An ainsliaea growth medium, tissue culture medium, tissue culture method and application

Abstract

The application belongs to the technical field of plant cultivation and particularly relates to a Blumea riparia growth medium, a tissue culture medium, a tissue culture method and application. The Blumea riparia growth medium takes MS medium as a basic medium and further comprises 0.2-0.3 mg / L NAA, 0.08-0.16 mg / L DA-6, 10 mg / L vitamin C, 20 mg / L myo-inositol, 30 g / L sucrose and 6 g / L agar. DA-6 and NAA are jointly applied to improve the rooting efficiency, root length and plant height of Blumea riparia. The Blumea riparia germplasm with high content of levorotatory borneol is used as an explant to carry out tissue culture and seedling raising, the Blumea riparia tissue culture seedling has high content of levorotatory borneol, and Blumea riparia stable genetic good seedlings can be produced.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, specifically relating to an Artemisia annua growth medium, tissue culture medium, tissue culture method, and application. Background Technology

[0002] *Blumea balsamifera* (L.) DC. is a perennial herb belonging to the genus *Blumea* in the family Compositae. It is the source plant for borneol (L-carnitine) in the Chinese Pharmacopoeia. The crystals obtained from its fresh leaves are extracted and processed into borneol (L-carnitine). *Blumea balsamifera* is a native herb of tropical and subtropical Asia with a medicinal history of over a thousand years. As a medicinal plant, it contains various chemical components, with volatile oils, especially L-carnitine, being its main chemical component. *Blumea balsamifera* possesses properties such as dispelling wind and dampness, warming the middle jiao and stopping diarrhea, promoting blood circulation and detoxifying. It also exhibits various physiological activities, including anti-inflammatory, anticancer, antifungal, and antimicrobial effects.

[0003] L-borneol in *Ageratum praecox* has high market value, but currently, there is a lack of stable genetically superior varieties and seedlings due to the rich variation in individual traits and susceptibility to environmental influences. The applicant previously collected a large number of *Ageratum praecox* germplasm resources from various producing areas across the country and preserved them in a germplasm resource nursery. Resource evaluation results showed significant differences in the L-borneol content among different germplasms. However, since each germplasm has only one mother plant, it is impossible to determine whether these differences are caused by genetic differences or by differences in the effects of environment or pests and diseases on the plants. Furthermore, the current propagation method of *Ageratum praecox* involves a mixture of sexual reproduction (cross-pollination to obtain heterozygous seeds and seedlings) and asexual reproduction (propagation of offspring), leading to phenotypic segregation in the offspring. These circumstances hinder the evaluation of *Ageratum praecox* germplasm and the breeding of superior varieties and seedlings.

[0004] Tissue culture is an asexual reproduction technique. In an artificial environment, tissue culture offspring can eliminate the segregation of traits in seedlings propagated from seeds and the influence of environmental factors. It is an efficient way to rapidly propagate seedlings and preserve the superior advantages of varieties. It is of great significance for the preservation and evaluation of excellent germplasm resources. At the same time, tissue culture rapid propagation technology can overcome the drawbacks of traditional seedling cultivation methods, improve the survival rate and propagation efficiency, and provide sufficient materials for the germplasm preservation, rapid propagation and subsequent regeneration research of high-quality seedlings.

[0005] As the investigation and breeding research of Artemisia annua germplasm resources progressed, we found that existing tissue culture techniques for Artemisia annua cannot meet the current research and production needs. These shortcomings are mainly reflected in two aspects: explants and culture effects.

[0006] Regarding explants, existing explants for *Artemisia argyi* tissue culture technology include stem segments with axillary buds (Li Lifeng, 2008. Tissue culture and GC-MS analysis of major chemical components of volatile oil in *Artemisia argyi*. Guangxi University, p. 49.), tender stem segments (specifically, stem segments 3-4 cm long containing 1-2 axillary or terminal buds, see: Xiao Yongfeng. Evaluation of *Artemisia argyi* germplasm resources and research on rapid propagation system of tissue culture [D]. Hainan University, 2023. DOI: 10.27073 / d.cnki.ghadu.2021.000970.), leaves (Yan Min, Liu Yan, Tang Hongmin, 2014. Tissue culture of *Artemisia argyi*. Jiangsu Agricultural Sciences 42, 33-35.; Chinese Patent CN105309315A), and roots (CN114375834B. A one-step method for inducing *Artemisia argyi* root cell differentiation to produce adventitious buds). The advantages of stem segments with axillary buds are their abundance, ease of acquisition, large size, ease of handling, and low mortality. However, the disadvantages are that these segments are mature, possessing well-developed vascular tissue and pith, making them susceptible to pathogenic microorganisms that can easily reach and infiltrate them via the vascular bundles, thus hindering detoxification. This is particularly problematic for important germplasm already affected by pests and diseases in the wild or field, where in vitro preservation and tissue culture result in high contamination rates and low success rates after a period of time. The second type of explant consists of 3-4 cm long stem segments containing 1-2 axillary or terminal buds, which also possess well-developed vascular bundles. One type of explant has no essential differences and loses the advantages of being abundant and readily available. The third type of explant is the leaf, which, like the first two types, suffers from a well-developed vascular bundle and the problems it brings. Furthermore, its culture requires inducing callus formation, then inducing embryoid differentiation, and finally developing into a shoot. This method inevitably involves a callus induction stage, potentially leading to uncontrollable somatic cell mutations. This could result in some or all of the tissue-cultured offspring from high-content mother plants becoming plants containing only low concentrations of L-borneol, and such mutations are often not visually detectable. The fourth type of explant is the root, which also suffers from vascular bundles and the problems they bring. Additionally, shoot tips are another viable explant option. Shoot tip culture specifically refers to the isolation and separate culture of a very small cluster of meristematic tissue (approximately 2-3 mm) at the shoot tip. Its advantage is good detoxification, but its disadvantages include high skill requirements for operators, small tissue size, and a high mortality rate. In summary, all explants used in current technologies have some problems: either the presence of vascular bundles leads to a high contamination rate, or the small tissue pieces result in a low survival rate. The technology disclosed in this invention addresses the aforementioned problems by combining the advantages of both methods. It selects a tender stem segment of 6-20 mm, including the stem tip, as the explant. This explant avoids all the disadvantages of the aforementioned explants. On the one hand, the tissue volume is larger than that of the stem tip meristem, making the operation easier and the survival rate higher. On the other hand, since the vascular tissue has not yet developed, pathogenic microorganisms have not yet arrived, resulting in better detoxification and a lower contamination rate.

[0007] Regarding cultivation effectiveness, existing technologies also have shortcomings: First, current technologies have not conducted research on tissue culture techniques for specific germplasms. With the development of *Artemisia argyi* breeding technology, we have found that old technologies can only support the tissue culture needs of some germplasms, while other germplasms require further research and supporting tissue culture technologies. Second, the rooting coefficient of *Artemisia argyi* using existing tissue culture techniques is low. For example, Chinese patent CN105309315A describes a tissue culture method for *Artemisia argyi* using an embryoid approach, which uses leaves as explants to induce callus tissue and embryoids, and then induces regenerated shoots. The average number of roots per *Artemisia argyi* tissue culture seedling is 5-7. For example, Tang Song and Yang Shiguan (see "Tang Song, Huang Yanfen, Kong Li, et al. Research on rapid propagation technology of Artemisia annua seedlings [J]. Journal of Guizhou Normal University, 2015, 31(03):38-40.; Yang Shiguan, Yang Meichun. Research on tissue culture technology of Artemisia annua [J]. Modern Agricultural Science and Technology, 2012, (18):151-153.") obtained sterile seedlings by inoculating stem segments into MS and 1 / 2 MS medium and inducing their proliferation, rooting, hardening and transplanting; the rooting coefficient is usually 4-18 segments / seedling, which is low. Third, in the process of tissue culture proliferation and subculture of Artemisia annua, plant hormones are used, and the concentration of hormones used is often high to achieve the highest proliferation efficiency, or by first inducing callus tissue and then inducing callus tissue embryogenesis, and obtaining regenerated shoots through the somatic embryo pathway, which may lead to uncontrollable somatic mutations and change the characteristics of the original germplasm. Since *Amaranthus praecox* is a medicinal plant with levorotatory borneol as its main active ingredient, somatic cell mutations that occur during rapid tissue culture propagation may cause some or all of the tissue culture offspring of the mother plant with high borneol content to become plants containing only low concentrations of levorotatory borneol, and such mutations are often not visible to the naked eye. However, all previous studies on *Artemisia annua* tissue culture have only aimed at obtaining tissue culture progeny, without considering specific germplasm or the combined effects on seedling rooting rate, root length, plant height, etc. They have also given little consideration to whether the chemical characteristics of the tissue culture progeny are consistent with those of the mother plant. In particular, existing tissue culture techniques have indeed found that the relative content of L-borneol in the volatile oil of tissue culture progeny is 17.2%, significantly lower than that of wild plants (46.7%) (Li Lifeng, 2008. Tissue culture and GC-MS analysis of major chemical components of volatile oil in *Artemisia annua*. Guangxi University, Abstract and pp. 24-25). This further strengthens the urgent need to develop new tissue culture techniques that can maintain the chemical characteristics of the mother plant. The *Artemisia annua* tissue culture seedlings obtained using the technology disclosed in this invention well inherit the original L-borneol content characteristics of the mother plant, overcoming the problems of existing techniques. Summary of the Invention

[0008] The purpose of this invention is to provide a growth medium for Artemisia annua. Using this growth medium for tissue culture results in a high rooting coefficient for the tissue-cultured seedlings. This invention provides an Artemisia annua tissue culture medium and a tissue culture method, enabling the acquisition of a large quantity of high-quality Artemisia annua tissue-cultured seedlings with maintained genetic stability.

