Artemisia annua tissue culture and one-step seedling formation method

Abstract

The present invention discloses a tissue culture and one-step seedling method of Artemisia annua, comprising the following steps: cutting Artemisia annua stem segments with axillary buds as explants, and performing sterilization and pre-inoculation treatment; inoculating the explants on an induction medium for cultivation, inducing them to generate cluster buds; transferring the germinated cluster buds to a proliferation medium for cluster bud proliferation culture; cutting the proliferated unrooted seedlings and transferring them to a rooting medium for rooting culture. The present invention uses a single high-concentration hormone 6-BA as a hormone in the induction medium, which can directly induce the axillary bud stem segment explant to generate cluster buds and significantly reduce the generation of callus tissue. At the same time, in the subculture proliferation culture, after flexibly adjusting the subculture stage and the concentration of the 6-BA used, the generation of callus tissue and the generation of vitrification can be further reduced. The method of the present invention can quickly root the cluster buds, thereby obtaining a large number of seedlings, which has the significant advantages of saving time, manpower and material costs in production.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and in particular relates to a one-step seedling formation method for Artemisia annua tissue culture technology. Background Art

[0002] Artemisia annua, also known as Artemisia Annua, is an annual herbaceous plant of the genus Artemisia in the Asteraceae family. It is a traditional Chinese herbal medicine for clearing heat and detoxifying, and holds a prominent position among medicinal plants due to its presence of artemisinin. Artemisinin, a novel active antimalarial ingredient, is widely used clinically and also plays an important role in anti-tumor, anti-arrhythmia, anti-cardiovascular disease, and anti-fibrosis effects.

[0003] However, traditional cultivation methods for Artemisia annua have problems such as long growth cycles and significant environmental impacts. Tissue culture, as a modern biotechnology, enables rapid plant propagation and genetic improvement, and is of great significance for increasing artemisinin content and yield.

[0004] During plant material culture, plant cells typically undergo a process of dedifferentiation and continuous cell division to form callus. Within the callus, clusters of meristematic cells form, which then differentiate into various organ primordia, even somatic embryos, and develop into seedlings or complete plants. Organogenesis in callus culture is divided into three distinct stages: ① Cell proliferation or dedifferentiation of explants to form callus. ② Formation of meristematic cells and tumor-like structures within the callus. ③ Under certain conditions, meristematic cells gradually transform into organ primordia exhibiting unidirectional polarity along the longitudinal axis, giving rise to buds and roots. In plant tissue culture, once callus is formed on an explant, the differentiation of buds and roots can be promoted by adjusting the ratio of plant growth regulators. Therefore, improving the callus induction rate and root differentiation from callus in Artemisia annua has become a hot topic in Artemisia annua tissue culture research.

[0005] Current tissue culture methods for Artemisia annua all use plant organs as explants, utilizing various organs and organ primordia of the plant for in vitro culture. Commonly used organ culture materials include roots (root tips, segments), stems (stem tips, axillary bud segments), leaves (leaf primordia, blades, cotyledons), flowers (petals, stamens), fruits, and seeds. Callus induction for rooting and seedling formation from explants typically involves inducing cluster buds after obtaining dense, green callus. The experimental process proceeds from explant to callus to cluster buds or adventitious buds to rooted seedlings. Adventitious buds are generally suitable for species difficult to propagate using axillary buds; cluster buds typically originate from axillary buds or stem tips and are cultured in vitro at specific locations on the plant to form multiple, densely packed buds. Adventitious buds can be further induced to develop into cluster buds, ultimately used for rooting and seedling formation.

[0006] Guo Xinrong et al. (Study on the Induction and Chemical Composition Changes of Artemisia annua Callus, Tianjin University of Traditional Chinese Medicine, Issue 4, 2014) investigated methods for inducing callus and rapidly propagating it using inflorescences, leaves, and petioles of Artemisia annua as explants. They found that a medium containing MS, 0.1 mg / L 2,4-D, and 0.5 mg / L 6-BA achieved a 100% induction rate for leaves, petioles, and inflorescences of Artemisia annua. Furthermore, they found that the hormone combination that induced clustered buds was 2.0 mg / L 6-BA and 0.15 mg / L NAA, while higher hormone concentrations were not conducive to clustered bud formation. The report found that 6-BA needed to be combined with other hormones to achieve optimal induction rates.

[0007] Yang Zhengxiu et al. (Study on Optimization of the In Vitro Regeneration System of Artemisia annua, Hubei Agricultural Sciences, Issue 6, 2017) reported that cotyledons of Artemisia annua are suitable explants for tissue culture. A medium containing MS + 1.5 mg / L 6-BA + 0.05 mg / L NAA is beneficial for inducing callus formation and differentiation, with callus and budding rates reaching 89.0% and 85.7%, respectively. A medium containing 1 / 2 MS + 0.05 mg / L NAA + 0.05 mg / L IAA is beneficial for inducing adventitious roots in regenerated plants, with a rooting induction rate of 94.0%. The paper further indicates that NAA is essential for callus formation, with concentrations of 1.0 to 2.0 mg / L 6-BA able to induce callus formation. Furthermore, a concentration of 3 mg / L 6-BA is prone to vitrification, while a concentration of 1.5 mg / L is more suitable. This suggests that high concentrations of 6-BA are inappropriate for callus induction.

