Artemisia apiacea tissue culture and one-step seedling forming method

Abstract

The invention discloses an artemisia apiacea tissue culture and one-step seedling forming method, which comprises the following steps: cutting stems with axillary buds of artemisia apiacea as explants, and carrying out sterilization and inoculation pretreatment; inoculating the explants on an induction culture medium for culturing, and inducing the explants to generate cluster buds; transferring the germinated cluster buds to a proliferation culture medium for proliferation culture of the cluster buds; and cutting the proliferated non-rooted seedlings, and transferring to a rooting culture medium for rooting culture. According to the method, single high-concentration excitation 6-BA is used as a hormone for inducing the culture medium, axillary bud stem explants can be directly induced to generate cluster buds, and the generation of calluses is remarkably reduced. Meanwhile, in the subculture multiplication culture, after the subculture stage and the concentration of the used 6-BA are flexibly adjusted, the generation of calluses and the generation of vitrification phenomena can be further reduced. According to the method, the cluster buds can be rapidly rooted, so that a large number of seedlings are obtained, and the method has the remarkable advantages of saving time and manpower and material resource cost in production.

Description

Technical Field

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

[0002] Artemisia annua, also known as sweet wormwood, is an annual herbaceous plant of the genus Artemisia in the Compositae family. It is a traditional Chinese medicine for clearing heat and detoxifying, and occupies an important position among medicinal plants due to the presence of artemisinin. As a new type of antimalarial active ingredient, artemisinin is widely used in clinical practice and also plays an important role in anti-tumor, anti-arrhythmia, anti-cardiovascular diseases, anti-tissue fibrosis and other aspects.

[0003] However, the traditional cultivation method of Artemisia annua has problems such as a long growth cycle and being greatly affected by the environment. Tissue culture technology, as a modern biotechnology, can achieve the rapid propagation and genetic improvement of plants, and is of great significance for increasing the artemisinin content and yield.

[0004] Generally, during the cultivation of plant materials, plant cells form callus through the process of dedifferentiation and continuous cell division. Some meristematic cell clusters are formed in the callus, and then various organ primordia, and even somatic embryos are redifferentiated, and then develop into seedlings or complete plants. Organogenesis in callus culture is divided into 3 different stages: ① Cell proliferation or dedifferentiation of explants in vitro to form callus. ② Some meristematic cells and tumor-like structures are formed in the callus. ③ Under certain conditions, the meristematic cells gradually transform into organ primordia showing unidirectional polarity on the longitudinal axis, that is, buds and roots are differentiated. In plant tissue culture, after the explant forms callus, the differentiation of buds and roots can be promoted by adjusting the proportion of plant growth regulators. Therefore, how to improve the induction rate of Artemisia annua callus and the differentiation of roots from callus has become a research hotspot in Artemisia annua tissue culture.

[0005] Currently, the methods for tissue culture of Artemisia annua are all culture methods using plant organs as explants, that is, methods for in vitro culture using various organs and organ primordia of Artemisia annua plants. Commonly used organ culture materials include roots (root tips, cut segments), stems (shoot tips, axillary bud stem segments), leaves (leaf primordia, leaves, cotyledons), flowers (petals, stamens), fruits, seeds, etc. For the process of inducing root formation and seedling growth from callus through explants, cluster bud induction is usually carried out after obtaining green and compact callus. The experimental process is explant → callus → cluster buds or adventitious buds → rooted seedlings. Among them, adventitious buds are generally suitable for species that are difficult to proliferate with axillary buds; cluster buds generally originate from axillary buds or shoot tips and are formed by in vitro culture at specific parts of the plant to form multiple densely arranged buds. Adventitious buds can be further induced to develop into cluster buds and ultimately used for rooting and seedling growth.

[0006] Guo Xinrong et al. (Research on the Induction of Artemisia annua Callus and the Variation Law of Chemical Constituents, Tianjin University of Traditional Chinese Medicine, No. 4, 2014) studied the method of inducing callus and rapid propagation using the inflorescence, leaves and petioles of Artemisia annua as explants. The results showed that the induction rate of the MS + 2,4-D 0.1 mg / L + 6-BA 0.5 mg / L medium for the leaves, petioles and inflorescences of Artemisia annua was 100%. In addition, it was also found that the hormone combination for inducing cluster buds was 6-BA 2.0 mg / L + NAA 0.15 mg / L, and higher hormone concentrations were not conducive to the formation of cluster buds. This report induced that 6-BA needs to cooperate with other hormones to achieve a better induction rate.

