

[0011] In a possible implementation, before obtaining the disinfected Angelica seeds, the process further includes obtaining the Angelica seeds and subjecting them to germination treatment.

[0012] In a possible implementation, the germination treatment includes soaking the Angelica seeds at a preset temperature for a preset time, wherein the preset temperature is 25-35°C and the preset time is 12-18 hours.

[0013] In a possible implementation, the induction medium in step S1 includes MS medium, agar, and sucrose.

[0014] In a possible implementation, the callus culture medium in step S2 includes a first basal culture medium and a first plant growth regulator, wherein the first plant growth regulator is 6-benzylaminopurine and / or naphthaleneacetic acid.

[0015] In a possible implementation, the callus culture medium satisfies at least one of the following conditions:

[0016] The first basal culture medium includes MS medium;

[0017] In the callus culture medium, the first plant growth regulator is 6-benzylaminopurine and naphthaleneacetic acid;

[0018] In the callus culture medium, the concentration of 6-benzylaminopurine is 3-4 mg / L;

[0019] In the callus culture medium, the concentration of naphthaleneacetic acid is 0.4–0.6 mg / L.

[0020] In a possible implementation, the subculture medium in step S3 includes a second basal medium and a second plant growth regulator, wherein the second plant growth regulator includes at least one of 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid.

[0021] In a possible implementation, the subculture medium satisfies at least one of the following characteristics:

[0022] The second basal culture medium includes 2 / 3 MS medium, which refers to the culture medium obtained by reducing the macroelements in MS medium to 2 / 3 of the original amount;

[0023] The second plant growth regulator is 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid;

[0024] In the subculture medium, the concentration of 6-benzylaminopurine is 0.4–0.6 mg / L;

[0025] In the subculture medium, the concentration of naphthaleneacetic acid is 0.05–0.15 mg / L;

[0026] In the subculture medium, the concentration of indolebutyric acid is 0.1–0.3 mg / L.

[0027] In a possible implementation, the rooting medium in step S4 includes a third basal medium and a third plant growth regulator, wherein the third plant growth regulator includes at least one of 6-benzylaminopurine, indolebutyric acid, and activated carbon.

[0028] In a possible implementation, the rooting medium satisfies at least one of the following characteristics:

[0029] The third basic culture medium includes 1 / 2MS culture medium, which is the culture medium obtained by halving the macroelements in MS culture medium;

[0030] In the rooting medium, the third plant growth regulator is 6-benzylaminopurine, indolebutyric acid, and activated carbon;

[0031] In the rooting medium, the concentration of 6-benzylaminopurine is 0.3–0.5 mg / L;

[0032] In the rooting medium, the concentration of indolebutyric acid is 0.1–0.2 mg / L;

[0033] In the rooting medium, the concentration of activated carbon is 0.4–0.6 g / L.

[0034] In a possible implementation, during the cultivation process in steps S1-S4, light cultivation is required, and the light cultivation satisfies at least one of the following characteristics:

[0035] The illumination duration during the light culture process was 12–16 hours.

[0036] The light intensity during the photoculture process was 3000–5000 lx.

[0037] The temperature during the light cultivation process was 20–26°C.

[0038] In a possible implementation, during the cultivation process in steps S1-S2, it is necessary to first cultivate in the dark and then cultivate under light. The method includes at least one of the following features:

[0039] During the cultivation process in steps S1-S2, the dark cultivation time is 5 to 7 days;

[0040] During the cultivation process in steps S1-S2, the temperature used for dark cultivation is 20-26℃;

[0041] The pH value of the culture medium in steps S1-S4 is maintained at 5-6.

[0042] In a possible implementation, obtaining the disinfected Angelica sinensis seeds includes: obtaining Angelica sinensis seeds and disinfecting the seeds to obtain disinfected Angelica sinensis seeds. During the disinfection process, the seeds are soaked in a sterilization solution for a preset time. The method satisfies at least one of the following characteristics:

[0043] The preset soaking time for the Angelica sinensis seeds is 8 to 16 minutes;

[0044] The concentration of the sterilization solution is 2-4%;

[0045] The sterilization solution is a sodium hypochlorite solution.

