Medium and method for cultivating artemisia annua callus

Abstract

The application discloses a culture medium and a cultivation method of artemisia annua callus. The formula of the culture medium comprises the following components: 2-6 g / L MS medium, 5-10 g / L agar, 5-30 g / L sucrose, 0.1-0.5 mg / L NAA, 1-2 mg / L 6-BA and 50-300 mL / L banana peel extract; and the pH is 5.5-6.0. The cultivation method comprises the step of proliferating the artemisia annua callus in the culture medium. It is found that the proliferation of the artemisia annua callus can be obviously improved when the cultivation of the artemisia annua callus is carried out in the culture medium containing the banana peel extract.

Description

Technical Field

[0001] This invention relates to a culture medium and cultivation method for Artemisia annua callus. Background Technology

[0002] Artemisia annua L., commonly known as yellow wormwood, is an annual herb belonging to the genus Artemisia in the family Asteraceae. The entire plant can be used medicinally; it has a bitter taste, is cold in nature, and is non-toxic. It possesses properties of clearing heat, relieving summer heat, cooling the blood, promoting urination, strengthening the stomach, stopping night sweats, and relieving itching. It is a major raw material for the antimalarial drug artemisinin. Artemisia annua thrives in warm, humid climates, prefers sunlight, does not tolerate shade, and dislikes waterlogging. When cultivated artificially, it is best grown in loose, fertile, humus-rich, and well-drained sandy loam soil. Propagation of Artemisia annua is primarily through seeds.

[0003] Plant tissue culture is a novel asexual reproduction technique developed based on the theory of plant cell totipotency. It is grounded in the biological principle that plant cells can restore totipotency through dedifferentiation, and callus culture is the core starting point and fundamental platform for most plant tissue culture techniques. Specifically, plant tissue culture involves using plant tissues or cells (such as petals, stem segments, leaves, roots, protoplasts, etc.) as explants under sterile conditions to induce callus formation. This callus is then cultured and grown on a medium containing appropriate nutrients and plant growth hormones to obtain plant tissue or complete plants. Therefore, utilizing plant tissue culture techniques (especially those based on callus culture) to achieve rapid and large-scale plant propagation is of great significance for expanding industrial production.

[0004] Plant tissue culture media (including callus culture media) are like soil, serving as the base for the survival and development of ex vivo materials in tissue culture. Currently, most tissue culture media use MS (Murashige and Skoog) medium as the most commonly used basic medium. To further improve the efficiency of inducing cell division and differentiation and to maintain plant growth, some plant growth regulators are added to the basic medium, such as (1) auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D), and (2) cytokinins such as zeatin (ZT), 6-benzylpurine (6-BA or BAP), and kinetin (KT).

[0005] However, for Artemisia annua tissue culture, especially for Artemisia annua callus culture, the existing culture media still have obvious shortcomings in terms of limited proliferation effect. Therefore, there is an urgent need for a culture medium and cultivation method that can be used for Artemisia annua callus culture and can ensure efficient and stable proliferation. Summary of the Invention

[0006] To address the technical limitations of existing plant tissue culture media in achieving effective proliferation of Artemisia annua callus, this invention provides a culture medium and cultivation method for Artemisia annua callus. Using this culture medium for cultivating Artemisia annua callus significantly improves its proliferation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides a culture medium for Artemisia annua callus, the formulation of which includes the following components: 2-6 g / L MS medium, 5-10 g / L agar, 5-30 g / L sucrose, 0.1-0.5 mg / L NAA (α-naphthaleneacetic acid), 1-2 mg / L 6-BA (6-benzylaminopurine), and 50-300 mL / L banana peel extract; pH 5.5-6.0.

[0009] In this invention, as is commonly understood by those skilled in the art, the content of each component in the culture medium formulation can be calculated based on the volume of the obtained culture medium, and water, such as sterile water, can usually be used to prepare the culture medium.