[0007] Yang Zhengxiu et al. (Optimization of the in vitro Regeneration System of Artemisia annua, Hubei Agricultural Sciences, No. 6, 2017) disclosed that the cotyledons of Artemisia annua were suitable as explants for tissue culture. The MS + 1.5 mg / L 6-BA + 0.05 mg / L NAA medium was beneficial to the induction and differentiation of callus, and the callus induction rate and bud induction rate were as high as 89.0% and 85.7% respectively. The 1 / 2MS + 0.05 mg / L NAA + 0.05 mg / L IAA medium was beneficial to the induction of adventitious roots of regenerated plants, and the rooting induction rate reached 94.0%. This literature further pointed out that NAA was necessary for the formation of callus induction. 1.0 - 2.0 mg / L 6-BA could induce callus, and when 6-BA reached 3 mg / L, it was easy to cause tissue vitrification, and the concentration of 1.5 mg / L 6-BA was more appropriate. That is, it taught that it was not suitable to use high-concentration 6-BA for induction.

[0008] Huang Hongfang et al. (Research on Tissue Culture of Artemisia annua, Hunan Agricultural Sciences, No. 7, 2012) studied the effects of various hormones and their combinations on the induction of callus and cluster buds, among which Figure 2 it was shown that the explant I of the stem segment with axillary buds was cultured on the medium M9 supplemented only with 6-BA (1.5 mg / L), and the axillary buds grew vigorously. However, adventitious buds were only induced from the cells around the axillary buds. When the concentration of 6-BA increased to a certain value, the number of adventitious buds produced on the explant decreased, and only the growth of adventitious buds occurred at the axillary buds without the differentiation of adventitious buds. Since the callus culture time was 30 days, this indicated that long-term culture with high-concentration 6-BA had an inhibitory effect on the differentiation of adventitious buds of the explant. That is, this literature taught that it was not suitable to use high-concentration 6-BA to induce the formation of cluster buds or adventitious buds, thus affecting rooting.

[0009] Chinese Patent Application CN202011644012.X, with the invention title of "A Tissue Culture Method and Medium Combination for Artemisia annua", discloses using the leaves of Artemisia annua as explants, and performing callus induction and differentiation culture for 21 - 25 days in a medium of MS + 2.0 - 2.5 mg / L 6 - BA + 0.2 - 0.3 mg / L NAA + 6.8 - 7.2 g / L agar + 28 - 32 g / L sucrose, then performing proliferation culture to form a large number of clustered buds, and finally performing rooting treatment on the clustered buds to obtain rapidly propagated seedlings. Although this literature reports that "during the process of forming callus, it is possible to induce the differentiation of callus into clustered bud buds at the initial stage of callus formation, that is, when very small callus dots appear on the explant", this process still needs to go through the process of inducing callus - inducing clustered buds, and the relative culture time is too long.

[0010] As the closest prior art, Yu Feifei et al. (Research on the in vitro rapid propagation technology of Artemisia annua, Journal of Southwest China Normal University, No. 1, 2008) studied the effects of different hormone ratios on the induction of clustered buds from axillary bud stem segments. After 14 days of culture, the combination of 6 - BA and NAA was better than the combination of KT and IAA, but too high overall hormone concentration was not conducive to growth. For example, in Medium No. 1 with 6 - BA 4.0 mg / L + NAA 0.1 mg / L, the explants swelled quickly but had few seedlings, and some callus had vitrification phenomenon. As the hormone concentration decreased, the growth basically showed an upward trend. The best hormone combination for inducing clustered buds was 6 - BA 1.0 mg / L + NAA 0.5 mg / L. This literature points out that the stem segments of Artemisia annua with axillary buds are ideal explants for in vitro rapid propagation, and can quickly form test - tube seedlings in a short time, and basically can maintain genetic stability. Hormones promote the differentiation of Artemisia annua stem segments into clustered seedlings or rooting, and the concentration requirements are moderate. Too high concentration is easy to cause plant deformity, form plates, and reduce the quality of test - tube seedlings. When the ratio of cytokinin to auxin is too high, it is easy to produce vitrified seedlings. In addition, during the induction of callus formation, the induction of callus differentiation, the differentiation of buds and roots, and sub - culture, the exogenous hormones gradually show a downward trend during the culture process, which is related to the formation of endogenous hormones. The use effect of multi - factor hormones is better than that of single hormones, which is related to the synergistic effect of hormones.