[0008] Huang Hongfang et al. (Study on tissue culture of Artemisia annua, Hunan Agricultural Sciences, Issue 7, 2012) studied the effects of various hormones and their combinations on callus induction and clustered buds. Figure 2 The study showed that stem segment explant I with axillary buds, cultured on medium M9 supplemented with only 6-BA (1.5 mg / L), showed vigorous axillary bud growth, but adventitious buds were induced only from cells surrounding the axillary buds. When the 6-BA concentration was increased to a certain value, the number of adventitious buds produced by the explant decreased, and only adventitious buds grew from the axillary buds, without differentiation into adventitious buds. Given that the callus culture lasted 30 days, this suggests that prolonged incubation with high 6-BA concentrations inhibited the differentiation of adventitious buds in the explants. This suggests that high 6-BA concentrations are inappropriate for inducing clustered or adventitious buds, as they can affect rooting.

[0009] Chinese patent application CN202011644012.X, entitled “A tissue culture method and culture medium combination for Artemisia annua”, discloses the use of Artemisia annua leaves as explants, and the induction and differentiation of callus culture for 21-25 days in a culture medium of MS+2.0-2.5mg / L 6-BA+0.2-0.3mg / L NAA+6.8-7.2g / L agar+28-32g / L sucrose, followed by proliferation culture to form a large number of clustered buds, and finally the clustered buds are rooted to obtain rapidly propagated seedlings. Although the document reports that “in the process of forming callus tissue, callus differentiation can be induced to form clustered buds in the early stage of callus formation, that is, when very small callus points appear on the explant”, the process still needs to go through the process of inducing callus tissue-inducing clustered buds, and the culture time is relatively long.

[0010] As the closest existing technology, Yu Feifei et al. (Research on In Vitro Rapid Propagation Technology of Artemisia annua, Journal of Southwest Normal University, Issue 1, 2008) studied the effects of different hormone ratios on the induction of clustered shoots from axillary bud stem segments. After 14 days of culture, the combination of 6-BA and NAA outperformed the combination of KT and IAA. However, excessively high overall hormone concentrations were detrimental to growth. For example, in Medium No. 1 containing 4.0 mg / L 6-BA and 0.1 mg / L NAA, explants expanded rapidly but produced few seedlings, with some calli exhibiting vitrification. Growth generally increased with decreasing hormone concentrations. The hormone combination with the best results in inducing clustered shoots was 1.0 mg / L 6-BA and 0.5 mg / L NAA. This article indicates that stem segments with axillary buds from Artemisia annua are ideal explants for in vitro rapid propagation, rapidly producing test tube seedlings with minimal genetic stability. Hormones promote the differentiation of Artemisia annua stem segments into clumps or roots, requiring a moderate concentration. Excessively high concentrations can easily lead to plant deformities and plate-like formation, reducing the quality of the test-tube seedlings. A high ratio of cytokinin to auxin can easily lead to the production of vitreous seedlings. Furthermore, exogenous hormone levels in callus formation induction, callus differentiation induction, bud and root differentiation induction, and subculture gradually decrease during the culture process, which is related to the formation of endogenous hormones. The effect of using multiple hormones is superior to that of a single hormone, which is related to the synergistic effect of hormones.

[0011] In summary, the existing technologies for tissue culture using Artemisia annua explants (especially axillary bud stem segment explants) all require the exploration of appropriate hormone combinations and ratios, and the callus induction cycle and rooting and seedling formation cycle are long, and it is difficult to solve the problem of callus tissue vitrification, which affects the rapid reproduction of Artemisia annua and thus affects the yield of artemisinin.

[0012] Therefore, developing a tissue culture technology for Artemisia annua and studying a one-step seedling method for Artemisia annua will help reduce the tedious steps in its tissue culture and is of great significance for promoting the sustainable development of the Artemisia annua industry. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a method for tissue culture and one-step seedling formation of Artemisia annua.

[0014] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0015] A method for culturing Artemisia annua tissue comprises the following steps:

[0016] (1) Cut the stem segments of Artemisia annua with axillary buds as explants and sterilize and treat them before inoculation;

[0017] (2) Inoculating the explants onto an induction medium and inducing them to form clustered buds;

[0018] The induction medium consists of MS medium, sucrose, agar and 6-BA, wherein the concentration of 6-BA is 3.5-4.5 mg / L;

[0019] (3) Transferring the germinated buds to a proliferation medium for bud proliferation culture;

[0020] The proliferation medium has the same components and contents as the induction medium except for the concentration of 6-BA, wherein the initial concentration of 6-BA is not higher than 3 mg / L and is gradually reduced;

[0021] (4) Cut the proliferated unrooted seedlings and transfer them to the rooting medium for rooting culture.

[0022] As a further improvement, the sterilization in step (1) is performed by immersing the explant in a mercuric chloride solution and continuously shaking the solution to ensure full contact between the solution and the explant.