[0009] To address the above problems, the present invention provides the following technical solution:

[0010] This invention provides a growth medium for promoting *Artemisia annua* growth. The growth medium uses MS medium as the basic medium and further includes: 0.2–0.3 mg / L NAA, 0.08–0.16 mg / L LDA-6, 10–50 mg / L vitamin C, 20–200 mg / L inositol, 30 g / L sucrose, and 4–10 g / L agar.

[0011] This invention provides the application of the *Artemisia annua* growth medium described in the above-mentioned technical solution in improving one or more of the following: 1) to 3) of *Artemisia annua* tissue culture seedlings.

[0012] 1) Rooting rate;

[0013] 2) Plant height;

[0014] 3) Root length.

[0015] This invention provides an agaricus tissue culture medium, comprising an adventitious shoot induction medium, a proliferation and subculture medium, and the growth medium described in the above technical solution;

[0016] The adventitious bud induction medium uses MS medium as the basic medium and also includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose and 4-10 g / L agar;

[0017] The proliferation and subculture medium uses MS medium as the basic medium and also includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose, and 4-10 g / L agar.

[0018] This invention provides a method for tissue culture of Artemisia annua, wherein the tissue culture method uses the growth culture medium described in the above-mentioned technical solution and includes the following steps:

[0019] Artemisia annua explants were inoculated onto adventitious bud induction medium for adventitious bud induction culture to obtain sterile adventitious buds of Artemisia annua.

[0020] The stem segments with axillary buds of the sterile adventitious buds of Artemisia annua were inoculated into the proliferation and subculture medium for subculture to obtain single buds of Artemisia annua.

[0021] The single shoots of Artemisia annua were inoculated into a growth medium for rooting culture to obtain tissue culture seedlings of Artemisia annua.

[0022] The tissue culture seedlings of *Artemisia annua* were hardened off and transplanted.

[0023] Preferably, the explant comprises at least one of the following:

[0024] (a) Stem segment with axillary buds;

[0025] (b) Tender stem segments with stem tips, 6–20 mm in length.

[0026] Preferably, the temperatures for adventitious bud induction culture, subculture proliferation culture, and rooting culture are 24–26°C.

[0027] Preferably, the adventitious bud induction culture, subculture proliferation culture and rooting culture are all carried out under alternating light and dark conditions, with the light duration being 11-13 h / d and the light intensity being 1000-1500 lx.

[0028] Preferably, the adventitious bud induction culture time is 28-32 days; the subculture proliferation culture time is 28-32 days; and the rooting culture time is 28-32 days.

[0029] Preferably, the proliferation and subculture medium uses MS medium as the basic medium and further includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose and 4-10 g / L agar;

[0030] The adventitious bud induction medium uses MS medium as the basic medium and also includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose, and 4-10 g / L agar.

[0031] This invention provides the application of the *Artemisia annua* tissue culture method described above in the breeding of *Artemisia annua* germplasm while maintaining the genetic stability of L-borneol content.

[0032] The beneficial effects of the present invention are as follows: The present invention provides a growth medium for Artemisia annua, wherein the growth medium is based on MS medium and further comprises: 0.2-0.3 mg / L NAA, 0.08-0.16 mg / L LDA-6, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose and 4-10 g / L agar.

[0033] In the growth medium provided by this invention, NAA (1-naphthaleneacetic acid) promotes rooting and increases the height of *Artemisia annua* tissue culture seedlings; DA-6 (aminoethyl acetate) promotes root length and increases the height of regenerated seedlings, thus improving rooting efficiency; the combined application of DA-6 and NAA synergistically improves the rooting rate of *Artemisia annua* germplasm. The results of the examples show that the rooting rate of *Artemisia annua* tissue culture seedlings obtained using the growth medium of this invention is 26.5–30.2 seedlings / seedling, with a high rooting coefficient. The growth medium provided by this invention solves the problem of low rooting coefficient in *Artemisia annua* tissue culture seedlings and has broad application prospects. Furthermore, the *Artemisia annua* tissue culture medium and method provided by this invention can obtain a large number of high-quality *Artemisia annua* tissue culture seedlings with maintained genetic stability. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0035] Figure 1 Comparison of adventitious shoot induction efficiency of three germplasms of Artemisia annua in MS medium supplemented with different concentrations of 6-BA;

[0036] Figure 2 Comparison of adventitious shoot rooting efficiency of three germplasms of Artemisia annua in MS medium supplemented with different concentrations of NAA;

[0037] Figure 3 Comparison of adventitious shoot rooting efficiency of three germplasms of Artemisia annua in MS medium supplemented with different concentrations of DA-6;

[0038] Figure 4 Hardening-off and transplanting process of tissue culture seedlings of three germplasm progeny of Artemisia argyi;

[0039] Figure 5 This is the ion chromatogram of levorotatory borneol reference standard; Figure 5 In the diagram, 1 represents methyl salicylate (internal standard), and 2 represents levoborneol.

[0040] Figure 6 Chromatograms of total ion extracts from leaves of three mother plants and their offspring of *Artemisia argyi*. Figure 6 In the diagram, 1 represents methyl salicylate (internal standard), and 2 represents levoborneol.

[0041] Figure 7 Comparison of L-carnitine content in three germplasm mother plants of *Artemisia argyi*;

[0042] Figure 8 Comparison of L-borneol content in three tissue culture progeny of *Ayssa argentea*. Detailed Implementation

[0043] This invention provides a growth medium for Artemisia annua, wherein the growth medium uses MS medium as the basic medium and further includes: 0.2-0.3 mg / L NAA, 0.08-0.16 mg / L LDA-6, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose and 4-10 g / L agar.

[0044] In this invention, the growth medium uses MS medium as the basal medium and further includes: 0.2–0.3 mg / L NAA, 0.08–0.16 mg / L DA-6, 10–50 mg / L vitamin C, 20–200 mg / L inositol, 30 g / L sucrose, and 4–10 g / L agar; preferably, MS medium is used as the basal medium and also contains only 0.2–0.3 mg / L NAA, 0.08–0.16 mg / L DA-6, 10–50 mg / L vitamin C, 20–200 mg / L inositol, 30 g / L sucrose, and 4–10 g / L agar; more preferably, MS medium is used as the basal medium and also contains only: 0.3 mg / L NAA, 0.16 mg / L DA-6, 10 mg / L vitamin C, 20 mg / L inositol, 30 g / L sucrose, and 6 g / L agar. In this invention, the concentration of DA-6 in the growth medium is 0.08–0.16 mg / L, preferably 0.10–0.16 mg / L, and more preferably 0.16 mg / L. The concentration of NAA in the growth medium is 0.2–0.3 mg / L, preferably 0.23–0.3 mg / L, and more preferably 0.3 mg / L. The concentration of vitamin C in the growth medium is 10–50 mg / L, preferably 10–30 mg / L, and more preferably 10 mg / L. The concentration of inositol in the growth medium is 20–200 mg / L, preferably 20–120 mg / L, more preferably 20–60 mg / L, and more preferably 20 mg / L. In this invention, the concentration of agar in the growth medium is 4–10 g / L, more preferably 5–8 g / L, and more preferably 6.0 g / L. In this invention, the pH value of the growth medium is preferably 6.0–6.3, and more preferably 6.2.

[0045] The growth medium provided by this invention contains NAA, which promotes rooting and increases the height of *Artemisia annua* tissue culture seedlings, while DA-6 promotes root length and increases the height of regenerated seedlings, thus improving the rooting efficiency of regenerated seedlings. The combined application of DA-6 and NAA in the growth medium of this invention improves the rooting rate of *Artemisia annua* germplasm. Furthermore, under the combined action of DA-6 and NAA, vitamin C directly participates in enzyme formation and protein and fat metabolism, addressing the problem of insufficient vitamin C synthesis by plant cells during culture, preventing browning. Inositol helps in the formation of pectin and cell walls, promoting bud formation and promoting tissue reproduction and differentiation. In addition, sucrose is an essential carbon source in the culture medium and also helps maintain the osmotic pressure, all contributing to the promotion of bud proliferation in *Artemisia annua*. This invention is the first to introduce the plant growth regulator DA-6 into the growth medium of *Artemisia annua* tissue culture, solving the problem of low rooting coefficient in existing technologies through the combined action of all components of the growth medium. The growth medium provided by this invention has good application effects and can simultaneously obtain a large number of tissue culture seedlings that can maintain genetic stability. It provides high-quality candidate germplasm and technical reference with promotion prospects for breeding superior seedlings of Artemisia annua through asexual reproduction.

[0046] This invention does not have any special limitations on the source of NAA, DA-6, inositol, vitamin C, sucrose, agar, and MS medium; conventional commercially available products can be used.

[0047] The growth medium described in this invention can be used to cultivate adventitious buds and original plant buds (original plant buds include terminal buds and lateral buds). Both adventitious buds and original plant buds can be cut off and grown and rooted in the growth medium.

[0048] In this invention, the source of the *Artemisia argyi* is not specifically limited; conventional *Artemisia argyi* can be used. In the embodiments of this invention, the *Artemisia argyi* is preferably from three germplasms with low, medium, and high L-borneol content. The low L-borneol content in this invention is 2.98–3.62 mg / g, the medium L-borneol content is 4.8–5.42 mg / g, and the high L-borneol content is 8.67–9.57 mg / g.