[0011] In summary, in the prior art, when using Artemisia annua explants (especially axillary bud stem segment explants) for tissue culture, it is necessary to explore appropriate hormone combinations and ratios, and the induction callus cycle and rooting and seedling - forming cycle are long, and it is difficult to solve the problem of callus vitrification, which affects the rapid propagation of Artemisia annua and thus affects the yield of artemisinin.

[0012] Therefore, developing a tissue culture technology for Artemisia annua and studying a method for Artemisia annua to form seedlings in one step is beneficial to reducing the cumbersome steps in its tissue culture and has important 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: A method for tissue culture of Artemisia annua, comprising the following steps: (1) Cut the stem segments of Artemisia annua with axillary buds as explants and sterilize and treat them before inoculation; (2) inoculating the explants onto an induction medium for cultivation to induce them to form clustered buds; 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; (3) transferring the germinated cluster buds to a proliferation medium for cluster 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 gradually decreases; (4) Cut the proliferated unrooted seedlings and transfer them to the rooting medium for rooting culture.

[0015] As a further improvement, the sterilization in step (1) is performed by immersing the explant in a mercuric chloride solution and shaking the solution continuously to ensure full contact between the solution and the explant.

[0016] As a further improvement, step (2) is cultured in the induction medium for 9-10 days.

[0017] As a further improvement, the concentration of 6-BA in the induction medium in step (2) is 4 mg / L.

[0018] As a further improvement, in step (2), the sucrose concentration in the induction medium is 20-40 g / L, and the agar concentration is 6-8 mg / L.

[0019] 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 20-25 days and 1 mg / L for 10 days for strong seedling culture; 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 10 days for strong seedling culture.

[0020] As a further improvement, the rooting medium described in step (4) consists of 1 / 2 MS medium, sucrose, agar, and NAA, and the concentration of NAA is 0.8 - 1.2 mg / L.

[0021] As a further improvement, the sucrose concentration in the rooting medium described in step (4) is 25 - 35 g / L, and the agar concentration is 6 - 8 mg / L.

[0022] A method for one-step seedling formation of rapid propagation of Artemisia annua provided by the present invention includes the following steps: (1) Tissue-culture the Artemisia annua explants according to the above method to generate tissue-cultured seedlings; (2) Pretreatment for hardening off: Prepare for hardening off the tissue-cultured seedlings; (3) Hardening off the tissue-cultured seedlings: Transplant the tissue-cultured seedlings into loose soil, thoroughly water the soil, and harden off the tissue-cultured seedlings; (4) Film-covered hardening off: Seal the tissue-cultured seedlings with a transparent plastic wrap or plastic bag and place them. Avoid direct sunlight at the placement location; (5) Open-air hardening off: Remove the plastic wrap or plastic bag, harden off in situ and then move to under the sun for hardening off; (6) Field transplantation: Conduct field transplantation and planting after the hardening off is completed.

[0023] As a further improvement, remove the plastic wrap or plastic bag 5 days after film-covered hardening off, harden off in situ for 3 days, then move to under the sun for hardening off for 20 days. When it grows to 15 cm, conduct field transplantation.

[0024] The present invention aims to achieve the efficient regeneration and rapid propagation of Artemisia annua plants by optimizing explant selection, culture medium formulation, hardening off and transplantation methods, etc.

[0025] The first principle of the present invention is that: in the case where the prior art teaches that a hormone combination is required for tissue culture of axillary bud stem segments of Artemisia annua, the present invention unexpectedly discovers that using a single high-concentration hormone (i.e., 4 mg / L 6-BA) as the hormone of the induction medium can directly induce axillary bud stem segment explants to generate cluster buds and significantly reduce the generation of callus, so as to rapidly root the cluster buds, thereby obtaining a large number of seedlings. This process of explants directly generating cluster buds by skipping the induction of callus will greatly shorten the culture cycle.