[0010] In this invention, the banana peel extract can refer to the extract obtained by extracting banana peel with an extraction reagent, that is, a solution containing banana peel extract.

[0011] In some embodiments, the extraction reagent comprises an aqueous solution of water and / or ethanol; the volume percentage of ethanol in the aqueous solution may be 70%-80%, for example 75%.

[0012] In some embodiments, the extraction process includes steps of primary extraction, secondary extraction, and concentration under ultrasonic conditions. The primary and secondary extractions are used only to distinguish different extraction steps and have no practical limiting effect.

[0013] In some specific implementations, the extraction process includes the following steps: (1) Mix a portion of the extraction reagent and the banana peel, and perform a single extraction under ultrasonic conditions. Centrifuge to obtain supernatant 1 and precipitate 1. (2) Mix the precipitate 1 with the remaining extraction reagent, perform a second extraction under the condition of secondary ultrasound, and centrifuge to obtain supernatant 2 and precipitate 2; (3) Combine the supernatant 1 and supernatant 2, and then concentrate them.

[0014] Among them, first ultrasound and second ultrasound, first extraction and second extraction, supernatant 1 and supernatant 2, precipitate 1 and precipitate 2 are only used to distinguish different steps or substances and have no actual limiting function.

[0015] In the first extraction, the mass ratio of the banana peel to the extraction reagent can be 1:(8-12), for example, 1:10.

[0016] The amount of extraction reagent used in the secondary extraction is the same as the amount of extraction reagent used in the primary extraction.

[0017] In the primary and secondary extractions, the power of the primary and secondary ultrasound can be independently 800-1200W, for example, both can be 1000W.

[0018] In the first and second extractions, the time for the first and second ultrasound can be 20-60 minutes each, for example, 30 minutes each.

[0019] In the primary and secondary extractions, the temperatures of the primary and secondary ultrasound can each be independently 24-26°C, for example, both 25°C.

[0020] The step of merging includes a step of centrifuging again to collect the supernatant; the centrifugation speed is, for example, 8000 r / min, and the time is, for example, 10 min.

[0021] In the concentration step, the concentration is carried out until the mass of the combined extract is reduced by half; for example, concentration is carried out using a rotary evaporator at 40°C until the mass of the extract is reduced by half.

[0022] In this invention, as is commonly understood by those skilled in the art, the banana peel extract can be sterilized by conventional procedures before being added to the culture medium, for example, by filtration sterilization using a 0.22 μm filter membrane.

[0023] In this invention, the MS culture medium can be conventional in the art.

[0024] In this invention, the content of the MS medium can be 4-6 g / L, for example 4.41 g / L.

[0025] In this invention, the content of agar can be 7-10 g / L, for example 7.5 g / L.

[0026] In this invention, the sucrose content can be 5-20 g / L, for example 10 g / L or 15 g / L.

[0027] In this invention, the NAA content can be 0.1-0.4 mg / L, for example 0.2 mg / L or 0.3 mg / L.

[0028] In this invention, the content of 6-BA can be 1.0-1.7 mg / L, for example 1.2 mg / L or 1.4 mg / L.

[0029] In this invention, the content of the banana peel extract can be 50-200 mL / L, for example 100 mL / L or 150 mL / L.

[0030] In this invention, the pH of the culture medium can be 5.6-5.9, for example 5.7 or 5.8.

[0031] In some preferred embodiments, the culture medium is formulated with the following components: 4-6 g / L MS medium, 5-20 g / L sucrose, 1.0-1.7 mg / L 6-BA, 0.1-0.4 mg / L NAA, 50-200 mL / L banana peel extract and 7-10 g / L agar; pH 5.5-6.0.

[0032] In some specific embodiments, the culture medium formulation includes the following components: 4.41 g / L MS medium, 5 g / L sucrose, 1.0 mg / L 6-BA, 0.1 mg / L NAA, 200 mL / L banana peel extract and 7.5 g / L agar; pH 5.8.