[0049] This invention provides the application of the *Artemisia annua* growth medium described in the above-mentioned technical solution in improving the growth of *Artemisia annua* tissue culture seedlings in one or more of the following 1) to 3).

[0050] 1) Rooting rate;

[0051] 2) Plant height;

[0052] 3) Root length.

[0053] This invention provides an agaricus tissue culture medium, comprising an adventitious shoot induction medium, a proliferation and subculture medium, and the growth medium described in the above technical solution;

[0054] The adventitious bud induction medium uses MS as the basic medium and also includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose and 4-10 g / L agar;

[0055] The proliferation and subculture medium uses MS medium as the basic medium and also includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose, and 4-10 g / L agar.

[0056] In this invention, the adventitious bud induction medium preferably uses MS as the basal medium and further includes: 0.1–0.5 mg / L NAA, 1.0–3.0 mg / L 6-BA, 10–50 mg / L vitamin C, 20–200 mg / L inositol, 30 g / L sucrose, and 4–10 g / L agar; more preferably, it uses MS as the basal medium and further contains only: 0.1–0.5 mg / L NAA, 1.0–3.0 mg / L 6-BA, 10–50 mg / L vitamin C, 20–200 mg / L inositol, 30 g / L sucrose, and 4–10 g / L agar. In this invention, the concentration of 6-BA in the adventitious bud induction medium is 1.0–3.0 mg / L, preferably 1.3–2.5 mg / L, and more preferably 2.0 mg / L. The concentration of NAA in the adventitious bud induction medium of the present invention is 0.1–0.5 mg / L, preferably 0.3 mg / L. The concentration of vitamin C in the adventitious bud induction medium of the present invention is 10–50 mg / L, preferably 10 mg / L. The concentration of inositol in the adventitious bud induction medium of the present invention is 20–200 mg / L, preferably 20 mg / L. In the present invention, the concentration of agar in the adventitious bud induction medium is 4–10 g / L, preferably 6.0–6.5 g / L, more preferably 6.0 g / L. In the present invention, the pH value of the adventitious bud induction medium is preferably 6.0–6.3, more preferably 6.2.

[0057] In this invention, the proliferation and subculture medium uses MS medium as the basic medium and further includes: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose, and 4-10 g / L agar; more preferably, MS medium is used as the basic medium and further contains only: 0.1-0.5 mg / L NAA, 1.0-3.0 mg / L 6-BA, 10-50 mg / L vitamin C, 20-200 mg / L inositol, 30 g / L sucrose, and 4-10 g / L agar.

[0058] In this invention, the concentration of 6-BA in the proliferation and subculture medium is 1.0–3.0 mg / L, preferably 1.3–2.0 mg / L, and more preferably 1.5 mg / L. The concentration of NAA in the proliferation and subculture medium is 0.1–0.5 mg / L, preferably 0.3 mg / L. The concentration of vitamin C in the proliferation and subculture medium is 10–50 mg / L, preferably 10 mg / L. The concentration of inositol in the proliferation and subculture medium is 20–200 mg / L, preferably 20 mg / L.

[0059] In this invention, the concentration of agar in the proliferation and subculture medium is 4-10 g / L, preferably 6.0-6.5 g / L, and more preferably 6.0 g / L.

[0060] In this invention, the pH value of the proliferation and subculture medium is preferably 6.0 to 6.3, and more preferably 6.2.

[0061] The proliferation and subculture medium provided by this invention contains 6-BA and NAA, which work together to promote the proliferation of *Artemisia annua* buds. Furthermore, under the combined action of 6-BA and NAA, vitamin C directly participates in enzyme formation and protein and fat metabolism, addressing the problem of insufficient vitamin C synthesis in plant cells during culture and preventing browning. Inositol helps in the formation of pectin and cell walls, promoting bud formation and promoting tissue reproduction and differentiation. In addition, sucrose is an essential carbon source in the medium and also helps maintain the osmotic pressure, all contributing to the promotion of *Artemisia annua* bud proliferation.

[0062] This invention provides a method for tissue culture of Artemisia annua, wherein the tissue culture method uses the growth culture medium described in the above-mentioned technical solution and includes the following steps:

[0063] Artemisia annua explants were inoculated onto adventitious bud induction medium for adventitious bud induction culture to obtain sterile adventitious buds of Artemisia annua.

[0064] The stem segments with axillary buds of the sterile adventitious buds of Artemisia annua were inoculated into the proliferation and subculture medium for subculture to obtain single buds of Artemisia annua.

[0065] The single shoots of Artemisia annua were inoculated into a growth medium for rooting culture to obtain tissue culture seedlings of Artemisia annua.

[0066] The tissue culture seedlings of *Artemisia annua* were hardened off and transplanted.

[0067] This invention involves inoculating *Artemisia argyi* explants onto an adventitious bud induction medium for adventitious bud induction culture to obtain sterile adventitious buds of *Artemisia argyi*. The explants used in this invention preferably include at least one of the following:

[0068] (a) Stem segment with axillary buds;

[0069] (b) Tender stem segments with stem tips, 0.6 to 2 cm in length.

[0070] The preferred length of the stem segment with axillary buds from *Artemisia annua* in this invention is 1–3 cm, more preferably 1.5 cm. The stem segment with axillary buds described in this invention is preferably obtained by removing lateral leaves from a branch with axillary buds. The reason for choosing stem segments with axillary buds is that these segments readily produce new buds.

[0071] Before cutting *Artemisia argyi* branches with buds into explant stem segments with axillary buds, the present invention preferably involves cleaning and disinfection. The cleaning process preferably includes detergent washing and rinsing with running water. The detergent washing time is preferably 15–25 minutes, more preferably 20 minutes; the running water rinsing time is preferably 15–25 minutes, more preferably 20 minutes. The present invention does not have any particular limitation on the detergent used; conventional products are acceptable. The present invention also does not have any particular limitation on the water used for rinsing; conventional tap water is sufficient.

[0072] The preferred disinfection method of this invention includes: after cleaning, soaking in a 1% sodium hypochlorite solution for 14-16 minutes, and rinsing with sterile water 4-6 times. When soaking in the 0.1% sodium hypochlorite solution, 2-3 drops of Tween 20 are added to enhance the sterilization effect of sodium hypochlorite as a surfactant. The preferred soaking time in the 1% sodium hypochlorite solution is 14-16 minutes, more preferably 15 minutes. After soaking in the 1% sodium hypochlorite solution, the preferred rinsing time is 4-6 times, more preferably 5 times, with sterile water.

[0073] The present invention describes the preparation of explants, namely, stem segments with axillary buds, from young shoots of *Artemisia argyi* with buds after cleaning and disinfection. Preferably, the disinfected stem segments with axillary buds are inoculated onto an adventitious bud induction medium to induce adventitious buds, thereby obtaining sterile adventitious buds of *Artemisia argyi*.

[0074] The present invention uses tender stem segments with buds from *Artemisia argyi* (with shoots) as explants, prepared after cleaning and disinfection, with a length of 0.6–2 cm and including the stem tip. The preferred length of the tender stem segments with stem tips is 0.6–2 cm, more preferably 1.0–1.5 cm, and even more preferably 1.5 cm. While some existing technologies use stem tips as explants and can achieve detoxification, the tissue used for stem tip culture is very small, generally less than 3 mm, resulting in low tissue survival rates and requiring high technical skills from the experimenter, making it difficult to promote. The present invention uses the stem tip and approximately 10–15 mm of tender stem tissue below it as explants. This area has not yet formed well-developed vascular bundles, thus achieving detoxification. Furthermore, the tissue block is larger, requiring less technical skill from the experimenter, and has a higher survival rate, offering certain advantages. Using tender stem segments with stem tips as explants can effectively increase the number of buds per explant, reduce contamination and mortality rates, and improve survival rates. The method for cleaning and disinfecting tender branches with buds of Artemisia argyi described in this invention is as described above and will not be repeated here.

[0075] After obtaining sterilized tender stem segments with stem tips, with a length of 0.6-2 cm, the present invention uses stem segments with axillary buds or tender stem segments with stem tips, with a length of 0.6-2 cm, as explants of Artemisia annua and inoculates them on adventitious bud induction medium to induce adventitious buds, thereby obtaining sterile adventitious buds of Artemisia annua.

[0076] In this invention, the temperature for adventitious bud induction culture is preferably 24–26°C, more preferably 25°C. The adventitious bud induction culture preferably includes dark culture and light culture stages, with the daily light culture time preferably being 11–13 days, more preferably 12 hours. The light intensity for the light culture is preferably 1000–1500 lx, more preferably 1100–1400 lx, and more preferably 1300 lx. The adventitious bud induction culture time is preferably 28–32 days, more preferably 30 days. The adventitious bud induction culture of this invention allows *Artemisia argyi* stem segments with axillary buds or tender stem segments with stem tips to further grow and propagate, obtaining *Artemisia argyi* adventitious buds. The composition of the adventitious bud induction culture medium is the same as the adventitious bud induction culture medium in the *Artemisia argyi* tissue culture medium described above, and will not be described again here.