[0023] As a further improvement, step (2) is cultured on induction medium for 9-10 days.

[0024] As a further improvement, the concentration of 6-BA in the induction medium in step (2) is 4 mg / L.

[0025] As a further improvement, the sucrose concentration in the induction medium in step (2) is 20-40 g / L, and the agar concentration is 6-8 mg / L.

[0026] As a further improvement, in the cluster bud proliferation culture of step (3): if large-scale seedling culture is required, the 6-BA concentration is adjusted to 3 mg / L during the first subculture and cultured for 10-12 days, and then adjusted to 2 mg / L for cultured for 20-25 days and 1 mg / L for cultured seedlings for 10 days; if rapid seedling formation is required, the 6-BA concentration is adjusted to 2 mg / L during the first subculture and cultured for 10-15 days, and then adjusted to 1 mg / L for cultured seedlings for 10 days.

[0027] As a further improvement, the rooting medium in step (4) consists of 1 / 2 MS medium, sucrose, agar, and NAA, and the NAA concentration is 0.8~1.2 mg / L.

[0028] As a further improvement, the sucrose concentration in the rooting medium in step (4) is 25-35 g / L, and the agar concentration is 6-8 mg / L.

[0029] The present invention provides a one-step method for rapid propagation of Artemisia annua seedlings, comprising the following steps:

[0030] (1) Tissue culture the Artemisia annua explants according to the above method to generate tissue culture seedlings;

[0031] (2) Pretreatment of seedling hardening: Prepare the tissue culture seedlings for hardening;

[0032] (3) Hardening of tissue culture seedlings: Transplant the tissue culture seedlings into loose soil and water the soil thoroughly to harden the tissue culture seedlings;

[0033] (4) Film covering and hardening seedlings: Seal the tissue culture seedlings with a light-transmitting plastic wrap or plastic bag and place them away from direct sunlight;

[0034] (5) Hardening the seedlings outdoors: Remove the plastic wrap or plastic bag, harden the seedlings in place, and then move them to the sun for hardening;

[0035] (6) Field transplanting: After the seedlings are hardened, they are transplanted and planted in the field.

[0036] As a further improvement, the plastic wrap or plastic bag is removed after 5 days of hardening the seedlings, the seedlings are hardened in situ for 3 days, then moved to the sun for hardening for 20 days, and then transplanted into the field when they grow to 15 cm.

[0037] The present invention aims to achieve efficient regeneration and rapid propagation of Artemisia annua plants by optimizing explant selection, culture medium formulation, seedling hardening and transplanting methods, etc.

[0038] The first principle of this invention lies in the fact that, while prior art suggests that a combination of hormones is required for tissue culture of axillary bud segments of Artemisia annua, the present inventors unexpectedly discovered that using a single high-concentration hormone (i.e., 4 mg / L 6-BA) as the hormone in the induction culture medium can directly induce the formation of clustered shoots from axillary bud segment explants while significantly reducing callus formation, allowing the clustered shoots to rapidly root, thereby yielding a large number of seedlings. This explant bypasses the callus induction process and directly generates clustered shoots, significantly shortening the culture cycle.

[0039] The second principle of the present invention is that in order to overcome the increased vitrification phenomenon of cluster buds caused by high hormone concentrations, the present invention significantly shortens the initial induction period of the cluster bud induction culture medium to about 9-10 days, then reduces the concentration of 6-BA, and then performs subculture proliferation culture (for example, according to the needs of rapid cultivation of seedlings or obtaining a large number of seedlings, the subculture stage and the concentration of 6-BA used are flexibly adjusted), which can further reduce the formation of callus tissue and the occurrence of vitrification.

[0040] The third principle of the present invention is that, based on the first and second principles, in addition to the optimization measure of shortening the induction time of high-concentration 6-BA to overcome tissue vitrification, the present invention unexpectedly discovered that placing the explants on a culture medium with a specific concentration of agar and sucrose can reduce the vitrification phenomenon.

[0041] The fourth principle of the present invention is that: the present invention optimizes the explant disinfection method and explores the seedling cultivation and hardening method. Combined with the above first to third principles, a method for rapidly propagating Artemisia annua into seedlings in one step is obtained, which can reduce the cost of seedling production, improve large-scale production efficiency, and meet the needs of Artemisia annua field cultivation plants.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention improves the sterilization method of Artemisia annua explants (increasing the concentration of mercuric chloride) to effectively reduce the infection rate of explants without affecting the inoculation effect, and can obtain a large amount of sterile materials for cluster bud induction in the early stage.

[0044] 2. Existing literature (e.g., Guo Xinrong et al., Yang Zhengxiu et al., Yu Feifei et al.) reports that the combination of 6-BA and NAA is highly beneficial for inducing clustered shoots from Artemisia annua explants, leading to rapid rooting and propagation. However, some reports (e.g., Huang Hongfang et al.) suggest that high concentrations of 6-BA are inappropriate for inducing clustered or adventitious shoots, as they impair rooting. However, the present inventors unexpectedly discovered that, using a single hormone, by increasing the 6-BA concentration in the bud-inducing medium, clustered shoots can be directly and rapidly induced in the early stages, minimizing callus formation. This facilitates one-step seedling formation, improves production efficiency, and reduces the cost of large-scale seedling production.