[0033] In some specific embodiments, the culture medium formulation includes the following components: 4.41 g / L MS medium, 10 g / L sucrose, 1.2 mg / L 6-BA, 0.2 mg / L NAA, 150 mL / L banana peel extract and 7.5 g / L agar; pH 5.8.

[0034] In some specific embodiments, the culture medium formulation includes the following components: 4.41 g / L MS medium, 15 g / L sucrose, 1.4 mg / L 6-BA, 0.3 mg / L NAA, 100 mL / L banana peel extract and 7.5 g / L agar; pH 5.8.

[0035] In some specific embodiments, the culture medium formulation includes the following components: 4.41 g / L MS medium, 20 g / L sucrose, 1.7 mg / L 6-BA, 0.4 mg / L NAA, 50 mL / L banana peel extract and 7.5 g / L agar; pH 5.8.

[0036] The present invention also provides a method for cultivating Artemisia annua callus, which includes the step of proliferating and culturing Artemisia annua callus in the culture medium as described above.

[0037] In this invention, the Artemisia callus refers to undifferentiated thin-walled cell clusters formed by the dedifferentiation of living plant cells in plant tissue culture, which are disordered, non-organic structures.

[0038] In this invention, the cultivation method may include the following steps: S1. Artemisia annua explants were cultured once in culture medium A to obtain callus 1; The culture medium A comprises the following components: 4-10 g / L basal medium, 20-40 g / L carbon source, 0.1-5 mg / L plant hormone, and 5-10 g / L solidifying agent; pH is 5.5-6.0. S2. The callus 1 is cultured a second time in culture medium B to obtain callus 2. The culture medium B comprises the following components: 4-10 g / L basal medium, 20-40 g / L carbon source and 0.1-5 mg / L plant hormone; pH is 5.5-6.0. S3. The callus 2 is cultured three times in culture medium C, which is the culture medium as defined above.

[0039] Among them, culture medium A, culture medium B and culture medium C are only used to distinguish different culture media; primary culture, secondary culture and tertiary culture are only used to distinguish different culture steps; callus 1 and callus 2 are only used to distinguish the callus culture steps obtained in different steps; none of them have any actual limiting function, and those skilled in the art will know their specific meaning.

[0040] The primary culture aims to induce callus formation, specifically to induce dedifferentiation of Artemisia annua explants into callus. The types and ratios of components in the culture medium formula, as well as culture conditions such as light, temperature, and time, can be selected accordingly based on the goal of "dedifferentiation." The secondary and tertiary cultures both aim to promote the rapid proliferation of the resulting callus, ensuring rapid proliferation without differentiation. Specifically, the proliferation culture can be conducted solely using the previously defined culture medium C, or the callus can be cultured in culture medium B to obtain callus with a certain morphology and activity, and then further cultured in culture medium C to obtain callus with higher activity. Those skilled in the art will understand the specific meaning of these methods.

[0041] The explants of Artemisia annua can refer to organs, tissue fragments or cells that are cut and separated from surviving Artemisia annua in Artemisia annua tissue culture and used for in vitro culture. They can generally be obtained according to the conventional explant preparation operating conditions in the field, such as cutting the corresponding part of surviving Artemisia annua and then performing steps such as cleaning, disinfection and cutting.

[0042] In some implementations, in step S1, the Artemisia annua explant is derived from one or more of the petals, leaves, and roots of Artemisia annua, such as the leaves.

[0043] In the culture medium A, the basal culture medium, carbon source, plant hormone and coagulant can all be conventional in the art, and their respective contents can be conventionally selected according to the actual situation.

[0044] In some specific embodiments, in step S1, the culture medium A is formulated with the following components: 4.41 g / L MS medium, 30 g / L sucrose, 1 mg / L 6-BA, 2 mg / L IBA and 7.5 g / L agar; pH is 5.8.

[0045] In some implementations, in step S1, the temperature of the first culture is 23-25°C; and / or, the time of the first culture is 20-25 days; and / or, the first culture is carried out in the dark.

