Method for efficient regeneration of artemisia stem tips

Abstract

The application discloses a kind of efficient regeneration methods of artemisia vulgaris stem tip, comprising the following steps: S1, with the current year's top bud of wanai as material, after surface disinfection, peel and take stem tip as explant material;S2, explant material is inoculated in adventitious bud induction culture medium, and adventitious bud is induced and cultured;S3, the explant material of adventitious bud induced in S2 is transferred to adventitious bud proliferation culture medium and is carried out to the proliferation and elongation culture of adventitious bud;S4, the elongated adventitious bud in S3 is carried out to obtain complete artemisia vulgaris regeneration plant outside adventitious root induction culture.This application provides a kind of fast, efficient, low-cost and can keep the excellent characteristics of maternal plant wanai propagation method, for the germplasm preservation, large-scale propagation and variety detoxification rejuvenation of wanai provides certain technical support.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, and particularly relates to a high-efficiency regeneration method of Artemisia argyi shoot tips. BACKGROUND

[0002] Artemisia argyi is a perennial herb of the genus Artemisia in the family Asteraceae, and is a large-scale medicinal plant with medicinal, edible and health values. The whole plant is used as medicine, and has the effects of warming meridians, removing dampness and cold, stopping bleeding, reducing inflammation, relieving asthma and cough, preventing miscarriage, resisting allergy and resisting blood clotting. In recent years, with the rapid development of traditional Chinese medicine health service industry, the market demand for Artemisia argyi industry is increasing day by day. The unique effects of Artemisia argyi and its products in preventing disease and skin care have been widely recognized by the society, and have broad market prospects.

[0003] Nanyang City in Henan Province is an internationally recognized important origin of Artemisia argyi industry, and is the largest Artemisia argyi raw material supply base in China. The Artemisia argyi produced in Nanyang City is collectively referred to as "Wanai" by the outside world. Wanai has attracted widespread attention due to its high medicinal value, multiple varieties, rich components and high yield. However, the lack of excellent varieties and effective variety multiplication techniques is a bottleneck problem restricting the development of Wanai industry.

[0004] At present, in production, the propagation of Artemisia argyi is mainly carried out by traditional methods such as rootstock branching, cutting and seeding. Although the asexual reproduction methods such as rootstock branching and cutting can maintain the excellent traits of the mother plant, they are limited by materials and seasons, and long-term asexual reproduction can easily lead to disease accumulation, yield and quality decline and other problems of variety degeneration. The use of seeding method for propagation is difficult to preserve the excellent traits of the mother plant. The above problems seriously limit the large-scale propagation, popularization and later variety improvement of Wanai high-quality germplasm resources.

[0005] The Chinese patent application document with the publication number CN106172002A discloses a production method of wormwood tissue culture seedlings, which has the advantages of high survival rate and high propagation coefficient, and can improve the survival rate by 15-20% compared with the traditional wormwood planting method in Guangxi region, can rapidly propagate a large number of wild wormwood in a short period of time, has the characteristics of high survival rate, rapid growth, and can realize large-scale planting and production rapidly; the Chinese patent application document with the publication number CN107549018A discloses a production method of Aiqi wormwood tissue culture seedlings, which has the advantages of high propagation coefficient, low cost and simple operation, and can be applied to factory production of Aiqi wormwood tissue culture seedlings; although plant tissue culture can overcome the above shortcomings, the difficulty of plant tissue culture for different varieties is very different; even for the same material, the regeneration capacity and required culture conditions are very different due to different explant sources and explant types. Therefore, in view of the problems existing in the breeding of Aiqi wormwood, an efficient regeneration method is found to maintain the genetic traits, so as to meet the needs of germplasm preservation, large-scale propagation, detoxification and rejuvenation, and variety improvement of Aiqi wormwood. SUMMARY

[0006] The technical problem to be solved by the present application is how to efficiently regenerate wormwood while maintaining the stability of genetic traits.

[0007] The present application solves the above technical problems by the following technical means:

[0008] A method for efficiently regenerating wormwood stem tips, comprising the following steps:

[0009] S1. Using the current year's top bud of Aiqi as the material, after surface disinfection, the stem tip is peeled off as the explant material;

[0010] S2. The explant material is inoculated into the adventitious bud induction medium for induction culture of adventitious buds;

[0011] S3. The explant material in S2 which induces adventitious buds is transferred to the adventitious bud proliferation medium for proliferation and elongation culture of adventitious buds;

[0012] S4. The elongated adventitious buds in S3 are subjected to ex-vitro adventitious root induction culture to obtain complete wormwood regenerated plants.