[0046] Based on the above technical solution, this application has the following beneficial effects: The technical solution of the present invention provides a method for plant tissue regeneration culture of Angelica sinensis. The method includes: obtaining sterilized Angelica sinensis seeds, germinating the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, obtaining the hypocotyl of the tissue culture seedlings as explants, then inducing callus culture on a callus medium to obtain callus tissue, then inducing adventitious bud culture on a subculture medium to obtain adventitious buds, and finally inducing rooting culture on a rooting medium to obtain complete plants. The Angelica sinensis seedlings cultivated by this method have a high survival rate and are not limited by season or region, that is, a large number of Angelica sinensis seedlings can be cultivated. Moreover, the method is easy to operate, has low production cost, and does not pollute the environment. The method establishes a stable and efficient tissue regeneration culture system, which can realize large-scale production. It also provides a flexible technical platform for the variety improvement and germplasm innovation of Angelica sinensis, and can easily introduce advanced technologies such as modern genetic engineering for the cultivation of new varieties. Attached Figure Description

[0047] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments will be briefly described below, wherein the same components are represented by the same reference numerals. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0048] Figure 1 This is a schematic flowchart of a plant tissue regeneration culture method for Angelica sinensis provided by the present invention.

[0049] Figure 2This is the growth process of Angelica sinensis seeds in the 14th experimental group of Example 1 of the present invention after being inoculated into the induction culture medium. In this example, serial number 1 is the inoculation period of Angelica sinensis, serial number 2 is the period when Angelica sinensis grows its first true leaf, and serial number 3 is the growth period of Angelica sinensis.

[0050] Figure 3 This describes the callus formation process in the first experimental group of Embodiment 2 of the present invention, wherein... Figure 3 A represents the callus inoculation period. Figure 3 B represents the stage of callus swelling and differentiation into buds.

[0051] Figure 4 This describes the differentiation and proliferation process of adventitious buds in the first experimental group of Example 3 of the present invention, where number 1 is the inoculation period of Angelica sinensis, number 2 is the proliferation period of Angelica sinensis, and number 3 is the growth period of Angelica sinensis.

[0052] Figure 5 This refers to the rooting culture and transplanting of Angelica sinensis tissue culture seedlings into the substrate in the second experimental group of Example 4 of the present invention, wherein... Figure 5 A represents a complete plant. Figure 5 B represents the transplanting of plants into the substrate.

[0053] Figure 6 This is a growth chart of the second experimental group of plants in Example 4 of the present invention after being transplanted into the substrate and growing for 3 months. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments, and therefore should not be construed as limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] It should be noted that the terms "first," "second," etc., in the specification, claims, and drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that embodiments of the invention can be implemented in orders other than those shown in the figures or descriptions below. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that includes a series of steps or units is not necessarily limited to those explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or devices.

[0056] This embodiment provides a method for the regeneration culture of Angelica sinensis plant tissue, such as... Figure 1 As shown, the method includes:

[0057] S1: Obtain sterilized Angelica sinensis seeds, germinate the sterilized Angelica sinensis seeds on induction medium to obtain tissue culture seedlings, and obtain the hypocotyl of the tissue culture seedlings as explants;

[0058] In some embodiments, obtaining sterilized Angelica sinensis seeds includes: obtaining Angelica sinensis seeds and sterilizing them to obtain sterilized Angelica sinensis seeds. By soaking the seeds in a sterilizing solution for disinfection, bacteria and fungi on the seed surface can be killed and inhibited, preventing diseases in the cultivated seedlings and thus reducing the seedling survival rate.

[0059] Specifically, the disinfection process includes soaking the Angelica seeds in a sterilization solution for a preset time to obtain disinfected Angelica seeds.

[0060] Specifically, after soaking the angelica seeds in a sterile solution for a preset time, the seeds need to be washed with sterile water to obtain disinfected angelica seeds. Furthermore, the washing process is repeated 2-3 times.

[0061] In some embodiments, before disinfecting the Angelica seeds, they need to be pre-disinfected with ethanol to obtain pre-disinfected Angelica seeds. During the pre-disinfection process, the Angelica seeds need to be soaked in ethanol for a preset time, with the ethanol concentration being 75% and the preset time being 30 seconds.

[0062] Specifically, in the pre-disinfection process of Angelica sinensis seeds, after soaking the seeds in ethanol for a preset time, they need to be washed with sterile water to obtain pre-disinfected seeds. Furthermore, the washing is performed 2-3 times.