[0046] In the culture medium B, the basal culture medium, carbon source, plant hormone and coagulant can all be conventional in the art, and their respective contents can be conventionally selected according to the actual situation.

[0047] In some specific embodiments, in step S2, the culture medium B is formulated with the following components: 4.41 g / L MS medium, 30 g / L sucrose, 1 mg / L 6-BA, 2 mg / L IBA, 1 mg / L ZT and water, with a pH of 5.8.

[0048] In some implementations, in step S2, the temperature of the secondary culture is 23-25°C; and / or, the secondary culture is a culture in culture medium B for 10-20 days followed by a subculture in a new culture medium B for 10-20 days.

[0049] In some specific implementations, step S2 includes the following steps: The callus tissue 2 was cultured in a shaker at 23-25℃ with a rotation speed of 100-120 rpm for 15-20 days, and then subcultured in a new culture medium B. For example, the callus tissue 2 could be obtained after 2-3 generations of culture.

[0050] In some implementations, step S3 includes the following steps: the temperature of the three cultures is 23-25°C; and / or the time of the three cultures is 20-25 days; and / or the three cultures are carried out in the dark.

[0051] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0052] The reagents and raw materials used in this invention are all commercially available.

[0053] The positive and progressive effects of this invention are as follows: In the cultivation of Artemisia annua callus, this invention unexpectedly discovered that partially replacing the carbon source and plant hormones in the existing plant callus culture medium with banana peel extract can significantly improve the proliferation of Artemisia annua callus. Attached Figure Description

[0054] Figure 1 This is a photograph of the Artemisia annua callus tissue obtained from proliferation culture in different culture media in Example 2.

[0055] Figure 2 The multiplication factor of Artemisia annua callus obtained by proliferation culture in different culture media in Example 2 is the multiplication factor. Detailed Implementation

[0056] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0057] Example 1: The present invention performed the acquisition and proliferation culture of callus tissue using the following steps, and verified the comparison of proliferation culture using culture media containing different banana peel extracts: S1, First culture: Cut leaves of *Artemisia annua* as explant material, place them in a clean beaker, add an appropriate amount of detergent and stir thoroughly until foam appears. Gently scrub the surface of the explant with a brush, and rinse with running water for 30-40 minutes. In a laminar flow hood, place the rinsed explants into a sterile small beaker, immerse in 75% alcohol for 50 seconds, rinse 1-3 times with sterile ultrapure water, immerse in 2% NaClO solution (mixed with 1-3 drops of Tween 20) for 6 minutes, and rinse 5-7 times with sterile ultrapure water. Blot the sterilized *Artemisia annua* explants dry with sterile filter paper and cut them into square tissue blocks with a sterile scalpel for later use.

[0058] The resulting square tissue blocks were placed on culture medium A for one culture.

[0059] Specifically, an aqueous solution containing 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331) was prepared. The pH was adjusted to 5.8 with NaOH aqueous solution, and the solution was autoclaved at 121℃ for 15 min. When the temperature of the medium dropped to 55-65℃, filtered and sterilized plant hormones were added to a final concentration of 1 mg / L 6-BA (6-Benzylaminopurine) and 2 mg / L IBA (3-Indolebutyric acid), resulting in medium A. Square tissue blocks were then placed in medium A and cultured in the dark at 23-25℃. After 20 days of culture, complete dedifferentiation was achieved, yielding callus tissue 1.

[0060] S2, Secondary Culture: The callus tissue 1 obtained in step S1 was transferred to culture medium B for secondary culture.