[0013] Preferably, S1 specifically comprises the following steps: the current year's top buds of the excellent health strain of Artemisia vulgaris are washed with running water for 10-20 min, then washed with sterile water for 3-5 times in a sterile operation table, then surface sterilized with 75% ethanol for 15-20 s, then washed with sterile water for 3-5 times, then surface sterilized with 75% ethanol for 15-20 s, then washed with sterile water for 3-5 times, then sterilized with 0.1% (w / v) mercuric chloride solution for 1-3 min, then washed with sterile water for 4-6 times, then the surface moisture is absorbed with sterile filter paper, then 1-3 mm stem tip meristems are peeled off with a pair of tweezers as explant materials.

[0014] Preferably, in S2, the adventitious bud induction medium is WPM medium added with 0.01-0.5 mg / L TDZ, 3.0% (w / v) sucrose and 0.7% (w / v) agar.

[0015] Preferably, in S2, the induction culture time of the adventitious buds is 4 weeks.

[0016] Preferably, in S3, the adventitious bud proliferation medium is WPM medium added with 0.1-1.0 mg / L TDZ, 0.1-1.0 mg / L ZT, 0.1-1.0 mg / L GA3, 3.0% (w / v) sucrose and 0.7% (w / v) agar.

[0017] Preferably, in S3, the proliferation and elongation culture time of the adventitious buds is 4 weeks.

[0018] Preferably, S4 specifically comprises the following steps: the elongated adventitious buds in S3 are separated from the bud cluster and then sterilized, then the morphological lower end of the adventitious buds is treated in a rooting promoting solution, then planted in a seedling tray, then subjected to adventitious root induction culture outside the bottle in a greenhouse to obtain complete A. vulgaris regenerated plants; wherein the rooting promoting solution is one of a KIBA aqueous solution with a concentration of 500-2000 mg / L, an IBA aqueous solution with a concentration of 500-1000 mg / L, an NAA aqueous solution with a concentration of 500-1000 mg / L and an IAA aqueous solution with a concentration of 500-1000 mg / L.

[0019] Preferably, the seedling tray is a seedling tray filled with a mixed substrate of nutrient soil and vermiculite.

[0020] Preferably, S4 specifically comprises the following steps: after separating the elongated adventitious buds from the bud cluster in S3, rinsing with running water, then sterilizing with 0.05%-0.2% (w / v) potassium permanganate solution for 5-10 min, absorbing surface moisture, placing the morphological lower end of the adventitious bud into a rooting promoting liquid for 5-10 s, then planting the morphological lower end into a seedling tray filled with mixed substrate of nutrient soil and vermiculite, and performing ex-vitro adventitious root induction culture in a greenhouse to obtain complete wormwood regenerative plants; wherein the rooting promoting liquid is one of KIBA aqueous solution with a concentration of 500-2000 mg / L, IBA aqueous solution with a concentration of 500-1000 mg / L, NAA aqueous solution with a concentration of 500-1000 mg / L, and IAA aqueous solution with a concentration of 500-1000 mg / L.

[0021] Preferably, the volume ratio of the nutrient soil to the vermiculite is 2:1.

[0022] Preferably, in S2, S3 and S4, the culture conditions are as follows: temperature is 25±2℃, light intensity is 2500-3000 lx, and photoperiod is 14 / 10 h (light / dark).