[0063] In some embodiments, the preset soaking time for Angelica sinensis seeds is 8–16 minutes. The preset time can be any value within this range, and will not be enumerated here. A longer soaking time will reduce the contamination rate of the Angelica sinensis seeds, but will result in a lower germination rate. Conversely, a shorter soaking time will not achieve the desired sterilization effect. Therefore, the above-mentioned time is limited to ensure that the seeds are completely sterilized while maintaining a high germination rate. Preferably, the preset soaking time for Angelica sinensis seeds is 16 minutes.

[0064] In some embodiments, the concentration of the sterilization solution is 2-4%. The concentration of the sterilization solution can be any value within this range, and will not be enumerated here. By limiting the concentration of the sterilization solution, the problem of low seed germination rate due to excessively high concentration is avoided, while the problem of high seed contamination rate due to excessively low concentration is also avoided. Preferably, the concentration of the sterilization solution is 4%.

[0065] In some embodiments, the disinfectant solution is a sodium hypochlorite solution. This invention uses sodium hypochlorite solution as the disinfectant solution, which is environmentally friendly and non-toxic to humans, and has a lower toxicity rate to seeds compared to using mercuric chloride for disinfection.

[0066] In some embodiments, before obtaining the sterilized Angelica sinensis seeds, the process includes obtaining the seeds and subjecting them to germination treatment. Germination treatment of the Angelica sinensis seeds can improve seed vigor and thus promote germination.

[0067] Specifically, the germination treatment includes soaking Angelica seeds at a preset temperature for a preset time, with the preset temperature being 25–35°C and the preset time being 12–18 hours. The preset temperature and preset time can be any value within the above range, and are not listed here. Preferably, the preset temperature is 30°C and the preset time is 16 hours. If the preset temperature is too high or too low, it will affect the seed's respiration and nutrient conversion, thus affecting the germination rate. A suitable preset time helps the seeds fully absorb water, break dormancy, and further improve the germination rate. Therefore, a suitable preset time and preset temperature range can improve the seed germination rate.

[0068] In some embodiments, the induction medium in step S1 includes MS medium, agar, and sucrose. The induction medium of this application is mainly MS medium, which can induce tissue culture seedlings without adding any plant growth regulators, and the germination rate is as high as 98%, significantly reducing costs.

[0069] Specifically, the concentration of agar in the induction medium is 5–7 g / L. Preferably, the concentration of agar is 6 g / L.

[0070] Specifically, the concentration of sucrose in the induction medium is 25–35 g / L. Preferably, the concentration of sucrose is 30 g / L.

[0071] S2: Explants are cultured on callus medium to induce callus formation and obtain callus tissue. The main purpose of inoculating explants onto callus medium is to form callus tissue, which allows cells in the hypocotyl to divide and differentiate, thereby producing more buds. That is, one hypocotyl can form multiple plants, resulting in a higher yield of seedlings.

[0072] In some embodiments, the callus medium includes a first basal medium and a first plant growth regulator, wherein the first plant growth regulator is 6-benzylaminopurine and / or naphthaleneacetic acid. 6-Benzylaminopurine can promote cell division and induce callus formation, and naphthaleneacetic acid can also promote cell division and growth, thereby promoting callus formation and growth. Using 6-benzylaminopurine and / or naphthaleneacetic acid as the first plant growth regulator can induce callus formation, growth, and proliferation, resulting in a greater number of buds and a higher yield of seedlings.

[0073] In some embodiments, the first basal culture medium includes MS medium.

[0074] Specifically, the first basal culture medium also includes agar at a concentration of 6 g / L and sucrose at a concentration of 30 g / L.

[0075] In some embodiments, the first plant growth regulator in the callus medium is 6-benzylaminopurine and naphthaleneacetic acid. The synergistic effect of 6-benzylaminopurine and naphthaleneacetic acid further stimulates callus formation and growth, avoiding the vitrification or browning of callus tissue that may occur due to a deficiency of either hormone.

[0076] In some embodiments, the concentration of 6-benzylaminopurine in the callus medium is 3–4 mg / L. The concentration of 6-benzylaminopurine can be any value within the above range, and will not be enumerated here. By limiting the concentration of 6-benzylaminopurine, excessively high concentrations can lead to excessive cell division and hinder growth, potentially causing browning or vitrification of the resulting callus. Conversely, excessively low concentrations can lead to a decrease in induction rate. Preferably, the concentration of 6-benzylaminopurine in the callus medium is 3 mg / L.