[0077] After obtaining aseptic adventitious buds of *Artemisia argyi*, the present invention preferably involves cutting the regenerated bud tissue culture seedlings obtained by further growth of the aseptic adventitious buds into stem segments with axillary buds and inoculating them into a proliferation and subculture medium for subculture to obtain single buds of *Artemisia argyi*. The length of the stem segments with axillary buds inoculated into the proliferation and subculture medium is preferably 1–2 cm, more preferably 1.5 cm. Each stem segment with axillary buds inoculated into the proliferation and subculture medium preferably contains one bud. The adventitious buds mentioned in the present invention are relative to fixed buds. Fixed buds are buds that are originally present in the plant, including terminal buds and lateral buds (i.e., axillary buds); adventitious buds are buds that were not originally present in the plant but were produced through dedifferentiation.

[0078] In this invention, the temperature for the subculture proliferation culture is preferably 24–26°C, more preferably 25°C. The subculture proliferation culture of this invention preferably involves both dark and light cultivation, with the daily light cultivation time preferably being 11–13 hours, more preferably 12 hours. The light intensity for the light cultivation of this invention is preferably 1000–1500 lx, more preferably 1100–1400 lx, and more preferably 1300 lx. The duration of the subculture proliferation culture of this invention is preferably 28–32 days, more preferably 30 days. The subculture proliferation culture of this invention includes one subculture. The subculture culture of this invention involves changing the culture medium for the subculture proliferation culture. The duration of the subculture culture of this invention is from day 14 to 16 of the proliferation and subculture culture, more preferably day 15. The purpose of the subculture proliferation culture of this invention is to promote further proliferation of the stem segments with buds to obtain more regenerated buds and branches, and to cut the regenerated buds and branches to obtain single buds of *Artemisia argyi*. The length of the single bud of this invention is preferably 2 cm. The composition of the proliferation and subculture medium described in this invention is the same as that of the proliferation and subculture medium in the *Artemisia annua* tissue culture medium described above, and will not be described again here.

[0079] To obtain single shoots of *Artemisia annua*, this invention involves inoculating these shoots into a growth medium for rooting culture to obtain *Artemisia annua* tissue culture seedlings. Preferably, the inoculation is performed by planting the single shoot vertically with the base facing down. One shoot is preferably inoculated into each rooting culture bottle. The rooting culture temperature is preferably 24–26°C, more preferably 25°C. The rooting culture is preferably performed using alternating dark and light cultures, with the daily light exposure time preferably 11–13 hours, more preferably 12 hours. The light intensity for the rooting culture is preferably 1000–1500 lx, further preferably 1100–1300 lx, and more preferably 1200 lx. The rooting culture time is preferably 28–32 days, more preferably 30 days. The composition of the growth medium is the same as described above and will not be repeated here.

[0080] The tissue culture seedlings obtained after rooting culture according to this invention meet the following requirements: the seedlings reach a height of 4cm, have 2-4 true leaves, and possess a healthy and well-developed root system. These seedlings are then subjected to hardening-off and transplanting culture to improve their survival rate. This invention involves hardening-off and transplanting the tissue culture seedlings of *Artemisia annua*.

[0081] The hardening-off process described in this invention is preferably completed in an artificial climate chamber, and the temperature for hardening-off is preferably suitable for the growth of *Artemisia annua*. The daytime temperature for hardening-off is preferably ≥20℃, more preferably 23℃; the nighttime temperature for hardening-off is preferably 18~20℃, more preferably 18℃.

[0082] The seedling hardening time described in this invention is preferably 3 to 15 days, more preferably 7 days.

[0083] In this invention, the transplanting culture is preferably carried out in an artificial climate chamber. During transplanting, the tissue culture seedlings, after being hardened off, are washed of their roots and transplanted into plug trays. When the tissue culture seedlings continue to grow and are repotted, they are transplanted into larger pots. The substrate in the plug trays is preferably small-particle peat moss, and the volume ratio of peat moss to sand in the larger pots is preferably 1:1. Before transplanting, the substrate is preferably sterilized. This invention does not have specific limitations on the sterilization method; conventional methods are acceptable.

[0084] This invention does not have any special limitations on the source and specifications of the planting pots used in the transplanting and cultivation; conventional products are sufficient. Preferably, each planting pot contains one hardened-off tissue culture seedling of *Artemisia annua*.

[0085] In the prior art, the germplasm of Artemisia argyi is genotype heterozygous, and sexual reproduction has the characteristic of genetic indeterminacy. The present invention uses Artemisia argyi stem segments with axillary buds as explants for tissue culture and rapid propagation, which solves the technical problem of germplasm degradation when using conventional seedlings of Artemisia argyi in the prior art. The tissue culture seedlings of Artemisia argyi obtained by the present invention have a high content of L-carnitine and can achieve high-quality seedlings with stable inheritance of Artemisia argyi.

[0086] This invention provides the application of the *Artemisia annua* tissue culture method described above in the breeding of *Artemisia annua* germplasm while maintaining the genetic stability of L-borneol content.

[0087] The embodiments of the present invention demonstrate that the mother plant has a high germination content of 9.12±0.45% L-carnitine. a The concentration of [mg / g] was significantly higher than that of [medium germplasm] (5.11 ± 0.31 mg / g). b mg / g) and low germplasm (3.30±0.0.32 mg / g) c The content of L-carnitine in the tissue culture seedlings was 7.28 ± 0.34 mg / g. a The concentration of [mg / g] was significantly higher than that of [medium germplasm] (5.71 ± 0.38 mg / g). bmg / g) and low germplasm (4.41±0.40 mg / g) c As can be seen, the leaf content of the progeny of *Ageratum argyi* tissue culture seedlings obtained by this invention is significantly different from that of the leaves of the three mother plants of *Ageratum argyi*, showing a consistent trend. Therefore, it can be concluded that the progeny of *Ageratum argyi* tissue culture seedlings obtained by the technology of this invention still exhibits the characteristic of high L-carnitine content and good growth; tissue culture can solve the problem of germplasm degradation in conventional culture of *Ageratum argyi*.

[0088] The applicant previously collected 235 *Artemisia annua* germplasm accessions from multiple provinces and cities in my country. After several years of agronomical and chemical trait analysis and comparison, it was found that there were significant differences in the content of L-borneol, and that high-content accessions generally exhibited poor growth. This invention selects mother plants with different L-borneol contents for research. Those with L-borneol contents of 2.98–3.62 mg / g are designated as low-content accessions (L), those with L-borneol contents of 4.8–5.42 mg / g as medium-content accessions (M), and those with L-borneol contents of 8.67–9.57 mg / g as high-content accessions (H). Specifically, the accessions are first preserved in vitro, then propagated, and the differences in tissue culture characteristics are compared. After hardening and transplanting, the chemical characteristics of the mother plants and corresponding tissue culture seedlings are detected using GC-MS technology. Using simultaneously cultured and transplanted low-content accessions as a control, the study investigates whether the selected accessions retain the high-content chemical characteristics of the mother plants. After verifying that its high content characteristics have been preserved, it is expected to be used for demonstration, expansion, cultivation and promotion of germplasm and supporting tissue culture technology, so as to promote the development of the industry.

[0089] This invention conducts tissue culture research on germplasm with different L-borneol contents, improving culture efficiency while using chemical detection techniques to confirm that the tissue-cultured progeny retain the chemical characteristics of the mother plant. This technique confirms that the chemical characteristics of the selected mother plant are not accidental manifestations caused by environmental or biotic stress, but are determined by genetic factors and possess a certain degree of stability, allowing for replication. This invention establishes an efficient seedling cloning technology system, enabling the acquisition of a large number of high-quality *Artemisia argyi* tissue-cultured seedlings with maintained genetic stability. It provides promising high-quality candidate germplasm and technical reference for the selection and breeding of superior *Artemisia argyi* seedlings through asexual reproduction.

[0090] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0091] The experimental materials and reagents used in the examples are as follows:

[0092] 1 Reagent

[0093] MS medium; 6-BA; NAA; aminoethyl ester (DA-6); inositol; vitamin C; L-Borneol standard; ethyl acetate and methyl salicylate are all acceptable as conventional chromatographically pure products, with no special restrictions on their source.

[0094] 2. Experimental Materials

[0095] Three two-year-old mother plants of Blumea balsamifera were preserved in the open air in the Blumea balsamifera germplasm resource nursery of the Institute of Tropical Crops Germplasm Resources, Chinese Academy of Tropical Agricultural Sciences, Danzhou City, Hainan Province. They were identified by researcher Yu Fulai as Blumea balsamifera (L.) DC. of the genus Blumea in the family Asteraceae, and were managed with unified water and fertilizer.

[0096] Based on previous research (Xiao Yongfeng, Huang Mei, Yu Fulai, et al. Analysis of phenotypic genetic diversity of Artemisia argyi germplasm resources [J]. Fujian Journal of Agricultural Sciences, 2021, 36(02):157-167.), it is known that there are significant differences among Artemisia argyi germplasms. In this invention, Artemisia argyi with a L-borneol content of 2.98–3.62 mg / g is designated as low germplasm (L), L-borneol content of 4.8–5.42 mg / g is designated as medium germplasm (M), and L-borneol content of 8.67–9.57 mg / g is designated as high germplasm (H). Healthy branches with buds from the three germplasms of Artemisia argyi were used for tissue culture in Examples 1–3 and Comparative Examples 1–2.