[0045] 3. The production advantage of rapid propagation of Artemisia annua explants depends primarily on the time it takes to propagate, rather than differences in induction rate (because the induction rate can be increased by increasing the number of explants to ensure sufficient clusters of buds). In the present invention, during the inoculation process of the explants, due to issues such as incomplete sterilization due to the explant's inherent characteristics or the death of relatively tender explants after sterilization, some explants may become infected or brown, preventing proper bud induction. This portion of material accounts for approximately 15%, resulting in a bud induction rate of approximately 70-80%. For example, the cluster bud induction rate (75.56±3.85) of the preferred embodiment of the present invention is lower than that reported by Yu Feifei (6-BA 1.0mg / L + NAA 0.5mg / L, induction rate 93%), but the average induction time is 9.67±0.58 days, significantly less than the 14 days reported by the latter. This significantly reduces time and costs, meeting the primary requirement of rapid tissue culture propagation.

[0046] 4. Compared to existing literature reports that "during callus formation, callus differentiation can be induced to form cluster buds at the early stages of callus formation, i.e., when a small callus point appears on the explant," this technique differs from the present invention in the location where cluster buds are generated: this technique requires inducing the production of a small patch of callus from the explant, which then directly differentiates into cluster buds; whereas the present invention uses the stem tip as the explant to directly induce cluster buds at the bud point itself (i.e., where the axillary buds are located). Although a small amount of callus is present in the wound, subsequent culture is focused on the proliferation of the cluster buds in the axillary bud area, rather than differentiation of the callus in the wound area. Therefore, compared to existing techniques, the present invention skips the process of inducing callus and directly generates cluster buds, meeting the needs of rapid propagation.

[0047] 5. The present invention improves the sterilization method and increases the concentration of plant growth hormone in the early stage, and appropriately reduces the concentration of plant growth hormone according to the situation of cluster buds in the later stage, thereby quickly inducing cluster buds and forming large-scale production. In the later stage, a large number of seedlings can be quickly obtained by covering the seedlings with films and then hardening them in the open air.

[0048] 6. The present invention uses the basic culture medium MS (containing agar + sucrose) and can induce the formation of clustered buds by simply adjusting the concentration of 6-BA, which greatly reduces the tedious process of preparing different culture media and improves production efficiency.

[0049] 7. The present invention only requires 9.67±0.58 days to induce clustered bud formation, 25-30 days for clustered bud proliferation and seedling growth, and 15 days for rooting, for a total average of 44-49 days. Compared to the traditional 2-3 month cultivation cycle required to obtain rooted seedlings, this method offers significant production advantages and reduces labor and material costs.

[0050] 8. Furthermore, it was demonstrated that the clustered buds directly induced by the present invention achieved consistent rooting efficiency when using the currently available optimal rooting medium. This demonstrates that the present one-step method for inducing clustered buds can be directly combined with the currently available optimal proliferation and subculture treatment methods and rooting and seedling induction methods. This greatly improves the operability of the present invention and offers advantages similar to modular replacement of production processes.

[0051] 9. At present, Zhangjiajie, Changde, Yongzhou and other areas in Hunan Province belong to important Artemisia annua planting bases. Artemisia annua planting model villages and cooperatives have been established in many counties and cities. By planting improved Artemisia annua varieties, and developing tissue culture technology to raise seedlings in combination with field cultivation, large-scale planting is formed, which is of great significance for helping the western region to develop and realize farmers' wealth and increase their income. According to incomplete statistics from the research group of the present invention, this method can achieve the seedling emergence rate to be increased to 95%, Artemisia annua yield to be increased by 20%, artemisinin content to be 1.8%, breeding time to be reduced by 1-2 years, nearly 25 kilograms per mu of increased production, Artemisia annua seedling purchase price to be increased by 3.6-4.8 yuan / jin, output value per mu to be increased from about 1200 yuan to about 2200 yuan, indirectly driving employment of 1000-1200 people, driving more than 20 cooperatives in 9 cities in Hunan Province to increase their income, and ultimately achieving rural revitalization with scientific and technological innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0053] Figure 1 This is the inoculation diagram of Artemisia annua explants;

[0054] Figure 2 This is a diagram showing the effect of directly producing clustered shoots from Artemisia annua explants, in which basically no callus tissue appears;

[0055] Figure 3 This is a picture of the subculture and proliferation culture of Artemisia annua;

[0056] Figure 4 This is a picture of the rooting culture of Artemisia annua clustered shoots;

[0057] Figure 5 This is a picture of film-covered and hardened Artemisia annua tissue culture seedlings;

[0058] Figure 6 This is a picture of the open-air hardening of Artemisia annua tissue culture seedlings;

[0059] Figure 7 This is a diagram showing the growth of tissue culture seedlings after transplanting into the field;

[0060] Figure 8 This is a diagram of callus induction cultured on conventional culture medium of Artemisia annua. DETAILED DESCRIPTION

[0061] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0062] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0063] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0064] Some specific implementations of the present invention include a method for tissue culture using an Artemisia annua stem segment with axillary buds explant, comprising the following steps:

[0065] (1) Cut the stem segments of Artemisia annua with axillary buds as explants and perform sterilization and pre-inoculation treatment:

[0066] In some embodiments, explant collection includes: using high-branch shears to cut tender shoots from the upper portion of an Artemisia annua plant into approximately 5 cm segments, each segment containing 2-3 axillary buds and retaining one leaflet. Explant sterilization and pre-inoculation treatment are then performed: after disinfection and sterilization, the explant is cut into 2-3 segments based on the location of the axillary buds, ensuring that each segment contains at least one axillary bud.