[0077] In some embodiments, the concentration of naphthaleneacetic acid (NAA) in the callus culture medium is 0.4–0.6 mg / L. The concentration of NAA can be any value within this range, and will not be enumerated here. By limiting the concentration range of NAA, the problem of reduced induction rate due to excessively low concentration is avoided, while the problem of easy browning of callus tissue due to excessively high concentration is also avoided. Preferably, the concentration of NAA is 0.5 mg / L.

[0078] S3: Induce adventitious shoots from the callus tissue on a subculture medium to obtain adventitious shoots.

[0079] In some embodiments, the subculture medium includes a second basal medium and a second plant growth regulator, the second plant growth regulator including at least one of 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid. Using the above-mentioned types of second plant growth regulators for adventitious shoot induction culture can promote the transformation of callus tissue into shoots, improve differentiation efficiency, increase proliferation rate, and avoid vitrification and browning.

[0080] In some embodiments, the second basal medium includes 2 / 3 MS medium, which refers to a medium obtained by reducing the macroelements in MS medium to two-thirds of their original value. During the adventitious bud differentiation stage, excessive macroelements in MS medium can cause the leaves of the emerging seedlings to turn yellow and inhibit the differentiation of callus tissue into buds, thus reducing the germination rate. Conversely, insufficient macroelements in MS medium can result in small leaves, slow growth rate, and may also be accompanied by vitrification and browning.

[0081] Specifically, the second basal culture medium also includes agar and sucrose, with the concentration of agar being 6 g / L and the concentration of sucrose being 30 g / L.

[0082] In some embodiments, the second plant growth regulator is 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid. The synergistic effect of 6-benzylaminopurine, naphthaleneacetic acid, and indolebutyric acid further promotes cell division and growth, increases the proliferation rate, and promotes callus differentiation into shoots, while preventing vitrification and browning.

[0083] In some embodiments, the concentration of 6-benzylaminopurine in the subculture medium is 0.4–0.6 mg / L. The concentration of 6-benzylaminopurine can be any value within the above range, and will not be enumerated here. Preferably, the concentration of 6-benzylaminopurine is 0.5 mg / L.

[0084] In some embodiments, the concentration of naphthaleneacetic acid (NAA) in the subculture medium is 0.05–0.15 mg / L. The concentration of NAA can be any value within the above range, and will not be enumerated here. Preferably, the concentration of NAA is 0.1 mg / L.

[0085] In some embodiments, the concentration of indolebutyric acid (IBA) in the subculture medium is 0.1–0.3 mg / L. The concentration of IBA can be any value within the above range, and will not be enumerated here. Preferably, the concentration of IBA is 0.2 mg / L. By limiting the concentration of the above-mentioned plant growth regulators, excessively high concentrations of 6-benzylaminopurine (6-Benzylaminopurine) can inhibit shoot differentiation, while limiting the concentration of naphthaleneacetic acid (NAA) to be lower than that of 6-Benzylaminopurine (6-Benzylaminopurine) is more conducive to shoot differentiation, resulting in better seedling growth. This also avoids the problem of excessively high IBA concentrations potentially reducing germination rate.

[0086] S4: Adventitious buds are induced to root on a rooting medium to obtain complete plants.

[0087] In some embodiments, the rooting medium includes a third basal medium and a third plant growth regulator, the third plant growth regulator including at least one of 6-benzylaminopurine, indolebutyric acid, and activated carbon. Indolebutyric acid can promote plant cell division and elongation, which helps the root growth of adventitious buds. At the same time, the addition of activated carbon during rooting culture can regulate the hormone content in the medium to maintain it at an appropriate level, thereby further promoting the rooting of adventitious buds. The addition of 6-benzylaminopurine further stimulates the growth and division of plant root cells, thereby increasing the number and growth rate of roots, and increasing the rooting rate to 89%.

[0088] In some embodiments, the third basal medium includes 1 / 2 MS medium, which is obtained by halving the macroelements in MS medium. Using MS medium with half the macroelements as the basal medium avoids excessively high salt concentrations that could inhibit root growth and development.

[0089] In some embodiments, the third plant growth regulator in the rooting medium is 6-benzylaminopurine, indolebutyric acid, and activated carbon. The synergistic effect of 6-benzylaminopurine, indolebutyric acid, and activated carbon promotes root growth in adventitious buds and significantly increases the rooting rate of the plant.

[0090] In some embodiments, the concentration of 6-benzylaminopurine in the rooting medium is 0.3–0.5 mg / L. The concentration of 6-benzylaminopurine can be any value within the above range, and will not be enumerated here. Preferably, the concentration of 6-benzylaminopurine in the rooting medium is 0.3 mg / L.