[0061] Specifically, an aqueous solution containing 4.41 g / L MS medium powder (Coolaber, PM1011) and 30 g / L sucrose (Coolaber, CS10581) was prepared. The pH was adjusted to 5.8 with NaOH aqueous solution, and the solution was autoclaved at 121℃ for 15 min. When the temperature of the medium dropped to 55-65℃, filtered and sterilized plant hormones were added to a final concentration of 1 mg / L 6-BA, 2 mg / L IBA, and 1 mg / L ZT (Zeatin), thus obtaining medium B. Callus 1 was then placed in a culture flask containing medium B and cultured in a shaker at a temperature of 23℃-25℃ and a rotation speed of 120 rpm. After 15 days of culture, the culture was subcultured with fresh medium B. After 15 days, a suspension containing suspended callus (denoted as callus 2) was obtained.

[0062] S3, Three-stage cultivation: The callus cultured in step S2 was cultured three times using 96-well culture dishes.

[0063] Specifically, 50 µL of suspension was pipetted into each well and spread evenly into different culture media C. The mixture was then incubated in the dark at 23-25°C for 20 days to obtain callus tissue 3. The differences in morphological and fresh weight differences among the callus tissue 3 indicate the variations in proliferation effects between different culture media.

[0064] As shown in Table 1, in this embodiment, based on the culture medium A in step S1, the sucrose content and the ratio of each hormone are adjusted, and different concentrations of banana peel water extract and banana peel alcohol extract are added (extraction process is as follows) to obtain the corresponding culture medium C.

[0065] The preparation process of culture medium C is the same as step S1: prepare an aqueous solution containing 4.41 g / L MS culture medium powder (Coolaber, PM1011) and 7.5 g / L agar (Coolaber, CA1331), adjust the pH to 5.8 with NaOH aqueous solution, and autoclave at 121℃ for 20 min; when the temperature of the culture medium drops to 55~65℃, add different concentrations of filtered sterilized sucrose solution, plant hormones (NAA (1-Naphthaleneacetic acid, α-naphthaleneacetic acid), 6-BA), banana peel aqueous extract and banana peel alcoholic extract, and the culture medium is obtained.

[0066] The callus proliferation coefficient for different culture media is calculated as: callus fresh weight 20 days after inoculation / callus fresh weight before inoculation.

[0067] The specific preparation methods for the water extract and alcohol extract of banana peel are as follows: (1) Extraction process one: water extraction and ultrasound-assisted extraction process - banana peel water extract Take chopped banana peel pieces and add ultrapure water at a material-to-liquid ratio of 1:10 (mass ratio of banana peel to ultrapure water is 1:10). Mix well and place in an ultrasonic extraction device at 1000W for extraction at room temperature (25℃). Each extraction cycle is 30 minutes. After one extraction, centrifuge to collect the supernatant 1 (extract 1). Add the resulting precipitate 1 (residual residue) back into the same amount of ultrapure water as the first extraction, repeat the ultrasonic extraction, and centrifuge again to collect the supernatant 2 (extract 2). Combine the two extracts and centrifuge again at 8000 rpm for 10 minutes. Collect all the supernatant and concentrate it to half its mass using a rotary evaporator at 40℃. This is the banana peel aqueous extract, which should be stored at low temperature and protected from light. After sterilization by filtration through a 0.22µm membrane, it can be used as a culture medium additive.

[0068] (2) Extraction process two: 75% ethanol ultrasonic-assisted extraction process - banana peel alcohol extract

[0069] Take the same mass of chopped banana peel pieces as in Process 1, add 75% ethanol solution as the extraction solvent at a material-to-liquid ratio of 1:10 (mass ratio), and place in an ultrasonic extraction device at 1000W for room temperature extraction. Each extraction cycle is 30 minutes. After one extraction, centrifuge to collect the supernatant 1 (extraction 1). Add the resulting precipitate 1 (residual residue) back to the same volume of 75% ethanol solution used in the first extraction, repeat the ultrasonic extraction, and centrifuge again to collect the supernatant 2 (extraction 2). Combine the two extracts and concentrate them to half their mass using a rotary evaporator at 40℃ to obtain the banana peel ethanol extract. Store at low temperature and protected from light. After sterilization by filtration through a 0.22µm membrane, it is used as a culture medium additive.