[0067] In some embodiments, disinfection and sterilization include: placing the treated explants into a clean Erlenmeyer flask, adding 0.2% mercuric chloride solution to immerse the explants, and shaking continuously to ensure full contact between the solution and the explants for thorough sterilization; after disinfection for 8 minutes, rinse with sterile water 4 times, and absorb the moisture with clean filter paper.

[0068] (2) Inoculate the explants on induction medium and culture them to induce them to produce clustered buds:

[0069] In some embodiments, the induction medium is prepared as follows: MS medium is used as the basic medium, and each liter of induction medium consists of MS medium, 20-40 g of sucrose, 6-8 mg of agar, and 3.5-4.5 mg (preferably 4 mg) of 6-BA (6-benzyladenine). Each bottle is filled with 40 ml of medium for primary clustered shoot induction.

[0070] Among them, MS refers to the MS component of the culture medium commonly used in the existing plant tissue culture field.

[0071] In some embodiments, the explants are induced to form clustered shoots by inserting the lower end of the explants into the culture medium, with 4-5 explants evenly distributed throughout each flask. The culture is then cultured for 9-10 days to induce clustered shoots. The culture conditions are as follows: temperature: 25±2°C, light intensity: 1500-2000 lux, and photoperiod: 12 hours / day.

[0072] In some embodiments, the callus formation rate of the explants is about 5-10%, and even if callus is generated, it can quickly differentiate into clustered shoots during subsequent culture. The clustered shoot induction rate of the explants is about 70-80%.

[0073] (3) Transfer the germinated buds to the proliferation culture medium for bud proliferation culture:

[0074] In some embodiments, the germinated cluster shoots are transferred to a proliferation medium for subculture. The culture conditions are as follows: temperature: 25±2°C, light intensity 1200 Lx, photoperiod: 14 h light, 10 h dark.

[0075] In some embodiments, except for the reduced 6-BA concentration, the other components and contents of the proliferation medium are the same as those of the induction medium. The initial 6-BA concentration is no higher than 3 mg / L and is gradually reduced.

[0076] The number of subcultures and the concentration of the subculture medium can be determined according to the number of seedlings required. If rapid seedling formation is required, the concentration can be adjusted to 2 mg / L 6-BA for the first subculture for 10-15 days, and then adjusted to 1 mg / L 6-BA for 10 days of seedling cultivation before continuing to root.

[0077] If large-scale cultivation of seedlings is required, the first subculture should be adjusted to 3 mg / L 6-BA for 10-12 days, followed by adjustment to 2 mg / L 6-BA for 20-25 days, and 1 mg / L 6-BA for 10 days before continuing to root.

[0078] (4) Cut the proliferated unrooted seedlings and transfer them to the rooting medium for rooting culture:

[0079] In some embodiments, rooting culture is performed when the clustered buds grow to approximately 4 cm, and the lower ends of the clustered buds are cut off and inoculated into the culture medium. Culture conditions are as follows: temperature: 25±2°C, light intensity 1200 Lx, photoperiod: 14 hours of light and 10 hours of darkness.

[0080] In some embodiments, each 1 L of rooting medium consists of 1 / 2 MS medium, 25-35 g of sucrose, 6-8 mg of agar, and 0.8-1.2 mg (preferably 1 mg) of NAA (naphthaleneacetic acid).

[0081] 1 / 2MS means that the macroelements in the MS component of the commonly used culture medium are halved, while the other components remain unchanged.

[0082] The method for rapidly propagating Artemisia annua into seedlings in one step according to some specific embodiments of the present invention comprises the following steps:

[0083] (1) Tissue culture the Artemisia annua explants according to the above method to quickly generate tissue culture seedlings;

[0084] (2) Pretreatment of seedling hardening: Select tissue culture seedlings with roots of 1-1.5 cm for hardening;

[0085] In some embodiments, the tissue culture seedlings are placed outdoors to acclimate to room temperature for 2-3 days while avoiding direct sunlight;

[0086] (3) Hardening of tissue culture seedlings: Transplant the tissue culture seedlings into loose soil and water the soil thoroughly to harden the tissue culture seedlings;

[0087] In some embodiments, the culture medium at the roots is rinsed clean with clean tap water and transplanted into loose soil. Approximately 8 to 9 plants are transplanted into a 20 cm diameter pot. After transplanting, the soil is thoroughly watered to harden the tissue culture seedlings.