[0091] In some embodiments, the concentration of indolebutyric acid (IBA) in the rooting medium is 0.1–0.2 mg / L. The concentration of IBA can be any value within the above range, and will not be enumerated here. Preferably, the concentration of IBA in the rooting medium is 0.2 mg / L.

[0092] In some embodiments, the concentration of activated charcoal in the rooting medium is 0.4–0.6 g / L. The concentration of activated charcoal can be any value within the above range, and is not listed here. Preferably, the concentration of activated charcoal in the rooting medium is 0.5 g / L. By limiting the concentrations of 6-benzylaminopurine, indolebutyric acid, and activated charcoal, cell division, enlargement, and elongation can be promoted, which helps in the division and differentiation of root cells, thereby promoting rooting. Furthermore, indolebutyric acid can, to some extent, increase the speed and quantity of rooting.

[0093] In some embodiments, light cultivation is required during the cultivation process in steps S1-S4.

[0094] In some embodiments, the illumination duration during the light cultivation process is 12–16 hours. The illumination duration can be any value within the above range, and will not be enumerated here. Preferably, the illumination duration during the light cultivation process is 13.5–14.5 hours.

[0095] In some embodiments, the light intensity during the photoculture process is 3000–5000 lx. The light intensity can be any value within this range, and is not enumerated here. Using appropriate light duration and intensity promotes plant growth and differentiation, improves plant reproduction speed and quality, and avoids excessively high light intensity or excessively long light duration, which may lead to photoinhibition and photooxidation in plant tissues, thereby affecting plant growth and development.

[0096] In some embodiments, the temperature during the light-induced culture process is consistently between 20 and 26°C. The temperature can be any value within this range, and will not be enumerated here. Preferably, the temperature during the light-induced culture process is between 22 and 23°C. Using a suitable temperature provides a suitable growth environment, allowing cell division and differentiation to proceed normally.

[0097] In some embodiments, during the cultivation process in steps S1-S2, it is necessary to first cultivate in the dark and then cultivate under light. During the cultivation process, cultivating in the dark first and then under light avoids direct light exposure to the callus tissue, which is prone to browning. At the same time, it can also promote the growth of the callus tissue, and the method of cultivating in the dark first and then under light helps to improve the germination rate of seeds.

[0098] Specifically, the dark culture time is 5 to 7 days. Preferably, the dark culture time is 6 days. Limiting the number of days of dark culture avoids slowing down the callus growth rate due to excessively long dark culture time, which may reduce the germination rate.

[0099] In some embodiments, the temperature used for dark culture is 20–26°C. The temperature can be any value within this range, and is not listed here. The temperature used for dark culture is 22–23°C. Using a suitable temperature provides a suitable growth environment, allowing cell division and differentiation to proceed normally.

[0100] In some embodiments, the pH of the culture medium in steps S1-S4 is maintained between 5 and 6. A suitable pH can inhibit the growth of harmful microorganisms and prevent browning of plant tissues during growth. Preferably, the pH of the culture medium in steps S1-S4 is maintained at 5.8.

[0101] The following describes specific embodiments of this application in conjunction with the above-described scheme.

[0102] Example 1: Seed induction culture.

[0103] (1) Seed germination and disinfection treatment: 1800 high-quality and plump Angelica sinensis seeds were selected as samples. The Angelica sinensis seeds were placed in a constant temperature water bath with a preset temperature and soaked for a preset time (germination time) to obtain germinated Angelica sinensis seeds. The germinated Angelica sinensis seeds were placed in a clean bench and soaked in 75% ethanol for 30 seconds. They were then rinsed with sterile water 2-3 times to obtain pre-disinfected Angelica sinensis seeds. The pre-disinfected Angelica sinensis seeds were soaked in sodium hypochlorite solution for a preset time and rinsed with sterile water 2-3 times to obtain 1800 disinfected Angelica sinensis seeds.

[0104] (2) Induction culture: 1800 sterilized Angelica sinensis seeds were set up as 18 experimental groups and cultured in 18 50 ml culture bottles for induction culture. Each culture bottle contained 100 sterilized Angelica sinensis seeds and an induction culture medium. The components of the induction culture medium were MS medium, 30 g / L sucrose and 6 g / L agar. The pH of the culture medium was 5.8. After the culture bottles containing Angelica sinensis seeds were first cultured in the dark for 7 days, they were transferred to light culture. After 28 days of light culture, the growth of tissue culture seedlings in each culture bottle was observed, and the germination rate and contamination rate in each culture bottle were calculated to obtain the corresponding experimental results. The conditions for light culture were 14±0.5 h / d of light, light intensity of 3000-5000 lx, and the temperature for both dark and light culture was 22-23℃. The composition of the induction medium was the same for each group, but the germination time, sodium hypochlorite concentration, and soaking time in sodium hypochlorite solution (sterilization time) were different for each experimental group, as detailed in Table 1.