[0026] The second principle of the present invention is that: in order to overcome the increase in the vitrification phenomenon of cluster buds caused by high-concentration hormones, the present invention significantly shortens the first induction period of the cluster bud induction medium to about 9 - 10 days, and then reduces the concentration of 6-BA. After subculture proliferation culture (for example, flexibly adjusting the subculture stage and the concentration of 6-BA used according to the need for rapid seedling formation or obtaining a large number of seedlings), the generation of callus and the vitrification phenomenon can be further reduced.

[0027] The third principle of the present invention is that, based on the first and second principles, in addition to the optimization measures 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.

[0028] The fourth principle of the present invention is that the present invention optimizes the explant disinfection method and explores a seedling cultivation and hardening method, and combines the above first to third principles to obtain a method for rapid propagation of Artemisia annua into seedlings in one step, which can reduce the cost of seedling production, improve large-scale production efficiency, and meet the needs of Artemisia annua field cultivation plants.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 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.

[0030] 2. Existing literature (such as Guo Xinrong, Yang Zhengxiu, Yu Feifei, etc.) has reported that the combination of 6-BA and NAA is very beneficial to the induction of clustered buds in Artemisia annua explants, and then the rooting and rapid propagation. There are reports (Huang Hongfang, etc.) that it is not suitable to use high concentrations of 6-BA to induce clustered buds or adventitious buds, which will affect rooting. However, the present invention unexpectedly found that in the case of using a single hormone, by increasing the concentration of 6-BA in the bud-inducing medium, clustered buds can be directly and quickly induced in the early stage, and callus tissue can be avoided as much as possible, which is beneficial to one-step seedling formation to improve production efficiency and reduce the cost of mass production of seedlings.

[0031] 3. The production advantage of rapid propagation of Artemisia annua explants mainly depends on the rapid propagation time, rather than the difference in induction rate (because the induction rate can ensure the acquisition of enough clustered buds by expanding the number of explants). As far as the present invention is concerned, during the inoculation of the explants of the present invention, due to the characteristics of the explants themselves, the sterilization is not thorough, or due to the death of some of the young explants after sterilization, there will be some explants infected, some explants browned and unable to induce buds normally, and this part of the material is about 15%, so the bud induction rate is about 70-80%. For example, the clustered bud induction rate (75.56±3.85) of the best embodiment of the present invention is lower than the clustered bud induction rate reported by Yu Feifei (6-BA 1.0mg / L+NAA 0.5mg / L, induction rate 93%), but the average time is 9.67±0.58 days, which is significantly less than the latter's 14 days, which will greatly reduce the time cost and meet the primary requirement of rapid propagation tissue culture.

[0032] 4. Compared with the description in the existing literature that "during the process of callus formation, callus differentiation to form cluster bud points can be induced at the initial stage of callus formation when very small callus dots appear on the explant", the site where cluster buds are generated by this technique is different from that of the present invention: this technique requires the induction of small pieces of callus from the explant, and the direct differentiation of the callus to form cluster buds; while the present invention uses the shoot tip as the explant to directly induce the generation of cluster buds at the site of its own bud points (i.e., where the axillary buds are located). Although there is a small amount of callus at the wound, during the subsequent culture process, the cluster buds at the axillary bud site are proliferated and cultured, rather than differentiating the callus at the wound site. Therefore, compared with the existing technology, the present invention realizes the process of directly generating cluster buds by skipping the induction of callus, meeting the requirements of rapid propagation.

[0033] 5. In the early stage of the present invention, while improving the sterilization method and increasing the concentration of plant growth hormone added, in the later stage, the concentration of plant growth hormone is appropriately reduced according to the situation of cluster buds. While quickly inducing cluster buds, large-scale production can be achieved. In the later stage, a large number of seedlings can be quickly obtained through the method of film covering and acclimatization and then open-air acclimatization.

[0034] 6. The present invention uses the basic medium MS (containing agar + sucrose), and only by adjusting the concentration of 6-BA, the generation of cluster buds can be induced, greatly reducing the cumbersome process of preparing different media and improving production efficiency.

[0035] 7. It only takes 9.67 ± 0.58 days to induce the generation of cluster buds by the present invention. The proliferation and strong seedling process of cluster buds only takes 25 - 30 days, and the rooting time only takes 15 days, with a total average of 44 - 49 days. Compared with the traditional culture cycle of 2 - 3 months to obtain rooted seedlings, it has significant production advantages and reduces the labor and material costs.