[0088] (4) Film covering and hardening seedlings: Seal the tissue culture seedlings with a light-transmitting plastic wrap or plastic bag and place them away from direct sunlight;

[0089] (5) Open-air hardening: After 5 days of hardening the seedlings with plastic film, remove the plastic film or plastic bag, and after 3 days of hardening the seedlings in situ, move them to the sun for hardening.

[0090] (6) Transplanting in the field: After the tissue culture seedlings have been hardened in direct sunlight for more than 20 days, they can be transplanted and planted in the field when they grow to 15 cm.

[0091] In some embodiments, the tissue culture seedlings are hardened in direct sunlight for more than 20 days and then grown to about 10 cm. At this time, attention should be paid to fertilizing. When they reach 15 cm, they can be transplanted into the field and then planted and managed as normal seedlings.

[0092] The present invention uses a single high-concentration hormone (i.e., 4 mg / L 6-BA) as the hormone in the induction culture medium, directly inducing the formation of clustered buds from axillary bud stem segment explants while significantly reducing callus formation. Furthermore, during subculture, flexible adjustment of the subculture stage and 6-BA concentration, depending on the need for rapid seedling development or large-scale seedling production, can further reduce callus formation and vitrification. This method can rapidly root clustered buds, thereby yielding large numbers of seedlings, significantly saving time, labor, and material costs in production.

[0093] Example 1 Tissue Culture Method of Artemisia annua Stem Segment Explants with Axillary Buds

[0094] (1) Select a single XH-1 Artemisia annua plant with healthy growth and good morphology, cut the tender stem at the top of the plant as an explant, trim the branches and leaves to obtain a stem segment with axillary buds, soak the stem end in a 5 wt% detergent aqueous solution for 10 minutes, then rinse with running water for 30 minutes, and dry it naturally to obtain an isolated stem end washed with running water.

[0095] (2) Place the detached stem end washed with running water in a clean bench for operation; soak and disinfect it with a 0.2wt% mercuric chloride aqueous solution for 8 minutes, shake it thoroughly during the process to ensure the sterilization effect, then rinse it with sterile water 4 times, and then use sterilized filter paper to absorb excess water under sterile conditions. Cut off about 0.1 cm wounds at both ends of the sterilized stem segment, and then cut it into 1.5 cm small segments, ensuring that there is at least one axillary bud on each stem segment. Inoculate its morphological lower end on a solid induction medium (MS+30g / L sucrose+7g / L agar+4.0mg / L 6-BA) and culture it at 25±2℃. The culture conditions are as follows: temperature: 25±2℃, light intensity 1500~2000 Lx, light duration 12 h / d, see Figure 1 The day of inoculation was designated as day 0. On day 8 of culture, clustered buds or callus appeared on the stem segments.

[0096] (3) Continue to culture after the buds appear. The day when the clustered buds appear is day 0. After 2-5 days of culture, clustered buds with a height of 0.5 to 1 cm appear. Figure 2 The incubation time with 4 mg / L 6-BA at this stage should not be too long, as vitrification will occur after 8-10 days.

[0097] (4) The cluster buds were cut into cubes with 1 to 2 cluster buds from the base and transferred to the cluster bud proliferation medium for cluster bud proliferation culture. Five cluster buds were inoculated into each culture dish. The culture conditions were as follows: temperature: 25±2℃, light intensity 1200Lx, photoperiod: 14h light, 10h dark. The day of transfer to the cluster bud proliferation medium was designated as day 0. After 10 days of culture, the proliferation of unrooted seedlings was observed. Figure 3 .

[0098] The 6-BA concentration in the cluster bud proliferation medium can be adjusted based on the actual number of seedlings required: if proliferation and expansion are required, continue culturing with 3 mg / L 6-BA, then replace it with 2 mg / L 6-BA after 10-12 days for subculture, or directly replace it with 1 mg / L 6-BA for subculture. If large-scale proliferation is not required, directly use 2 mg / L 6-BA for 15-20 days, then use 1 mg / L 6-BA for subculture for 10 days before rooting. The components of the cluster bud proliferation medium, except for hormones, are the same as those of the induction medium. The types and dosages of hormones can be found in Table 3.

[0099] (5) Cut a single seedling from the base of the proliferated unrooted seedling and transfer it to a test tube seedling bottle filled with 45 ml of adventitious rooting medium (1 / 2 MS + 7 g / L agar + 30 g / L sucrose + 1 mg / L NAA, pH 5.8-6.0) for cultivation. The culture conditions are as follows: temperature: 25 ± 2 ° C, light intensity 1200 Lx, photoperiod: light 14 h, dark 10 h. The day of transfer to the adventitious rooting medium is day 0. After 10-12 days of cultivation, rooted seedlings are obtained. Figure 4 .

[0100] Example 2 Testing the effects of different hormone ratios on the induction of clustered shoots

[0101] According to the method of Example 1 and Table 1, a cluster bud induction medium was prepared, wherein the difference between the experiments of each serial number was only the different hormone combination ratios.

[0102] Among them, according to reports by Guo Xinrong et al., Yang Zhengxiu et al., and Yu Feifei et al., the combination of 6-BA and NAA is very beneficial for inducing clustered buds in Artemisia annua explants and then rooting and rapid propagation, so this combination was selected.