[0105] Germination rate calculation method: Germinated Angelica seeds / Total number of inoculated Angelica seeds;

[0106] When the inoculated Angelica seeds are infected by fungi or bacteria, they are considered contaminated. The contamination rate is calculated as: contaminated Angelica seeds / total number of inoculated Angelica seeds.

[0107] (3) Experimental Results: As shown in Table 1 below, the contamination rate of Angelica sinensis seeds gradually increased with the decrease of sodium hypochlorite solution concentration. However, excessively long disinfection time led to a low germination rate. Therefore, in order to achieve disinfection while ensuring a certain germination rate, a 4% sodium hypochlorite concentration and a soaking time of 16 min were selected. The germination time affects the germination rate of seeds, and 16 h was selected as the optimal germination time. In addition, as... Figure 2 As shown, Figure 2This is the induction culture process of tissue culture seedlings in the 14th experimental group. Serial number 1 represents the inoculation period of *Angelica sinensis*, serial number 2 represents the emergence of the first true leaf, and serial number 3 represents the growth period. It can be seen that the sterilized seeds began to germinate and grow, with robust seedlings, bright green leaves, and white, straight, and smooth roots.

[0108] Table 1: Effects of different germination times, sodium hypochlorite concentrations, and soaking times in sodium hypochlorite solution on seed germination rates.

[0109]

[0110]

[0111] Example 2: Selection of callus culture medium

[0112] (1) Experimental method: The tissue culture seedlings obtained under the culture conditions of the 14th experimental group were used as samples. The hypocotyls of the samples were used as explants. Three experimental groups were set up. The number of hypocotyls in the first experimental group was 120, the number of hypocotyls in the second experimental group was 92, and the number of hypocotyls in the third experimental group was 105. The hypocotyls in the three experimental groups were inoculated into callus culture medium. After being cultured in the dark for 7 days, they were transferred to light culture. After 28 days of culture, the induction rate of each experimental group was calculated and the corresponding experimental results were obtained. The conditions for light culture were 14 ± 0.5 h / d, light intensity 3000-5000 lx, and the temperature for both dark and light culture was 22-23℃. The callus medium for each group consisted of MS medium + 30 g / L sucrose + 6 g / L agar, 6-benzylpurine, and 0.5 mg / L naphthaleneacetic acid. However, the concentration of 6-benzylpurine in the callus medium differed among the experimental groups: 3 mg / L in the first group, 5 mg / L in the second group, and 2 mg / L in the third group. The experimental results are shown in Table 2. The pH of the callus medium was 5.8.

[0113] Induction rate (%) = Number of hypocotyls producing callus / Total number of inoculated hypocotyls × 100%

[0114] (2) Experimental results: As shown in Table 2, callus culture medium in each experimental group was able to induce callus tissue. When the concentration of 6-benzylpurine was 2 mg / L, 3 mg / L to 5 mg / L, the callus induction rate of Angelica sinensis was 37.14%, 69.16% and 45.65%, respectively. It can be seen that a higher concentration of 6-benzylpurine or a lower concentration of 6-BA is not conducive to the formation of callus tissue. Therefore, the callus culture medium obtained when the concentration of 6-benzylpurine was 3 mg / L had a better callus culture effect. Figure 3 For the callus formation process in the first experimental group, from Figure 3 As can be seen, with the extension of culture time, the callus tissue grows and expands relatively rapidly, among which... Figure 3 A represents the callus inoculation period. Figure 3 B represents the stage of callus swelling and differentiation into buds.