[0070] The results are shown in Table 1 and below. Figures 1-2 As shown.

[0071] Table 1. Culture medium C with different formulations and their corresponding proliferation coefficients

[0072]

[0073]

[0074] The experimental results showed that, in MS basal medium (4.41 g / L MS medium powder, 7.5 g / L agar, pH 5.8), by adjusting the concentration combinations of 6-BA (1-2 mg / L), NAA (0.1-0.5 mg / L), and sucrose (5-30 g / mL), and then compounding with different concentrations of banana peel extract (50-200 ml / L), the resulting callus tissues showed significant differences in proliferation 20 days after inoculation in step S3.

[0075] Furthermore, specific comparisons revealed that banana peel extract has a significant promoting effect on callus proliferation, and it is expected to be used for the large-scale culture of Artemisia annua callus and the efficient synthesis of subsequent metabolites.

Claims

1. A culture medium for Artemisia annua callus, characterized in that, The culture medium formulation includes the following components: 2-6 g / L MS medium, 5-10 g / L agar, 5-30 g / L sucrose, 0.1-0.5 mg / L NAA, 1-2 mg / L 6-BA and 50-300 mL / L banana peel extract; pH 5.5-6.

0.

2. The culture medium for Artemisia annua callus as described in claim 1, characterized in that, The culture medium satisfies one or more of the following conditions (1)-(8): (1) The banana peel extract is an extract obtained by extracting banana peel with an extraction reagent; (2) The content of the banana peel extract is 50-200 mL / L; (3) The concentration of the MS medium is 4-6 g / L; (4) The content of the agar is 7-10 g / L; (5) The sucrose content is 5-20 g / L; (6) The content of NAA is 0.1-0.4 mg / L; (7) The content of 6-BA is 1.0-1.7 mg / L; (8) The pH of the culture medium is 5.6-5.

9.

3. The culture medium for Artemisia annua callus as described in claim 2, characterized in that, The culture medium satisfies one or more of the following conditions (1)-(9): (1) The extraction reagent includes aqueous solutions of water and / or ethanol; (2) The extraction process includes the steps of primary extraction, secondary extraction and concentration under ultrasonic conditions; (3) The content of the banana peel extract is 100 mL / L or 150 mL / L; (4) The concentration of the MS medium is 4.41 g / L; (5) The content of the agar is 7.5 g / L; (6) The sucrose content is 10 g / L or 15 g / L; (7) The NAA content is 0.2 mg / L or 0.3 mg / L; (8) The content of 6-BA is 1.2 mg / L or 1.4 mg / L; (9) The pH of the culture medium is 5.7 or 5.

8.

4. The culture medium for Artemisia annua callus as described in claim 3, characterized in that, The banana peel extract meets the following conditions (1) and / or (2): (1) The volume percentage of ethanol in the ethanol-water solution is 70%-80%; (2) The extraction process includes the following steps: ① Mix a portion of the extraction reagent with the banana peel, and perform a single extraction under ultrasonic conditions. Centrifuge to obtain supernatant 1 and precipitate 1. ② Mix the precipitate 1 with the remaining extraction reagent, and perform a second extraction under the condition of secondary ultrasound. Centrifuge to obtain supernatant 2 and precipitate 2. ③ Combine the supernatant 1 and supernatant 2, and then concentrate them.

5. The culture medium for Artemisia annua callus as described in claim 4, characterized in that, The banana peel extract meets one or more of the following conditions (1)-(6): (1) In the first extraction, the mass ratio of the banana peel to the extraction reagent is 1:(8-12); (2) The amount of extraction reagent used in the secondary extraction is the same as the amount of extraction reagent used in the primary extraction; (3) In the first extraction and the second extraction, the power of the first ultrasound and the second ultrasound is 800-1200W each independently; (4) In the first extraction and the second extraction, the time for the first ultrasound and the second ultrasound are each 20-60 min independently; (5) In the first extraction and the second extraction, the temperature of the first ultrasound and the second ultrasound are each independently 24-26℃; (6) In the concentration step, the concentration is carried out until the mass of the combined extract is reduced by half.