[0103]

[0104] Each hormone combination was treated in three parallel groups, with 15 explants in each group. The callus formation rate (induction rate), cluster bud induction rate and bud formation time were then calculated.

[0105] The results are shown in Table 2.

[0106]

[0107] As shown in Table 2, combination No. 13 achieved the best results, demonstrating a high bud induction rate and a short bud formation time. The callus induction time was low, allowing for direct seedling development, significantly reducing the time required for early differentiation and formation of clustered buds. Clustered buds were generated directly during the culture process. Although individual explants produced callus, callus formation was followed by rapid differentiation into clustered buds, with the overall clustered bud differentiation time exceeding 12 days. Complete differentiation was achieved in the bud induction medium without the need for medium changes, resulting in high efficiency and rapid growth.

[0108] In contrast, combination No. 14, which also received high-concentration 6-BA treatment, added NAA to the bud-inducing medium. This resulted in a higher callus induction rate than combination No. 13. While the calli also differentiated into clustered buds during culture, the time required for clustered bud formation was prolonged. Furthermore, due to the prolonged callus differentiation period, vitrification increased in plants exposed to high 6-BA cytokinin concentrations for extended periods.

[0109] At the same time, low concentration 6-BA treatment, such as No. 1-3, will produce a lot of brown callus (see Figure 8 ), and the same culture medium cannot be used for successful differentiation. Other culture media need to be replaced for differentiation induction during the subsequent culture process. The overall time for forming clustered buds is long, and the operation is too complicated compared to sequence number 13.

[0110] And by comparing the combinations of serial numbers 1, 5, 9, and 13, it can be found that the higher the 6-BA concentration, the shorter the time it takes to form cluster buds, which is conducive to the direct formation of cluster buds in the early stage of tissue culture.

[0111] Example 3 Testing and Analysis of the Effects of Different Concentrations of 6-BA in Proliferation Medium on Proliferation

[0112] According to the results of Example 2, the clustered seedlings of the best induction medium (i.e., the 4 mg / L 6-BA group of medium number 13) were selected, and the effects of proliferation medium with different concentrations of 6-BA on the proliferation effect were tested and analyzed according to Table 3 below.

[0113]

[0114] According to the above table, the proliferation efficiency of using high concentration 6-BA alone is higher than that of the same concentration of 6-BA combined with NAA. Therefore, 6-BA was added alone for culture during the differentiation and proliferation process.

[0115] According to No. 1, it can be found that the germination medium uses 4 mg / L 6-BA for culture, and it is necessary to replace it with a lower concentration medium as soon as possible. Continuous use of 4 mg / L for culture is very likely to cause vitrification. After replacing with low-concentration 6-BA, such as No. 4, No. 7, and No. 10, the vitrification phenomenon is significantly alleviated, and the culture time for vitrification phenomenon continues to prolong. Therefore, after the buds are successfully induced, it is necessary to replace the low-concentration 6-BA medium as soon as possible for proliferation culture.

[0116] During the proliferation culture process without adding NAA, the proliferation coefficient decreased continuously from the higher concentration No. 4 to the lowest No. 10, but the vitrification phenomenon continued to decrease. After the buds were successfully induced, 1 mg / L 6-BA was directly used for proliferation culture and no vitrification phenomenon was found.

[0117] Therefore, considering the need for large-scale seedling cultivation, after successful bud induction, the concentration is reduced and sequence number 4 is used for continued proliferation culture. The culture time for this process is 10-12 days. Sequence number 7 is used for the second subculture and this process is about 20-25 days. After that, sequence number 10 is used for seedling strengthening and finally for rooting. If large-scale seedlings are not required, sequence number 7 can be used directly for the first subculture for about 10-15 days. Sequence number 10 is used for the second subculture and cultured for 10-15 days, and finally for rooting and hardening.

[0118] Example 4: Testing and Analysis of the Rooting Efficiency of the Present Invention

[0119] According to the method of Example 1 and Table 4, the cluster root culture medium was prepared, wherein the difference between the experiments of each serial number was only the different hormone combination ratios.

[0120]

[0121] When NAA was added alone for rooting, the experimental results in Table 4 showed that 1 mg / L NAA had the best rooting effect. As the NAA concentration increased, the rooting rate continued to decrease. The average rooting rate and number of roots at 2 mg / L NAA were significantly lower than those at 1 mg / L NAA. Therefore, 1 mg / L NAA was the best choice for rooting treatment.

[0122] As shown in Table 4, the rooting effect of using NAA alone was better than that of the combination with 6-BA at the same concentration, such as No. 1, No. 4, and No. 7. In addition, using NAA alone can also reduce the initial cost loss. Therefore, the subsequent large-scale seedling hardening used the culture medium 1 / 2 MS + 7g / L agar + 30g / L sucrose + 1mg / L NAA, which had the best rooting induction effect, with an average rooting rate of 97.78±3.85.