[0115] Table 2: Effects of different combinations of primary plant growth regulator concentrations on callus induction

[0116]

[0117] Example 3: Selection of Subculture Culture Medium

[0118] (1) Experimental Method: The callus tissue obtained under the culture conditions in Example 2 was used as the sample and divided into three experimental groups. The number of callus tissues in each experimental group was 60. The experiment was repeated 3 times. The samples were inoculated into subculture medium with a pH of 5.8 and cultured under light conditions of 22-23℃, light duration of 14±0.5h / d and light intensity of 3000-5000lx for 28 days. The growth potential of adventitious shoots in each experimental group was observed and the proliferation ratio was calculated to obtain the experimental results. The subculture medium of the three experimental groups consisted of 30g / L sucrose + 6g / L agar + 0.5mg / L 6-benzylpurine + 0.1mg / L naphthaleneacetic acid + 0.2mg / L indolebutyric acid. The difference was that the first experimental group also included 2 / 3 MS medium, the second experimental group also included MS medium, and the third experimental group also included 1 / 2 MS medium. The formula for calculating the proliferation ratio was: proliferation ratio = number of adventitious shoots / total amount of callus tissue.

[0119] (2) Experimental Results: As shown in Table 3, higher or lower concentrations of macroelements in the subculture medium reduced or slowed the germination rate of adventitious buds, resulted in lower fold increases, and also caused vitrification or browning. Therefore, 2 / 3 MS medium was the optimal basal medium for subculture. Figure 4 As shown, the adventitious buds obtained in the first experimental group have dark green leaves and good growth. Among them, number 1 is the inoculation period of Angelica sinensis, number 2 is the proliferation period of Angelica sinensis, and number 3 is the growth period of Angelica sinensis.

[0120] Table 3 Effects of different subculture media on the subculture proliferation of adventitious shoots of Angelica sinensis

[0121]

[0122]

[0123] Note: The results of the ANOVA in the list are represented by letters, and different capital letters indicate extremely significant differences (P<0.01).

[0124] Example 4: Selection of Rooting Culture Medium

[0125] (1) Experimental method: The adventitious buds obtained under the culture conditions in Example 3 were used as samples and divided into three experimental groups. The number of samples in each experimental group was 100. The samples were inoculated into rooting medium with pH 5.8 and cultured under light conditions of 22-23℃, light duration of 14±0.5h / d and light intensity of 3000-5000lx. The growth of the plants was observed after 28 days and the rooting rate was calculated. The rooting medium for the first experimental group consisted of 1 / 2 MS medium + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L 6-benzylpurine + 0.1 mg / L indolebutyric acid + 0.5 g / L activated carbon. The rooting medium for the second experimental group consisted of 1 / 2 MS medium + 30 g / L sucrose + 6 g / L agar + 0.3 mg / L 6-benzylpurine + 0.2 mg / L indolebutyric acid + 0.5 g / L activated carbon. The rooting medium for the third experimental group consisted of MS medium + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L 6-benzylpurine + 0.1 mg / L indolebutyric acid + 0.5 g / L activated carbon.

[0126] The rooting rate is calculated using the formula: (Number of rooted adventitious buds / Total number of adventitious buds in the sample) × 100%.

[0127] (2) Experimental results: As can be seen from Table 4, the rooting rate of the 1 / 2MS medium was significantly higher than that of the conventional MS medium. With the 1 / 2MS medium as the control, the rooting rate of the second experimental group was significantly higher than that of the first and third experimental groups. Figure 5 For the plants cultivated in the second experimental group, such as Figure 5 As shown in Figure A, the seedlings that underwent rooting culture showed good growth and had vigorous, robust root systems. Figure 5 B represents the transplanted plants. The transplanting process was as follows: After hardening off the plants obtained from the second experimental group for 3-5 days, they were removed, and the roots were rinsed with water to remove any residual culture medium. The plants were then soaked in a systemic fungicide solution for 1-2 minutes before transplanting. The transplanting substrate consisted of peat moss: coconut coir: perlite = 5:3:1 (volume ratio). Figure 5 B represents the plant growth after transplanting into the substrate and growing for 45 days. Figure 6 The growth of the plants after transplanting them into the substrate and growing for 3 months is shown to be good. As can be seen, the transplanted plants obtained by the cultivation method of this application are growing well.