[0097] Example 1

[0098] (1) Disinfection of explants

[0099] Three robust stem cuttings with buds from *Artemisia argyi* germplasm were selected. The cuttings were first washed with clean water, then soaked in a 2g / L detergent solution for 20 minutes, followed by rinsing under running water for 20 minutes. Under a clean bench, 2-3 drops of Tween 20 were added to a 1% sodium hypochlorite solution, and the mixture was soaked for 15 minutes. The cuttings were then rinsed five times with sterile water. Lateral leaves were removed using a sterile scalpel and forceps, and the stems were divided into 3-4cm segments with axillary buds, which were used as explants for inducing *Artemisia argyi* tissue culture seedlings.

[0100] (2) Primary induction-adventitious shoot induction culture

[0101] The explants of the three germplasms of *Artemisia argyi* were cut into 1.5 cm stem segments with axillary buds and inoculated into culture media to initiate induction. MS medium was used as the basal medium, supplemented with 2.0 mg / L 6-BA, 0.3 mg / L NAA, 20 mg / L inositol, 10 mg / L vitamin C, 30 g / L sucrose, and 6 g / L agar, at pH 6.2. The culture was carried out for 30 days to obtain aseptically regenerated bud tissue culture seedlings. Culture conditions: temperature 25℃, 12 h photoperiod, light intensity 1000–1500 lx (the culture conditions for the following tissue culture experiments were consistent).

[0102] (3) Subculture

[0103] Regenerated shoot tissue culture seedlings from three germplasms obtained through adventitious bud induction culture were cut into 1.5cm stem segments with axillary buds, each segment containing one axillary bud. These segments were then vertically planted into proliferation and subculture medium according to the bud's growth direction for subculture. The regenerated shoot tissue culture seedlings were required to be robust and uniformly growing. Subculture yielded three germplasm (germstone L, germplasm M, and germplasm H) subcultured tissue culture seedlings.

[0104] The proliferation and subculture medium used MS medium as the basal medium, containing only NAA 0.3 mg / L, inositol 20 mg / L, vitamin C 10 mg / L, 6-BA 1.5 mg / L, sucrose 30 g / L, and agar 6 g / L, with a pH of 6.2. Subculture was performed on day 15, with the proliferation and subculture medium being replaced once. After 30 days of co-culture, the number and length of shoots per unit explant were counted.

[0105] Example 2

[0106] Same as Example 1, except that in the subculture, the 6-BA concentration in the proliferation and subculture medium was 1.0 mg / L.

[0107] Example 3

[0108] Same as Example 1, except that in the subculture, the 6-BA concentration in the proliferation and subculture medium was 2.0 mg / L.

[0109] Comparative Example 1

[0110] Same as Example 1, except that 6-BA was not added to the proliferation and subculture medium in the subculture.

[0111] Comparative Example 2

[0112] Same as Example 1, except that in the subculture, the 6-BA concentration in the proliferation and subculture medium was 3.0 mg / L.

[0113] The number of explant shoots per unit was statistically analyzed for Examples 1-3 and Comparative Examples 1-2. The formula was: Number of explant shoots per unit = Total number of shoots / Total number of explants. The results are shown in Table 1 and 2. Figure 1 .

[0114] Table 1 Comparison of adventitious shoot induction efficiency of three germplasms of *Artemisia argyi* in MS medium with different concentrations of 6-BA.

[0115]

[0116]

[0117] Note: Duncan's method was used to perform multiple comparisons between different groups. The significance level was p < 0.05. Different letters at the end of the same column indicate significant differences between different groups of the same type (the same applies to Tables 2 to 6 below).

[0118] According to Table 1 and Figure 1 It was found that the addition of different concentrations of 6-BA had a significant effect on the shoot proliferation of the three explants of *Artemisia argyi*. Firstly, regarding the number of shoots per unit explant, when the 6-BA concentration was 1.5 mg / L, the number of shoots per unit explant in the three explants was 4.54 ± 0.67. a M: 4.41 ± 0.71 a H: 4.00±0.65 a The number of explants per unit of explant (L) was significantly higher than the other four groups under optimal culture conditions. Among the three germplasms, L was the highest, M was in the middle, and H was the lowest, but the differences between groups were not significant. When the 6-BA concentration was 0 mg / L, the number of shoots per unit of explant (L: 0.94 ± 0.12) was [missing value]. e M: 0.97±0.08 d H: 0.95±0.10 e At the lowest concentration, although existing buds can continue to grow without the addition of 6-BA, no new buds will proliferate, and there is no promoting effect on the proliferation of Artemisia argyi buds. Therefore, the appropriate addition of cytokinin 6-BA is necessary for the proliferation of Artemisia argyi buds. When the concentration of 6-BA is 1.0 to 3.0 mg / L, it has a certain promoting effect on the proliferation of Artemisia argyi buds. As the concentration of 6-BA increases, the number of buds per unit explant shows a trend of first increasing and then decreasing, reaching a peak at a concentration of 1.5 mg / L, and significantly decreasing at 3.0 mg / L. This indicates that when the concentration of 6-BA is too high, it will inhibit the proliferation of Artemisia argyi buds.

[0119] Secondly, regarding shoot length, when the 6-BA concentration was 1.5 mg / L, the shoot length of the three germplasms was (L: 2.02 ± 0.22). a cm, M: 2.07±0.12 a cm, H: 1.88±0.12 a The bud length of the two groups was significantly higher than that of the other three groups at 6-BA concentrations of 0 and 1.0 mg / L. Secondly, there was no significant difference in bud length between the two groups at 6-BA concentrations of 2.0 mg / L or even higher. When the 6-BA concentration was 2.0 mg / L or even higher, the bud length of both groups gradually decreased with increasing 6-BA concentration, reaching its lowest point at a concentration of 3 mg / L (L: 0.49 ± 0.10). d cm, M: 0.41±0.13 d cm, H: 0.39±0.10 eWhen the bud length is less than 1 cm, bud growth is inhibited by high concentrations of hormones. Such buds are not suitable for subculture and rooting. When they are transferred to a new culture medium, the buds do not grow taller or root as the culture time progresses, and they gradually die. Therefore, it is essential to select a hormone concentration suitable for the proliferation of *Artemisia annua* tissue culture seedlings.

[0120] In summary, when the concentration of 6-BA was 1.5 mg / L, the number of explant buds and the average bud length of the three germplasm tissue culture seedlings of *Artemisia argyi* reached their highest levels, and the plants appeared bright green and robust at this concentration.

[0121] In Example 1, three germplasm types (germstone L, germplasm M, and germplasm H) were subcultured and propagated to obtain tissue culture seedlings. Eleven healthy and uniform seedlings from each germplasm were selected, totaling 33 seedlings, which were used to prepare explants (buds) for the next stage of rooting induction experiments. The subcultured and propagated seedlings from the three germplasms were cut into 2cm long single buds, randomly assigned, and used in the experiments described in Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-2.

[0122] Example 4-1

[0123] Single buds were inoculated into growth medium for rooting culture. The growth medium was MS medium as the basal medium, containing only NAA 0.3 mg / L, inositol 20 mg / L, vitamin C 10 mg / L, sucrose 30 g / L, agar 6 g / L, and pH 6.2. Twenty bottles were inoculated for each germplasm, with one single bud inoculated per bottle. Rooting rate, root length, and plant height were recorded after 30 days of culture. Culture conditions: temperature 25℃, 12h photoperiod, light intensity 1000–1500 lx.

[0124] Example 4-2

[0125] Same as Example 4-1, except that the NAA concentration in the growth medium is 0.2 mg / L.

[0126] Example 4-3

[0127] Same as Example 4-1, except that the NAA concentration in the growth medium is 0.4 mg / L.

[0128] Comparative Example 4-1

[0129] Same as Example 4-1, except that the NAA concentration in the growth medium is 0 mg / L.

[0130] Comparative Example 4-2

[0131] Same as Example 4-1, except that the NAA concentration in the growth medium is 0.5 mg / L.

[0132] The results of rooting rate, root length, and plant height for Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-2 are shown in Table 2 and 3. Figure 2 .

[0133] Table 2 Comparison of growth effects of three *Artemisia annua* germplasms in MS medium supplemented with different concentrations of NAA.

[0134]

[0135] In plant tissue culture, the main function of auxin is to promote cell division and elongation; different auxin concentrations have different effects on plant growth and development. (Refer to Table 2 and...) Figure 2 It can be seen that different concentrations of NAA have a significant impact on the rooting of three germplasm tissue culture seedlings of *Artemisia argyi*. Firstly, regarding the rooting rate, when the NAA concentration is 0.3 mg / L, the rooting rate of the three germplasms is (L: 25.25 ± 4.39) a Seedlings / sprout, M: 28.60±1.64 a Seedlings / strips, H: 20.55±2.07 a The rooting rates of the three germplasms (M: 3.45 ± 0.94 mg / L) were significantly higher than the other four groups. Under optimal culture conditions, the rooting rates of the three germplasms were highest in group M, intermediate in group L, and lowest in group H. When the NAA concentration was in the range of 0.2–0.5 mg / L, it significantly increased the rooting rate of the three germplasms of *Artemisia argyi*. Furthermore, as the NAA concentration increased, the rooting rate showed a trend of gradually increasing and then decreasing, reaching its highest point at 0.3 mg / L and significantly decreasing at 0.5 mg / L. Therefore, selecting an appropriate concentration of NAA is beneficial for the rooting of *Artemisia argyi* tissue culture seedlings, while excessively high concentrations have a certain inhibitory effect on the rooting rate. When the NAA concentration was 0 mg / L, the rooting rate of the three germplasms (L: 3.45 ± 0.94 mg / L) was significantly higher than the other four groups. d Seedlings / sprout, M: 3.85±1.14 e Seedlings / strips, H: 4.10±1.02 e The number of roots per seedling was significantly lower than that of the other four groups. Although roots could still germinate without the addition of NAA, the number of roots was small and the rooting rate was low. This would inhibit the growth of the plants during hardening-off and transplanting stages, making it difficult for them to survive in the later stages. Therefore, the presence of auxin NAA is essential in the growth medium of *Ageratum argyi*.