[0023] The present application has the following advantages:

[0024] The present application provides a method for efficient regeneration of wormwood stem tips by using plant tissue culture technology, which has the following advantages: first, the stem tips are taken from the current year's terminal buds of excellent wormwood strains, and the direct induction of adventitious buds from the stem tips is adopted in the breeding process, so that the germplasm characteristics of the mother plants can be maintained, and the rapid breeding of excellent single wormwood plants can be realized; second, the elongated cluster buds are directly subjected to ex-vitro rooting technology, so that the rooting process and the domestication and transplantation are synchronized, which improves the survival rate of the regenerated plants after transplantation and reduces the cost of seedling raising; third, the regeneration efficiency is high, the induction rate of the cluster buds is up to 100%, and the ex-vitro rooting rate of the adventitious buds is up to 100%, which provides important technical support for the germplasm preservation, large-scale breeding, detoxification and rejuvenation, and later genetic improvement of excellent wormwood strains. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Wormwood stem tips inoculated on the adventitious bud induction medium in Example 2 of the present application;

[0026] Figure 2 Wormwood stem tip adventitious buds obtained after 2 weeks of illumination culture on the adventitious bud induction medium in Example 2 of the present application;

[0027] Figure 3 Wormwood stem tip adventitious buds obtained after 4 weeks of culture on the adventitious bud induction medium in Example 2 of the present application;

[0028] Figure 4 Artemisia vulgaris elongated adventitious shoots obtained after culturing for 4 weeks in the proliferation medium in Example 2 of the present application;

[0029] Figure 5 Artemisia vulgaris elongated shoots obtained after culturing for 4 weeks in the proliferation medium in Example 2 of the present application;

[0030] Figure 6 Artemisia vulgaris elongated shoots obtained after culturing for 4 weeks in the proliferation medium in Example 2 of the present application;

[0031] Figure 7 Artemisia vulgaris elongated shoots obtained after culturing for 4 weeks in the proliferation medium in Example 2 of the present application; DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in connection with the embodiments of the present application. Obviously, the described embodiments are only some but not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0033] In the following examples, the test materials and reagents used, unless otherwise specified, can be obtained through commercial channels.

[0034] If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions can be used.

[0035] Example 1

[0036] A method for efficient regeneration of Artemisia vulgaris shoot tips, the specific operation is as follows:

[0037] (1) Using the current year's top bud of the excellent strain of Wannai as the material, after washing with running water for 10 min, in a sterile operation table, washing with sterile water for 3 times, then surface sterilizing with 75% ethanol for 15 s, then washing with sterile water for 3 times, then surface sterilizing with 75% ethanol for 15 s; after washing with sterile water for 3 times, then sterilizing with 0.1% (w / v) mercury solution for 1 min, then washing with sterile water for 6 times. After absorbing the surface water with sterile filter paper, using forceps to peel off 1-3 mm stem tips meristem tissue for standby.

[0038] (2) The shoot tip meristems extracted in step (1) were inoculated into WPM medium supplemented with 0.01 mg / L TDZ, 3.0% (w / v) sucrose, and 0.7% (w / v) agar. Adventitious shoots were induced in a constant temperature culture room at 25±2℃, light intensity of 2500-3000 lx, and light duration of 14 h / d. After 4 weeks of light culture, the shoot tip contamination rate was 21.4%, the shoot tip adventitious shoot induction rate was 67.3%, and an average of 3.6 adventitious shoots were produced per explant.

[0039] (3) The adventitious shoots induced in step (2) were transferred to WPM medium supplemented with 0.1 mg / L TDZ, 0.1 mg / L ZT, 1.0 mg / L GA3, 3.0% (w / v) sucrose and 0.7% (w / v) agar. The adventitious shoots were cultured in a constant temperature incubator at 25±2℃, with a light intensity of 2500–3000 lx and a photoperiod of 14 h / d for proliferation and elongation. After 4 weeks of photoperiod culture, the adventitious shoot proliferation rate was 84.8%, with an average of 5.1 adventitious shoots per explant and an average shoot height of 2.4 cm.

[0040] (4) After separating the elongated adventitious buds from the bud cluster in step (3), rinse them with running water, then disinfect them with 0.05% (w / v) potassium permanganate solution for 10 min, and then dry the surface moisture. Place the morphological lower end of the adventitious bud in an aqueous solution containing 500 mg / L KIBA for 10 s, and then plant the morphological lower end in a seedling tray containing a mixture of Pints ​​potting soil and vermiculite (v:v = 2:1). Cover with a plastic lid and incubate in a constant temperature culture room at 25±2℃, light intensity of 2500~3000lx, and light duration of 14h / d for exorooting. After 2 weeks of culture, adventitious root primordia were produced at the base of the stem segment. After 4 weeks of culture, the adventitious root induction rate reached 85.8%, with an average of 4.3 adventitious roots produced per explant.