[0036] 8. In addition, it has also been proved that for the cluster buds directly induced by the present invention, when using the existing optimal rooting medium, consistent rooting efficiency can be achieved. It shows that the one-step method for inducing cluster buds of the present invention can directly combine the existing best proliferation and subculture treatment methods and rooting and seedling induction methods, which greatly improves the operability of the present invention and has the advantages similar to modular replacement in the production process.

[0037] 9. Currently, regions such as Zhangjiajie, Changde, and Yongzhou in Hunan Province are important Artemisia annua planting bases. Model villages and cooperatives for Artemisia annua planting have been established in multiple counties and cities. Through planting improved Artemisia annua varieties and developing tissue culture technology for seedling raising combined with field cultivation, large-scale planting has been formed, which is of great significance for helping the large-scale development of the western region and realizing the increase of farmers' income. According to the incomplete statistics of the research group of the present invention, this method can achieve a seedling emergence rate of 95%, an increase in Artemisia annua yield by 20%, an artemisinin content of 1.8%, a reduction in breeding time by 1 - 2 years, an increase in yield per mu by nearly 25 kg, an increase in the purchase price of Artemisia annua seedlings by 3.6 - 4.8 yuan per catty, an increase in the output value per mu from about 1200 yuan to about 2200 yuan, indirectly driving employment of 1000 - 1200 people, and driving the income increase of more than 20 cooperatives in 9 cities within Hunan Province, ultimately realizing the promotion of rural revitalization with scientific and technological innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is a diagram of inoculating Artemisia annua explants; Figure 2 It is an effect diagram of directly generating cluster buds from Artemisia annua explants, in which callus basically does not appear; Figure 3 It is a diagram of subculture proliferation culture of Artemisia annua; Figure 4 It is a diagram of rooting culture of Artemisia annua cluster buds; Figure 5 It is a diagram of film covering and hardening of Artemisia annua tissue culture seedlings; Figure 6 It is a diagram of open-air hardening of Artemisia annua tissue culture seedlings; Figure 7 It is a diagram of the growth situation of tissue culture seedlings transplanted in the field; Figure 8 It is a diagram of inducing callus by culturing Artemisia annua in a conventional medium. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in combination with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0041] Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention.

[0042] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0043] Some specific methods for tissue culture using Artemisia annua stem segments with axillary buds as explants in the present invention include the following steps: (1) Cut the Artemisia annua stem segments with axillary buds as explants and perform sterilization and pre-inoculation treatment: In some embodiments, the explant material collection includes: using high branch shears to cut the upper tender branches of Artemisia annua plants, cutting them into small segments about 5 cm long, with 2 - 3 axillary buds in each small segment, and retaining one small leaf. Then perform sterilization and pre-inoculation treatment on the explants: after disinfecting and sterilizing the explants, cut them into 2 - 3 parts according to the location of the axillary buds, ensuring that there is at least one axillary bud on each stem segment.

[0044] In some embodiments, the disinfection and sterilization include: putting the processed explants into a clean triangular flask, adding a 0.2% mercuric chloride solution to submerge the explants, and shaking continuously to make the solution and the explants fully contact to achieve thorough sterilization; after disinfecting for 8 minutes, rinse with sterile water 4 times, and dry the moisture with a clean filter paper.

[0045] (2) Inoculate the explants on the induction medium for culture to induce the formation of multiple shoots: In some embodiments, the induction medium is prepared as follows: using MS medium as the basic medium, each 1 L of the 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 contains 40 ml of the medium for primary multiple shoot induction.

[0046] Among them, MS refers to the MS component of the medium commonly used in the existing plant tissue culture field.

[0047] In some embodiments, to induce multiple shoots from the explants: insert the morphological lower end of the explants into the medium, with 4 - 5 explants in each bottle, evenly distributed into the culture bottle, and culture for 9 - 10 days to induce the formation of multiple shoots from the explants. The culture conditions are as follows: temperature: 25 ± 2 °C, light intensity 1500 - 2000 Lx, light duration 12 h / d.

[0048] In some embodiments, the formation rate of explant callus is about 5-10%. Even if callus is generated, it can rapidly differentiate into cluster buds during subsequent culture. The induction rate of cluster buds of the explant is about 70-80%.

[0049] (3) Transfer the germinated cluster buds to a proliferation medium for proliferation culture of cluster buds: In some embodiments, the germinated cluster buds are transferred to a proliferation medium for subculture. The culture conditions are as follows: Temperature: 25±2°C, light intensity 1200 Lx, light cycle: 14 h of light and 10 h of darkness.