[0136] In terms of root length, when the NAA concentration was 0 mg / L, the root length of the three germplasms of *Artemisia argyi* was 4.86 ± 0.23 L. a cm, M: 4.65±0.37 a cm, H: 4.06±0.23 aThe root length was significantly higher than the other four groups at 0.2 mg / L; secondly, the root length was highest at 0.3 mg / L. It was also observed that when the NAA concentration was in the range of 0.2–0.5 mg / L, the root length gradually increased and then decreased with increasing NAA concentration, reaching its lowest point at 0.5 mg / L. At this concentration, the root lengths of the three germplasms were (L: 1.25 ± 0.21) cm. d cm, M: 1.18±0.18 e cm, H: 1.01±0.14 d cm).

[0137] Regarding plant height, when the NAA concentration was 0.3 mg / L, the plant height of two germplasms was 3.22 ± 0.43 L. a cm, H: 3.08±0.17 a The concentration of the germplasm at 0 mg / L (plant height: 2.95 ± 0.25 cm) was significantly higher than that of other groups. a (cm), 0.3 mg / L (plant height: 3.02±0.17) a There were no significant differences in plant height at different concentrations (cm), but overall, a NAA concentration of 0.3 mg / L had the most significant effect on promoting plant height. It was observed that NAA concentrations within the range of 0–0.3 mg / L all significantly increased plant height, indicating that adding lower concentrations of NAA in the growth medium was more suitable for the three *Ageratum* germplasms. At a NAA concentration of 0.5 mg / L, the plant height of the three germplasms (L: 1.41 ± 0.19) was [not specified in the original text]. d cm, M: 1.38±0.20 d cm, H: 1.20±0.16 e The significant decrease in plant height (cm) indicates that higher concentrations of NAA have a significant inhibitory effect on plant height.

[0138] Considering the rooting rate, root length, and plant height, NAA concentrations in the range of 0.2–0.5 mg / L all promoted rooting in all three *Ageratum tectorum* germplasms. The highest rooting rate was observed at a concentration of 0.3 mg / L. Without NAA, the root length was relatively longer than other groups, but the rooting rate was low at this concentration. When the NAA concentration was in the range of 0–0.3 mg / L, there seemed to be little difference in plant height among the three germplasms, as all concentrations within this range increased plant height. However, at a concentration of 0.3 mg / L, the plant height of all three germplasms increased significantly. Therefore, a NAA concentration of 0.3 mg / L was selected as the most suitable concentration for the growth of the three *Ageratum tectorum* germplasms.

[0139] In Example 1, after adventitious bud induction culture and subculture proliferation culture, three germplasm (germplasm L, germplasm M, and germplasm H) were obtained through subculture proliferation culture of tissue culture seedlings. These seedlings were then cut into 2cm long single buds for use in Examples 5-1 to 5-2 and Comparative Examples 5-1 to 5-2. Examples 5-1 to 5-2 and Comparative Examples 5-1 to 5-2 were conducted in the same batch. The subculture proliferation culture seedlings were required to be of uniform growth and healthy.

[0140] Example 5-1

[0141] Single buds are planted vertically with the base of the bud facing down in a growth medium containing DA-6 for rooting culture.

[0142] The growth medium consisted of MS medium as the basal medium, and also contained only inositol 20 mg / L, vitamin C 10 mg / L, sucrose 30 g / L, agar 6 g / L, NAA 0.3 mg / L, and DA-6 0.16 mg / L. Culture conditions were: temperature 25℃, 12 h photoperiod, and light intensity 1000–1500 lx.

[0143] Example 5-2

[0144] Same as Example 5-1, except that the concentration of DA-6 in the growth medium is 0.08 mg / L.

[0145] Comparative Example 5-1

[0146] Same as Example 5-1, except that the concentration of DA-6 in the growth medium is 0 mg / L.

[0147] Comparative Example 5-2

[0148] Same as Example 5-1, except that the concentration of DA-6 in the growth medium is 0.32 mg / L.

[0149] The rooting rates of Examples 5-1 to 5-2 and Comparative Examples 5-1 to 5-2 were compared, and the results are shown in Table 3. Figure 3 .

[0150] In Examples 5-1 to 5-2 and Comparative Examples 5-1 to 5-2, the NAA concentration in the growth medium was 0.3 mg / L. The addition of different concentrations of DA-6 had a certain effect on the rooting of various types of *Artemisia argyi*. The results are shown in Table 3. Figure 3 .

[0151] Table 3 Comparison of the growth effects of Artemisia annua in MS medium with different concentrations of DA-6

[0152]

[0153] According to Table 3 and Figure 3It can be seen that adding different concentrations of DA-6 can improve the root rate, root length, and plant height. Firstly, regarding the rooting rate, when the DA-6 concentration is 0.16 mg / L, the rooting rate of the three germplasms (L: 29.8 ± 2.80) is... a Seedlings / sprout, M: 30.2±2.03 a Seedlings / sprout, H: 29.9±2.30 a The rooting rate of the three germplasms was significantly higher at 0.08 mg / L than the other three groups. Secondly, at 0.32 mg / L, the rooting rate of the three germplasms initially increased and then decreased with increasing DA-6 concentration, reaching its lowest point at 0.32 mg / L (L: 15.6 ± 3.56). d Seedlings / sprout, M: 17.5±3.15 d Seedlings / strips, H: 16.25±3.35 d The rooting rate of the three germplasms was significantly lower than that of other groups when the DA-6 concentration was 0 mg / L. When the DA-6 concentration was 0 mg / L, the rooting rate was significantly lower than that of 0.08 mg / L and 0.16 mg / L. In other words, when a certain concentration of DA-6 and NAA was added, the rooting rate of the three germplasms was better than that of NAA alone.

[0154] In terms of root length, when the DA-6 concentration was 0.16 mg / L, the root length of the three germplasms was 2.41 ± 0.20 L. a cm, M: 2.33±0.16 a cm, H: 2.24±0.20 a The root lengths of the three germplasm groups were significantly higher than those of the other three groups (cm). When the DA-6 concentration was 0 or 0.8 mg / L, there was no significant difference in root length among the three germplasm groups, possibly because lower DA-6 concentrations resulted in less effective rooting than higher concentrations. The root lengths of the three germplasm groups gradually decreased after the DA-6 concentration increased, reaching their lowest point at 0.32 mg / L, at which point the root length (L: 1.27 ± 0.17) was [not specified]. c cm, M: 1.01±0.10 c cm, H: 1.26±0.19 c The root length of *Ageratum chinense* was significantly lower than that of other groups, indicating that the appropriate addition of a certain concentration of DA-6 can increase the root length of *Ageratum chinense*.

[0155] Regarding plant height, when the DA-6 concentration was 0.16 mg / L, the plant height of two germplasms was 3.46 ± 0.27 L. a cm, M: 3.17±0.21 a The concentration of high-quality germplasm (cm) was significantly higher than that of other groups, with the highest concentration being 0.08 mg / L (plant height: 2.91 ± 0.23 cm). a (cm), 0.16 mg / L (plant height: 3.03±0.19) aThere were no significant differences in plant height at different concentrations (cm), but overall, the DA-6 concentration of 0.16 mg / L had the most significant effect on promoting plant height. It was observed that without DA-6, the plant height of the three germplasms was significantly lower than that with DA-6, therefore DA-6 is essential in the *Ageratum argyi* growth medium. At a concentration of 0.32 mg / L, the plant height of the three germplasms (L: 1.95 ± 0.24) was significantly higher. d cm, M: 2.34±0.23 d cm, H: 2.22±0.18 c The significant decrease in height (cm) indicates that higher concentrations of DA-6 have a significant inhibitory effect on plant height.

[0156] In summary, when the concentration of DA-6 is in the range of 0.08 to 0.16 mg / L, it can significantly improve the rooting effect of plants. However, the growth effect is best when 0.16 mg / L of DA-6 is added. At this concentration, the rooting rate, root length and plant height of plants are significantly increased.

[0157] Example 1: Regenerated shoot tissue culture seedlings obtained after explant disinfection and adventitious bud induction culture were cut into 1-2 cm stem segments with axillary buds, each segment containing one axillary bud, and inoculated into proliferation and subculture medium for subculture. Examples 1-1, Comparative Examples 5-3, 5-4, and 5-5 were conducted in the same batch.

[0158] Comparative Example 5-3

[0159] The induction and proliferation media did not contain vitamin C or inositol. The proliferation and subculture media consisted of MS as the basal medium, containing only NAA 0.3 mg / L, 6-BA 1.5 mg / L, sucrose 30 g / L, and agar 6 g / L. Other conditions for subculture proliferation were the same as in Example 1.

[0160] Comparative Example 5-4

[0161] Inositol was not present in the induction and proliferation media. The proliferation and subculture media consisted of MS as the basal medium, containing only NAA 0.3 mg / L, vitamin C 10 mg / L, 6-BA 1.5 mg / L, sucrose 30 g / L, and agar 6 g / L. Other conditions for subculture proliferation were the same as in Example 1.