[0041] Example 2

[0042] A method for efficient regeneration of Artemisia argyi stem tips, the specific operation of which is as follows:

[0043] (1) Using the current year's terminal buds of superior Wan'ai strains as material, after rinsing with running water for 20 minutes, rinse five times with sterile water in a sterile operating table, then disinfect the surface with 75% ethanol for 20 seconds, then rinse five times with sterile water, then disinfect the surface with 75% ethanol for 15 seconds; continue rinsing with sterile water five times, then disinfect with 0.1% (w / v) mercuric chloride solution for 2 minutes, and then rinse six times with sterile water. Absorb the surface moisture with sterile filter paper, and then use tweezers to peel off 1-3 mm of stem tip meristem for later use.

[0044] (2) The stem tip meristem peeled in step (1) is inoculated in WPM medium added with 0.2 mg / L TDZ, 3.0% (w / v) sucrose and 0.7% (w / v) agar (as shown in Figure 1 ), and is subjected to adventitious bud induction in a constant temperature culture room at a temperature of 25±2°C, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 2 weeks of illumination culture, the stem tip contamination rate is 0.0%, and the stem tip induces adventitious bud clusters (as shown in Figure 2 ). After 4 weeks of illumination culture, the induction rate of adventitious buds is as high as 100%, and an average of 8.1 adventitious buds is generated per explant (as shown in Figure 3 ).

[0045] (3) The adventitious buds induced in step (2) are transferred to WPM medium added with 0.5 mg / L TDZ, 0.5 mg / L ZT, 0.1 mg / L GA3, 3.0% (w / v) sucrose and 0.7% (w / v) agar, and are subjected to proliferation and elongation culture of adventitious buds in a constant temperature culture room at a temperature of 25±2°C, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 4 weeks of illumination culture, the proliferation rate of adventitious buds is 97.6%, an average of 11.3 adventitious buds is generated per explant, and the average height of adventitious buds is 3.5 cm (as shown in Figure 4 ).

[0046] (4) The elongated adventitious buds in step (3) are separated from the bud clusters, washed with running water, then disinfected with 0.1% (w / v) potassium permanganate solution for 5 min, the surface water is absorbed, the morphological lower end of the adventitious buds is treated in 1000 mg / L KIBA aqueous solution for 5 s, and then is planted in a seedling raising tray filled with mixed medium of perlite and peat (v:v=2:1) (as shown in Figure 5 ), and is covered with a plastic cover, and is subjected to ex-vitro rooting induction in a constant temperature culture room at a temperature of 25±2°C, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 10 d of culture, adventitious root primordia are generated at the base of the stem segments, and after 4 weeks of culture, the induction rate of adventitious roots is as high as 100%, and an average of 5.7 adventitious roots is generated per explant (as shown in Figure 6 ). After 3 weeks of continuous culture, robust mugwort complete regenerated plants are obtained (as shown in Figure 7 ).

[0047] Example 3

[0048] A method for efficient regeneration of mugwort stem tips, the specific operation is as follows:

[0049] (1) Take the current year's top bud of the fine strain of Artemisia vulgaris as the material, wash it with running water for 20 min, then wash it with sterile water for 5 times in a sterile operation table, then sterilize the surface with 75% ethanol for 20 s, then wash it with sterile water for 5 times, then sterilize the surface with 75% ethanol for 20 s again, continue to wash it with sterile water for 5 times, then sterilize it with 0.1% (w / v) mercury chloride solution for 3 min, then wash it with sterile water for 6 times. Dry the surface water with sterile filter paper, then use forceps to peel off 1-3 mm stem tip meristem for standby.

[0050] (2) Inoculate the stem tip meristem peeled off in step (1) into WPM medium added with 0.5 mg / L TDZ, 3.0% (w / v) sucrose and 0.7% (w / v) agar, and carry out adventitious bud induction culture in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 4 weeks of illumination culture, the stem tip induces adventitious bud clusters, the induction rate of adventitious buds is 84.5%, and an average of 5.3 adventitious buds are generated per explant.