[0123] There is no essential difference in the rooting treatment of the buds obtained from different parts of the Artemisia annua explants, and the rooting rate mainly depends on the type and ratio of hormones. Therefore, the early proliferation method used in the present invention is highly adaptable and can be used with different rooting media for root induction in the later stage.

[0124] Example 5: Field cultivation of rapid propagation tissue culture seedlings

[0125] Hardening: When the roots of the tissue culture seedlings are about 1-1.5 cm, harden them. Open the cap of the test tube seedling bottle and acclimate in the tissue culture room for 1 day. Remove the rooted seedlings and wash away any residual culture medium attached to the roots. Place the tissue culture seedlings outdoors to acclimate to room temperature, avoiding direct sunlight. This takes about 2-3 days.

[0126] Hardening of tissue culture seedlings: Gently remove the tissue culture seedlings from the culture medium, rinse the culture medium at the roots with clean tap water, and transplant them into loose soil. Transplant about 8 to 9 plants in a 20 cm diameter flower pot. After transplanting, water the soil thoroughly to harden the tissue culture seedlings.

[0127] Filming and hardening seedlings: To ensure water demand, the newly transplanted tissue culture seedlings should be sealed with light-transmitting plastic wrap or plastic bags and placed away from direct sunlight. Figure 5 ;

[0128] Open air hardening: After 5 days of hardening, remove the plastic wrap or plastic bag, harden the seedlings in the original place for 3 days, and then move them to the sun for hardening. Figure 6 ;

[0129] Transplanting in the field: After more than 20 days of hardening in direct sunlight, the tissue culture seedlings grow to about 10 cm. At this time, pay attention to fertilization. When they grow to 15 cm, they can be transplanted into the field. After that, they can be planted and managed as normal seedlings. Figure 7 .

[0130] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form. Therefore, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical solution of the present invention and are based on the technical essence of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for tissue culture of Artemisia annua, characterized in that: The following steps are included: (1) Cut the stem segments of Artemisia annua with axillary buds as explants and sterilize and treat them before inoculation; (2) Inoculate the explants on an induction medium and culture for 9-10 days to induce them to form clustered buds; The induction medium consists of MS medium, sucrose, agar and 6-BA, wherein the concentration of sucrose is 20-40 g / L, the concentration of agar is 6-8 mg / L, and the concentration of 6-BA is 3.5-4.5 mg / L; (3) Transferring the germinated buds to a proliferation medium for bud proliferation culture; The proliferation medium has the same components and contents as the induction medium except for the concentration of 6-BA, wherein the initial concentration of 6-BA is not higher than 3 mg / L and is gradually reduced; (4) Cut the proliferated unrooted seedlings and transfer them to the rooting medium for rooting culture.

2. The method for culturing Artemisia annua tissue according to claim 1, wherein: The sterilization in step (1) is performed by immersing the explant in a mercuric chloride solution and continuously shaking the solution to ensure full contact between the solution and the explant.

3. The method for tissue culture of Artemisia annua according to claim 1, wherein: The concentration of 6-BA in the induction medium in step (2) is 4 mg / L.

4. The method for tissue culture of Artemisia annua according to claim 1, wherein: Step (3) The cluster bud proliferation culture: if large-scale seedling culture is required, the 6-BA concentration is adjusted to 3 mg / L during the first subculture and cultured for 10-12 days, and then adjusted to 2 mg / L for cultured for 20-25 days and 1 mg / L for seedling culture for 10 days; if rapid seedling formation is required, the 6-BA concentration is adjusted to 2 mg / L during the first subculture and cultured for 10-15 days, and then adjusted to 1 mg / L for seedling culture for 10 days.

5. The method for culturing Artemisia annua tissue according to claim 1, wherein: The rooting medium in step (4) consists of 1 / 2 MS medium, sucrose, agar, and NAA, and the NAA concentration is 0.8~1.2 mg / L.

6. The Artemisia annua tissue culture method according to claim 5, wherein: The sucrose concentration in the rooting medium in step (4) is 25-35 g / L, and the agar concentration is 6-8 mg / L.

7. A one-step method for rapid propagation of Artemisia annua seedlings, characterized in that: The following steps are included: (1) Tissue culture the Artemisia annua explants according to the method of any one of claims 1 to 6 to produce tissue culture seedlings; (2) Pretreatment of seedling hardening: Prepare the tissue culture seedlings for hardening; (3) Hardening of tissue culture seedlings: Transplant the tissue culture seedlings into loose soil and water the soil thoroughly to harden the tissue culture seedlings; (4) Film covering and hardening seedlings: Seal the tissue culture seedlings with a light-transmitting plastic wrap or plastic bag and place them away from direct sunlight; (5) Hardening the seedlings outdoors: Remove the plastic wrap or plastic bag, harden the seedlings in place, and then move them to the sun for hardening; (6) Field transplanting: After the seedlings are hardened, they are transplanted and planted in the field.

8. The one-step seedling-forming method for rapid propagation of Artemisia annua according to claim 7, characterized in that: After 5 days of hardening the seedlings with film, remove the plastic wrap or plastic bag, harden the seedlings in situ for 3 days, move them to the sun for hardening for 20 days, and transplant them into the field when they grow to 15 cm.

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