[0162] Comparative Example 5-5

[0163] The induction and proliferation media were vitamin-free. The proliferation and subculture media consisted of MS as the basal medium, containing only NAA 0.3 mg / L, inositol 20 mg / L, 6-BA 1.5 mg / L, sucrose 30 g / L, and agar 6 g / L. Other conditions for subculture proliferation were the same as in Example 1.

[0164] Example 1-1

[0165] The proliferation and subculture culture medium and subculture conditions in Example 1-1 are exactly the same as in Example 1.

[0166] The results of the adventitious bud proliferation rate of single buds from Examples 1-1 and Comparative Examples 5-3 to 5-5 in growth media with different concentrations of inositol and vitamin C are shown in Table 4.

[0167] Table 4 Comparison of adventitious shoot proliferation efficiency in MS medium supplemented with inositol and vitamin C

[0168]

[0169]

[0170] Table 4 shows that adding appropriate concentrations of inositol and vitamin C can improve the proliferation efficiency of adventitious buds in plants. Firstly, considering the number of buds per explant, the treatment group with simultaneous addition of inositol and vitamin C in all three germplasms (L: 3.95±0.94) showed the highest efficiency. a M: 3.68 ± 0.83 a H: 3.80 ± 0.77 a The number of adventitious shoots in the groups with added inositol and vitamin C was significantly higher than that without the addition of either. Secondly, the number of explant shoots in the groups with added inositol and vitamin C alone was significantly higher than that without the addition of either. When inositol and vitamin C were not added simultaneously, the number of shoots in all three germplasms was significantly lower than in any other group. In summary, adding appropriate amounts of inositol and vitamin C can promote the proliferation of adventitious shoots in all three germplasms, but the effect is optimal when both are added simultaneously.

[0171] Examples 5-3 and Comparative Examples 5-6 to 5-8 were conducted in the same batch of experiments. The subcultured tissue culture seedlings obtained after subculture in Example 1 were cut into 2cm long single buds, which were used in the experiments of Examples 5-3 and Comparative Examples 5-6 to 5-8. The subcultured tissue culture seedlings were required to be uniformly grown and healthy.

[0172] Comparative Examples 5-6

[0173] The growth medium consisted of MS as the basal medium, and also contained only 30 g / L sucrose, 6 g / L agar, 0.3 mg / L NAA, and 0.16 mg / L LDA-6. Other conditions for rooting culture were the same as in Example 5-1.

[0174] Comparative Examples 5-7

[0175] The growth medium consisted of MS as the basal medium, and also contained only vitamin C 10 mg / L, sucrose 30 g / L, agar 6 g / L, NAA 0.3 mg / L, and DA-6 0.16 mg / L. Other conditions for rooting culture were the same as in Example 5-1.

[0176] Comparative Examples 5-8

[0177] The growth medium consisted of MS as the basal medium, and also contained only 20 mg / L inositol, 30 g / L sucrose, 6 g / L agar, 0.3 mg / L NAA, and 0.16 mg / L LDA-6. Other conditions for rooting culture were the same as in Example 5-1.

[0178] Example 5-3

[0179] The rooting culture conditions in Example 5-3 were exactly the same as those in Example 5-1.

[0180] Table 5 Comparison of adventitious shoot growth effects in MS medium supplemented with inositol and vitamin C

[0181]

[0182]

[0183] Table 5 shows that adding appropriate concentrations of inositol and vitamin C can significantly improve the growth of adventitious buds in plants. Firstly, regarding the rooting rate, when both are added simultaneously, the rooting rate of the three germplasms (L: 30.05 ± 3.41%)... a Seedlings / sprout, M: 28.95±3.10 a Seedlings / strips, H: 31.00±2.88 a The rooting rate of the groups with the highest number of scions / seedlings was significantly higher than that of other groups. The rooting rate of the groups with added inositol and vitamin C alone was significantly higher than that of the groups without added inositol and vitamin C, indicating that adding appropriate amounts of inositol and vitamin C can promote the rooting rate of the three germplasms, but the effect is best when both are added at the same time.

[0184] In terms of root length, when both were added simultaneously, the root length of the three germplasms was (L: 1.17±0.09). a cm, M: 1.21±0.14 a cm, H: 1.31±0.11 a The rooting rate of the groups with inositol and vitamin C added individually was significantly higher than that of the groups without either addition, but the effect was best when both were added at the same time.

[0185] In terms of plant height, when both were added simultaneously, only the L germplasm plant height was 2.64±0.21 mm. aThe inositol (cm) group was significantly higher than the group without the addition of either substance; in the M and H germplasms, there was no significant difference among the four groups, indicating that the addition of inositol and vitamin C may not have a significant promoting effect on plant height, but it also has no negative effect.

[0186] In summary, the simultaneous addition of appropriate amounts of inositol and vitamin C significantly promoted the growth of the three germplasms of *Artemisia argyi*, indicating that inositol and vitamin C are necessary in the growth medium of *Artemisia argyi*.

[0187] Example 6-1

[0188] The explants were tender stem segments with stem tips, 1.5 cm in length. The disinfection and primary induction culture conditions for the explants were the same as in Example 1.

[0189] Comparative Example 6-1

[0190] The explants were stem tips with a length of 2 mm. The disinfection and primary induction culture conditions of the explants were the same as in Example 1.

[0191] Comparative Example 6-2

[0192] The explants were stem segments with axillary buds, 1.5 cm in length. The disinfection and primary induction culture conditions of the explants were the same as in Example 1.

[0193] The explant induction efficiency results of Example 6-1 and Comparative Examples 6-1 to 6-2 are shown in Table 6.

[0194] Table 6 Comparison of induction efficiency of different explants

[0195]

[0196]

[0197] Table 6 shows that, firstly, regarding the contamination rate and mortality rate, when the explants for the three germplasms were 1.5cm long tender stem segments with stem tips, the contamination rate and mortality rate were significantly lower than other groups; secondly, regarding the survival rate, the 1.5cm long tender stem segments with stem tips had a significantly higher survival rate than other groups; finally, regarding the number of buds per explant, when the explants were 1cm long tender stem segments with stem tips, the survival rate for the three germplasms (L: 5.74±2.60) was significantly higher. a M: 5.69±2.80 a H: 6.34±2.72 aThe contamination rate and mortality rate were significantly higher in the first two groups than in the other groups. Among them, the 1.5cm stem segment with axillary buds had the highest contamination rate and mortality rate. The contamination rates of stem tips and 1.5cm tender stem segments containing stem tips were similar, possibly because the vascular bundles of the former two were not yet fully differentiated, and pathogens had not yet reached the apical meristem via the well-developed vascular bundles. In contrast, the 1.5cm stem segment with axillary buds had already formed well-developed vascular bundles, allowing pathogens to remain dormant within them and grow during culture. Therefore, the contamination rate of the former two was significantly lower than that of the 1.5cm stem segment with axillary buds. In summary, selecting 1.5cm buds containing stem tips as explants achieves a balance of low contamination rate, high survival rate, and high induction efficiency, resulting in the best culture effect.

[0198] Example 7 Seedling hardening and transplanting

[0199] The culture flasks containing tissue culture seedlings from all three germplasm progeny strains of *Ageratum argyi* obtained in Example 5-1, which showed robust and healthy root growth, were placed in an artificial climate chamber for hardening-off. The artificial climate chamber conditions were: 23°C during the day, 18°C ​​at night, 12-hour photoperiod, and 29000 lx light intensity. The hardening-off and transplanting process for the tissue culture seedlings of the three germplasm progeny strains of *Ageratum argyi* is described below. Figure 4 Transplanting was carried out in an artificial climate chamber, and the substrate used for transplanting was a mixture of peat moss and sand in a volume ratio of 1:1.

[0200] After in vitro preservation, sterile seedlings were obtained from the above-mentioned germplasm. Three germplasm clones were obtained through tissue culture propagation. After hardening off and transplanting, the seedlings were planted in flower pots and cultured in artificial climate chambers with uniform light, temperature, water and fertilizer management.

[0201] Example 8: Comparison of L-borneol content in three mother plants and their offspring tissue culture seedlings of *Artemisia argyi*

[0202] Three germplasm mother plants of Artemisia: Three healthy, disease-free Artemisia germplasm mother plants of low (L), medium (M) and high (H) germplasm were collected. Nine functional leaves were picked from the same position of each plant. Nine parallel experiments were set up. All functional leaves were dried in the shade for 7 days and then stored in a -80℃ refrigerator for testing.

[0203] Three germplasm tissue culture seedlings of *Ageratum truncatum*: In Example 7, after 338 days of hardening, 9 healthy plants with uniform growth and free from pests and diseases were selected from each germplasm. One functional leaf was picked from the same position, dried in the shade for 7 days, and then stored in a -80℃ refrigerator for testing.

[0204] The method for determining the content of levorotatory borneol was based on (Pang Yuxin, Huang Mei, Yu Fulai, et al. Analysis of the differences in the content of major chemical components in Artemisia argyi from Guizhou and Hainan [J]. Journal of Guangdong Pharmaceutical University, 2014, 30(04):448-452.), with minor adjustments. The specific method for determining the content of levorotatory borneol is as follows:

[0205] (1) Preparation of the test solution

[0206] Take leaves of *Artemisia argyi*, grind them rapidly with liquid nitrogen, accurately weigh 0.2 g of powder (passing through a 20-mesh sieve), add 2.5 mL of ethyl acetate, and weigh to a final mass m1; extract ultrasonically for 30 min at a frequency of 40 kHz and a power of 420 W. Cool and weigh to a final mass m2; add ethyl acetate to make up the mass to m1, then shake well, filter, take 1 mL of the filtrate and place it in a 10 mL volumetric flask, add 1 mL of internal standard solution, dilute to volume with ethyl acetate, shake well, and filter through a 0.45 μm microporous membrane to obtain the final product.