[0051] (3) Transfer the induced adventitious buds in step (2) into WPM medium added with 1.0 mg / L TDZ, 1.0 mg / L ZT, 1.0 mg / L GA3, 3.0% (w / v) sucrose and 0.7% (w / v) agar, and carry out adventitious bud proliferation and elongation culture in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 4 weeks of illumination culture, the proliferation rate of adventitious buds is 79.5%, an average of 7.6 adventitious buds are generated per explant, and the average height of adventitious buds is 2.8 cm.

[0052] (4) After separating the elongated adventitious buds in step (3) from the bud clusters, wash them with running water, then sterilize them with 0.2% (w / v) potassium permanganate solution for 5 min, dry the surface water, place the morphological lower end of the adventitious buds into 750 mg / L KIBA aqueous solution for 10 s, then plant the morphological lower end into a seedling raising tray filled with mixed substrate of perlite and vermiculite (v:v=2:1), cover it with a plastic cover, and carry out ex-vitro rooting induction in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx and a light time of 14 h / d. After 10 d of culture, adventitious root primordia are generated at the base of the stem segments, and after 4 weeks of culture, the induction rate of adventitious roots is as high as 100%, and an average of 5.1 adventitious roots are generated per explant.

[0053] Example 4

[0054] This example tests the effects of exogenous KIBA concentration and treatment time on the ex-vitro rooting of elongated buds of Artemisia vulgaris, and the specific steps are as follows:

[0055] (1) Elongated shoots were obtained on the basis of Example 2, and the elongated shoots with uniform growth and a height of about 3.5 cm obtained in Example 2 were separated from the culture bottles, washed with running water, sterilized with 0.1% (w / v) potassium permanganate solution for 5 min, and then dried, and the morphological lower end of the shoots was treated with different concentrations of KIBA (0-2000 mg / L) for different times (0, 5 and 10 s). Then the morphological lower end of the treated shoots was planted in a seedling raising tray containing a mixture of Pindan soil and vermiculite (v:v=2:1), covered with a plastic cover, and subjected to ex vitro adventitious root induction in a constant temperature incubator at a temperature of 25±2°C, a light intensity of 2500-3000 lx, and a light time of 14 h / d. After 4 weeks of culture in a greenhouse, the effects of the exogenous KIBA concentration and treatment time on the ex vitro rooting of the elongated shoots of wormwood were determined, and the results are shown in Table 1.

[0056] Table 1 Effects of exogenous KIBA concentration and treatment time on the ex vitro rooting of elongated shoots of wormwood

[0057]

[0058]

[0059] Note: The data are average values, each treatment contained 120 explants, and each treatment was repeated three times

[0060] Table 1 shows that the exogenous KIBA concentration and treatment time have important effects on the ex vitro rooting of elongated shoots of wormwood. Within a certain range, as the treatment concentration and treatment time are increased, the induction rate of adventitious roots, the number of adventitious roots and the average length of adventitious roots gradually increase. When the exogenous KIBA concentration is 1000-2000 mg / L, the induction rate of adventitious roots is 100%, but as the treatment concentration and treatment time are increased, the length of adventitious roots gradually decreases. It is speculated that as the concentration and treatment time are increased, the elongation of adventitious roots is inhibited to some extent.

[0061] In addition, the present embodiment also tests the effect of exogenous IBA, NAA and IAA concentration on the elongated bud of wormwood outside the bottle. The elongated adventitious bud of wormwood with uniform growth and a height of about 3.5 cm obtained in Example 2 is separated from the tissue culture bottle. After being washed with running water, the adventitious bud is sterilized with 0.1% (w / v) potassium permanganate solution for 5 min, and the surface water is absorbed. The morphological lower end of the adventitious bud is treated in an aqueous solution containing different concentrations (500, 750 and 1000 mg / L) of IBA, NAA and IAA for 10 s. Then the morphological lower end of the treated adventitious bud is planted in a seedling tray containing a mixed substrate (v:v = 2:1) of nutrient soil and vermiculite, covered with a plastic cover, and placed in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a light time of 14 h / d for bottle outside adventitious root induction. After 4 weeks of culture, the effect of wormwood elongated bud on bottle outside root is counted, and the results are shown in Table 2.

[0062] Table 2 Effect of exogenous IBA, NAA and IAA concentration on the elongated bud of wormwood outside the bottle

[0063]

[0064]

[0065] The results of the above Tables 1 and 2 show that the application of exogenous auxin (IBA, NAA, IAA and KIBA) can greatly improve the effect of bottle outside root of wormwood elongated bud. Among the four auxins tested, KIBA has the best effect on the induction of bottle outside adventitious root of wormwood elongated bud, followed by NAA.