[0128] Table 4: Effects of different rooting media on adventitious shoot rooting

[0129]

[0130] As can be seen from the above embodiments, the present invention has the following beneficial effects:

[0131] This invention significantly reduces the contamination rate by setting the germination time in the seed germination and disinfection process to 16 hours, limiting the sodium hypochlorite solution concentration to 4%, and limiting the soaking time in the sodium hypochlorite solution to 16 minutes. This maintains the contamination rate between 2-4%. Furthermore, the induction medium is primarily MS-based, enabling the induction of sterile tissue culture seedlings without the addition of any plant growth regulators, and increasing the seed germination rate to 98%. Subsequently, adjusting the concentration of 6-benzylpurine in the callus medium allows for better callus formation from the hypocotyl, increasing the callus induction rate to 69.16%. Based on this, the composition of the subculture medium was further adjusted to achieve a high germination rate and a high multiplication rate of adventitious buds, with a multiplication coefficient of over 5. Further adjustment of the concentrations of 6-benzylpurine and indolebutyric acid in the rooting medium significantly improved the rooting rate of adventitious buds, reaching as high as 89%. The resulting plants exhibited excellent growth and vigorous, robust root systems. The Angelica seedlings cultivated using this method have a high survival rate after transplanting and are not limited by season or region, enabling the cultivation of large quantities of Angelica seedlings. This effectively solves the problem of large-scale production of Angelica seedlings. Furthermore, this method is easy to operate, has low production costs, and does not pollute the environment.

[0132] The above description is merely some embodiments of the present invention and is not intended to limit the present invention. Those skilled in the art should understand that the present invention can have various changes and modifications, and any modifications, equivalent substitutions and improvements made in accordance with the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for regenerating plant tissues of Angelica sinensis, characterized in that, The method includes: S1: Obtain sterilized Angelica sinensis seeds, germinate the sterilized Angelica sinensis seeds on an induction medium to obtain tissue culture seedlings, and obtain the hypocotyl of the tissue culture seedlings as explants; S2: The explants are cultured on callus culture medium to induce callus formation and obtain callus tissue; S3: The callus tissue is subjected to adventitious shoot induction culture on a subculture medium to obtain adventitious shoots; S4: The adventitious buds are induced to root on a rooting medium to obtain complete plants; The process of obtaining disinfected Angelica seeds includes: obtaining Angelica seeds and disinfecting the Angelica seeds to obtain disinfected Angelica seeds. During the disinfection process, the Angelica seeds need to be soaked in a 4% sodium hypochlorite solution for 8-16 minutes. The callus medium in step S2 consists of MS medium, 30 g / L sucrose, 6 g / L agar, 3 mg / L 6-benzylaminopurine, and naphthaleneacetic acid. In the callus medium, the concentration of sucrose is 30 g / L, the concentration of agar is 6 g / L, the concentration of 6-benzylaminopurine is 3 mg / L, and the concentration of naphthaleneacetic acid is 0.5 mg / L. The subculture medium in step S3 consists of 2 / 3MS medium, sucrose, agar, 6-benzylpurine, naphthaleneacetic acid, and indolebutyric acid. In the subculture medium, the concentration of sucrose is 30 g / L, the concentration of agar is 6 g / L, the concentration of 6-benzylpurine is 0.5 mg / L, the concentration of naphthaleneacetic acid is 0.1 mg / L, and the concentration of indolebutyric acid is 0.2 mg / L. The rooting medium in step S4 consists of 1 / 2 MS medium, sucrose, agar, 6-benzylpurine, indolebutyric acid, and activated carbon. In the rooting medium, the concentration of sucrose is 30 g / L, the concentration of agar is 6 g / L, the concentration of 6-benzylpurine is 0.3 mg / L, the concentration of indolebutyric acid is 0.2 mg / L, and the concentration of activated carbon is 0.5 g / L.

2. The method according to claim 1, characterized in that, Before obtaining the disinfected Angelica seeds, the method further includes obtaining the Angelica seeds and performing a germination treatment on the Angelica seeds.

3. The method according to claim 2, characterized in that, The germination treatment includes soaking the Angelica seeds at a preset temperature for a preset time, wherein the preset temperature is 25~35℃ and the preset time is 12~18h.

4. The method according to any one of claims 1-3, characterized in that, During the cultivation process in steps S1-S4, light cultivation is required, and the light cultivation must satisfy at least one of the following characteristics: The illumination duration during the light culture process was 12-16 hours. The light intensity during the photoculture process was 3000~5000 lx; The temperature during the light cultivation process was 20~26℃.

5. The method according to any one of claims 1-3, characterized in that, In the cultivation process of steps S1-S2, dark cultivation must be performed first, followed by light cultivation. The method includes at least one of the following features: the dark cultivation time is 5 to 7 days in the cultivation process of steps S1-S2. During the cultivation process in steps S1-S2, the temperature used for dark cultivation is 20~26℃; The pH value of the culture medium in steps S1-S4 is maintained at 5-6.

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