[0207] (2) Preparation of reference solution

[0208] Accurately weigh an appropriate amount of L-borneol reference standard, dissolve it in ethyl acetate and dilute to volume to prepare a reference standard stock solution with a mass concentration of 0.998 mg / mL.

[0209] (3) Preparation of internal standard solution

[0210] Accurately weigh 100 mg of methyl salicylate, place it in a 100 mL volumetric flask, add ethyl acetate to make up to volume, shake well, and prepare an internal standard solution with a mass concentration of 1.016 mg / mL.

[0211] (4) Plotting the standard curve

[0212] Accurately measure 0.1, 0.2, 0.5, 0.7, 1, and 2 mL of the reference solution and place them in separate 10 mL volumetric flasks. Add 1 mL of internal standard solution, dilute to volume with ethyl acetate, shake well, and filter through a 0.45 μm microporous membrane. Plot a standard curve with the mass concentration of the reference solution as the abscissa (X) and the peak area ratio of the reference solution to the internal standard methyl salicylate as the ordinate (Y). The linear regression equation obtained is Y = 61.586X + 0.8878, R0. 2 =0.9992, with a linear range of 0.0101 to 0.202 mg / mL.

[0213] (5) GC-MS analysis conditions

[0214] The chromatographic column was an Agilent CYCLOSIL-B (30m × 0.250mm × 0.25μm) capillary column. Mass spectrometry conditions: EI source, solvent delay 6 min, ion source temperature 230℃, injection port temperature 220℃, scan range 45–500 amu.

[0215] L-borneol reference standard ion chromatogram ( Figure 5 Chromatograms of total ions from leaf extracts of three mother plants and their offspring tissue culture seedlings of *Artemisia argyi* (Hymenochloa chinensis). Figure 6The figure shows that the retention times of the internal standard solutions and L-borneol in the three germplasms are consistent with those of the control. Calculations were performed to compare the L-borneol content in the leaves of the three mother plants and tissue culture seedlings of *Ageratum argyi*. Figure 7 and Figure 8 The results showed that there were significant differences in the content of L-carnitine between the mother plants and tissue culture progeny among the three germplasms. The content of L-carnitine in the mother plants with higher germplasm content was 9.12±0.45%. a The mg / g value was significantly higher than that of M (5.11 ± 0.31). b mg / g) and L (3.30±0.0.32) c The content of L-carnitine in the tissue culture seedlings was 7.28 ± 0.34 mg / g. a The mg / g value was significantly higher than that of M (5.71 ± 0.38 mg / g). b mg / g) and L (4.41±0.40) c (mg / g). The tissue culture protocol described in this invention enables the preservation of high-quality L-borneol content in Artemisia annua. Figure 7 and Figure 8 The corresponding raw data is shown in Table 7.

[0216] Table 7. Raw data on the content of levorotatory borneol in leaves of three germplasm mother plants and tissue culture seedlings of *Artemisia argyi*.

[0217]

[0218] Note: Duncan's method was used to perform multiple comparisons between different groups. The significance level was p < 0.05. Different letters at the end of the same column indicate significant differences between different germplasms of the mother plant or between different germplasms of the offspring.

[0219] In summary, the most suitable culture medium for the propagation of the three *Artemisia argyi* germplasms is MS medium supplemented with NAA 0.3 mg / L, 6-BA 1.5 mg / L, Vitamin C 10 mg / L, Inositol 20 mg / L, Sucrose 30 g / L, and Agar 6 g / L. The most suitable culture medium for the rooting of the three *Artemisia argyi* germplasms is MS medium supplemented with NAA 0.3 mg / L, DA-6 0.16 mg / L, Vitamin C 10 mg / L, Inositol 20 mg / L, Sucrose 30 g / L, and Agar 6 g / L.

[0220] This invention, based on conventional in vitro preservation and tissue culture techniques for *Artemisia argyi*, refines the culture medium and obtains a population of cloned seedlings. Then, GC-MS analysis is used to detect the chemical characteristics of individual plants and cloned progeny populations from three *Artemisia argyi* germplasm mother plants. Using the simultaneously cultured low-content germplasm (L) as a control, the high-content chemical characteristics of the other two germplasms (M and H) mother plants are investigated to determine whether they are determined by genetic factors. Mother plants whose high L-borneol content might be due to external factors such as biotic stress, or whose high L-borneol content trait cannot be continued through tissue culture, are excluded. The results show that the L-borneol content of the three *Artemisia argyi* mother plants differs significantly, with values ​​of (L: 3.30 ± 0.32) c mg / g; M: 5.11±0.31 b mg / g; H: 9.12±0.45 a The content of L-borneol in the progeny of tissue culture seedlings from three germplasms after tissue culture cloning (i.e., primary culture), proliferation, induced rooting, hardening, and transplanting also showed consistent low, medium, and high significant differences, respectively (L: 4.41±0.40 mg / g). c (mg / g), (M: 5.71±0.38) b mg / g), (H: 7.28±0.34) a The concentration of borneol (mg / g) was consistent with the difference in borneol content between the mother plants, thus confirming that the three germplasm tissue culture progeny obtained through this scheme continued the difference in borneol content between the three germplasm mother plants.

[0221] This result not only verifies that the difference in L-borneol content among the three mother plants is determined by genetic factors, but also confirms that this genetic characteristic did not cause mutations or other factors during the culture process provided in this invention, thus preventing changes in the L-borneol content difference in the offspring. There is no comparability between the various mother plants and their tissue-cultured offspring. There are two reasons for this: First, their growth states differ. The former is affected by environmental differences and pests and diseases, which may promote or inhibit L-borneol synthesis, while the latter has a more uniform growth environment in the incubator and is free from pests and diseases. Second, their plant ages differ. Previous studies have shown that the L-borneol content in *Ageratum argyi* varies significantly depending on the plant age, with lower content in the seedling stage. Therefore, only by comparing the differences between different germplasms at the same growth state and plant age, as described in this embodiment, can the differences between germplasms be more accurately reflected. In conclusion, the tissue culture scheme described in this invention allows the trait of *Ageratum argyi* germplasm with high L-borneol content to be preserved.

[0222] This invention yields high-quality germplasm of *Ayssa arvense* with high borneol content, which has promising prospects for commercial application. It can obtain superior seedlings and maintain genetic advantages through asexual reproduction. Furthermore, it establishes a complete and efficient tissue culture technology that can maintain the genetic stability of the germplasm's superior quality. In the future, it is expected to promote the germplasm and its supporting tissue culture technology, which has good industrial prospects.

[0223] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for tissue culture of Artemisia annua, characterized in that, Includes the following steps: Artemisia annua explants were prepared by cleaning and disinfecting tender shoots with buds. The cleaning consisted of detergent cleaning and rinsing with running water. The detergent cleaning time was 15-25 minutes, and the running water rinsing time was 15-25 minutes. The disinfection method was as follows: after cleaning, the plant was soaked in a 1% sodium hypochlorite solution for 14-16 minutes and rinsed with sterile water 4-6 times. 2-3 drops of Tween 20 were added to the 1% sodium hypochlorite solution during the soaking process. The explants of *Artemisia argyi* are tender stem segments with stem tips, 6-20 mm in length; The *Artemisia annua* explants were inoculated onto an adventitious bud induction medium for adventitious bud induction culture to obtain sterile adventitious buds of *Artemisia annua*. The adventitious bud induction medium consisted of MS medium, 0.3 mg / L NAA, 2.0 mg / L 6-BA, 10 mg / L vitamin C, 20 mg / L inositol, 30 g / L sucrose, and 6 g / L agar. Axillary buds of aseptic adventitious buds of *Artemisia argyi* were inoculated into proliferation and subculture medium for subculture to obtain single buds of *Artemisia argyi*. The proliferation and subculture medium consisted of MS medium, 0.3 mg / L NAA, 1.5 mg / L 6-BA, 10 mg / L vitamin C, 20 mg / L inositol, 30 g / L sucrose and 6 g / L agar. The single shoots of *Artemisia argyi* were inoculated into a growth medium for rooting culture to obtain *Artemisia argyi* tissue culture seedlings; the growth medium consisted of MS medium, 0.3 mg / L NAA, 0.16 mg / L DA-6, 10 mg / L vitamin C, 20 mg / L inositol, 30 g / L sucrose and 6 g / L agar; The tissue culture seedlings of *Artemisia annua* were hardened off and transplanted.

2. The tissue culture method according to claim 1, characterized in that, The temperatures for adventitious bud induction culture, subculture proliferation culture, and rooting culture were 24~26℃, respectively.

3. The tissue culture method according to claim 1, characterized in that, The adventitious bud induction culture, subculture proliferation culture and rooting culture were all carried out under alternating light and dark conditions, with light duration of 11-13 h / d and light intensity of 1000-1500 lx.

4. The tissue culture method according to any one of claims 1 to 3, characterized in that, The adventitious bud induction culture time is 28-32 days; the subculture proliferation culture time is 28-32 days; and the rooting culture time is 28-32 days.

5. The application of the *Artemisia annua* tissue culture method according to any one of claims 1 to 4 in the breeding of *Artemisia annua* germplasm while maintaining the genetic stability of L-borneol content.

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