[0066] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for regenerating Artemisia argyi stem tips, characterized in that: Includes the following steps: S1. Using the current year's terminal buds of Artemisia argyi as material, after surface disinfection, the stem tips are peeled off as explant material; S2. The explant material is inoculated into an adventitious shoot induction medium to induce adventitious shoots; the adventitious shoot induction medium is WPM + 0.01-0.5 mg / L TDZ + 3.0% w / v sucrose + 0.7% w / v agar; S3. The explant material from which adventitious shoots were induced in S2 was transferred to an adventitious shoot proliferation medium for adventitious shoot proliferation and elongation culture; the adventitious shoot proliferation medium was WPM + 0.1-1.0 mg / L TDZ + 0.1-1.0 mg / L ZT + 0.1-1.0 mg / L GA3 + 3.0% w / v sucrose + 0.7% w / v agar; S4. Obtain complete Artemisia argyi regenerated plants by in vitro adventitious root induction culture of the elongated adventitious buds in S3; specifically, the following steps are included: after separating the elongated adventitious buds from the bud cluster in S3 and disinfecting them, the morphological lower end of the adventitious buds is placed in a rooting promotion solution for treatment, and after treatment, they are planted in seedling trays and in vitro adventitious root induction culture is carried out in a greenhouse to obtain complete Artemisia argyi regenerated plants; wherein, the rooting promotion solution is one of the following: KIBA aqueous solution with a concentration of 500-2000 mg / L, IBA aqueous solution with a concentration of 500-1000 mg / L, NAA aqueous solution with a concentration of 500-1000 mg / L, or IAA aqueous solution with a concentration of 500-1000 mg / L.

2. The method for regenerating Artemisia argyi stem tips according to claim 1, characterized in that: S1 specifically includes the following steps: After rinsing the current year's terminal buds of superior and healthy *Artemisia argyi* strains with running water for 10-20 minutes, rinse them 3-5 times with sterile water in a sterile operating table, then disinfect the surface with 75% ethanol for 15-20 seconds, rinse again with sterile water 3-5 times, disinfect again with 75% ethanol for 15-20 seconds, continue rinsing with sterile water 3-5 times, then disinfect with 0.1% w / v mercuric chloride solution for 1-3 minutes, then rinse 4-6 times with sterile water, absorb the surface moisture with sterile filter paper, and then use tweezers to peel off 1-3 mm of stem tip meristem as explant material.

3. The method for regenerating Artemisia argyi stem tips according to claim 1, characterized in that: In S2, the induction culture time for the adventitious buds is 4 weeks.

4. The method for regenerating Artemisia argyi stem tips according to claim 1, characterized in that: In S3, the proliferation and elongation culture time of the adventitious buds is 4 weeks.

5. The method for regenerating Artemisia argyi stem tips according to claim 1, characterized in that: S4 specifically includes the following steps: After separating the elongated adventitious buds from the bud cluster in S3, rinse them with running water, then disinfect them with 0.05%–0.2% w / v potassium permanganate solution for 5–10 minutes, absorb the surface moisture, place the morphological lower end of the adventitious bud in a rooting promotion solution for 5–10 seconds, and then plant the morphological lower end in a seedling tray containing a mixture of nutrient soil and vermiculite substrate. In vitro adventitious root induction culture is carried out in a greenhouse to obtain complete Artemisia argyi regenerated plants. The rooting promotion solution is one of the following: a KIBA aqueous solution with a concentration of 500–2000 mg / L, an IBA aqueous solution with a concentration of 500–1000 mg / L, a NAA aqueous solution with a concentration of 500–1000 mg / L, or an IAA aqueous solution with a concentration of 500–1000 mg / L.

6. The method for regenerating Artemisia argyi stem tips according to claim 5, characterized in that: The volume ratio of the nutrient soil to vermiculite is 2:

1.

7. The method for regenerating Artemisia argyi stem tips according to any one of claims 1-6, characterized in that: In S2, S3 and S4, the cultivation conditions were as follows: temperature 25±2℃, light intensity 2500~3000lx, and photoperiod 14h light / 10h dark.

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