6. The culture medium for Artemisia annua callus as described in any one of claims 1-5, characterized in that, The culture medium formulation includes the following components: 4-6 g / L MS medium, 5-20 g / L sucrose, 1.0-1.7 mg / L 6-BA, 0.1-0.4 mg / L NAA, 50-200 mL / L banana peel extract and 7-10 g / L agar; pH 5.5-6.

0.

7. The culture medium for Artemisia annua callus as described in any one of claims 1-5, characterized in that, The culture medium formulation includes the following components: 4.41 g / L MS medium, 5 g / L sucrose, 1.0 mg / L 6-BA, 0.1 mg / L NAA, 200 mL / L banana peel extract and 7.5 g / L agar; pH 5.8; Alternatively, the culture medium may be formulated with the following components: 4.41 g / L MS medium, 10 g / L sucrose, 1.2 mg / L 6-BA, 0.2 mg / L NAA, 150 mL / L banana peel extract and 7.5 g / L agar; pH 5.8; Alternatively, the culture medium may be formulated with the following components: 4.41 g / L MS medium, 15 g / L sucrose, 1.4 mg / L 6-BA, 0.3 mg / L NAA, 100 mL / L banana peel extract and 7.5 g / L agar; pH 5.8; Alternatively, the culture medium may be formulated with the following components: 4.41 g / L MS medium, 20 g / L sucrose, 1.7 mg / L 6-BA, 0.4 mg / L NAA, 50 mL / L banana peel extract and 7.5 g / L agar; pH 5.

8.

8. A method for cultivating Artemisia annua callus, characterized in that, The cultivation method includes the step of proliferating and culturing Artemisia annua callus tissue using the culture medium as described in any one of claims 1-7.

9. The method for cultivating Artemisia annua callus as described in claim 8, characterized in that, The cultivation method includes the following steps: S1. Artemisia annua explants were cultured once in culture medium A to obtain callus 1; The culture medium A comprises the following components: 4-10 g / L basal medium, 20-40 g / L carbon source, 0.1-5 mg / L plant hormone, and 5-10 g / L solidifying agent; pH is 5.5-6.

0. S2. The callus 1 is cultured a second time in culture medium B to obtain callus 2. The culture medium B comprises the following components: 4-10 g / L basal medium, 20-40 g / L carbon source and 0.1-5 mg / L plant hormone; pH is 5.5-6.

0. S3. The callus 2 is cultured three times with culture medium C, wherein culture medium C is the culture medium.

10. The method for cultivating Artemisia annua callus as described in claim 9, characterized in that, The cultivation method satisfies one or more of the following conditions (1)-(11): (1) In step S1, the explants of Artemisia annua are derived from one or more of the petals, leaves and roots of Artemisia annua; (2) In step S1, the formulation of the culture medium A includes the following components: 4.41 g / L MS medium, 30 g / L sucrose, 1 mg / L 6-BA, 2 mg / L IBA and 7.5 g / L agar; pH is 5.8; (3) In step S2, the formulation of the culture medium B includes the following components: 4.41 g / L MS medium, 30 g / L sucrose, 1 mg / L 6-BA, 2 mg / L IBA and 1 mg / L ZT; pH is 5.8; (4) In step S1, the temperature of the first culture is 23-25℃; (5) In step S1, the culture time for one culture is 20-25 days; (6) In step S1, the first culture is carried out in a dark environment; (7) In step S2, the temperature of the secondary culture is 23-25℃; (8) In step S2, the secondary culture is to culture in culture medium B for 10-20 days and then subculture in new culture medium B for 10-20 days. (9) In step S3, the temperature of the three cultures is 23-25℃; (10) In step S3, the three culture times are 20-25 days; (11) In step S3, the three cultures are carried out in a dark environment.

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