[0050] In some embodiments, except for the reduced concentration of 6-BA, the other components and contents of the proliferation medium are the same as those of the induction medium. The initial concentration of 6-BA is not higher than 3 mg / L and gradually decreases.

[0051] The subculture times and the concentration of the subculture medium can be determined according to the required number of seedlings. If rapid seedling formation is needed, it can be adjusted to 2 mg / L 6-BA for 10-15 days during the first subculture, and then adjusted to 1 mg / L 6-BA for 10 days for strong seedling culture, and then rooting can continue.

[0052] If large-scale seedling culture is needed, it is adjusted to 3 mg / L 6-BA for 10-12 days during the first subculture, and then sequentially adjusted to 2 mg / L 6-BA for 20-25 days and 1 mg / L 6-BA for 10 days for strong seedling culture, and then rooting can continue.

[0053] (4) Cut the proliferated unrooted seedlings and transfer them to a rooting medium for rooting culture: In some embodiments, rooting culture is carried out when the cluster buds grow to about 4 cm. Cut the lower end of the cluster buds and inoculate them into the medium. The culture conditions are as follows: Temperature: 25±2°C, light intensity 1200 Lx, light cycle: 14 h of light and 10 h of darkness.

[0054] In some embodiments, each 1 L of the rooting medium is composed 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 (naphthalene acetic acid).

[0055] 1 / 2 MS means that the macroelements in the commonly used medium MS components are halved, and the other components remain unchanged.

[0056] The method for rapid propagation of Artemisia annua to form seedlings in one step in some specific embodiments of the present invention includes the steps of: (1) Carry out tissue culture on Artemisia annua explants according to the above method to rapidly generate tissue culture seedlings; (2)Acclimatization pretreatment: Select tissue culture seedlings with roots of 1 - 1.5 cm for acclimatization preparation; In some embodiments, the tissue culture seedlings are placed under outdoor conditions to adapt to room temperature conditions for 2 - 3 days while avoiding direct sunlight; (3)Acclimatization of tissue culture seedlings: Transplant the tissue culture seedlings into loose soil and thoroughly water the soil for acclimatization of the tissue culture seedlings; In some embodiments, the culture medium on the roots is rinsed clean with clean tap water, and then transplanted into loose soil. Approximately 8 - 9 plants are transplanted into a flower pot with a diameter of 20 cm. After transplantation, thoroughly water the soil for acclimatization of the tissue culture seedlings; (4)Film - covering acclimatization: Seal the tissue culture seedlings with a transparent plastic wrap or plastic bag with good light transmittance, and the placement should avoid direct sunlight; (5)Open - air acclimatization: Remove the plastic wrap or plastic bag 5 days after film - covering acclimatization, and after acclimatizing in place for 3 days, move it to direct sunlight for acclimatization; (6)Field transplantation: When the tissue culture seedlings grow to 15 cm after acclimatizing under direct sunlight for more than 20 days, they can be transplanted and planted in the field.

[0057] In some embodiments, the tissue culture seedlings grow to about 10 cm after acclimatizing under direct sunlight for more than 20 days. At this time, pay attention to fertilization. When they grow to 15 cm, they can be transplanted to the field, and then managed according to normal seedlings.

[0058] The present invention uses a single high - concentration hormone (i.e., 4 mg / L 6 - BA) as the hormone in the induction medium, which can directly induce adventitious buds from axillary bud stem explants and significantly reduce the formation of callus. At the same time, in the subculture proliferation culture, according to the need of rapid seedling formation or obtaining a large number of seedlings, after flexibly adjusting the subculture stage and the concentration of 6 - BA used, the formation of callus and vitrification can be further reduced. The method of the present invention can rapidly root adventitious buds, thereby obtaining a large number of seedlings, having the significant advantages of saving time, labor, and material costs in production.

[0059] Example 1 Tissue culture method for Artemisia annua axillary bud - bearing stem explants (1) Select a single plant of XH - 1 Artemisia annua with strong growth and good morphology, cut the tender stem at the top of the plant as the explant, prune the branches and leaves to obtain an axillary bud - bearing stem segment, soak the stem tip in a 5 wt% aqueous solution of dishwashing liquid for 10 min, then rinse with running water for 30 min, and air - dry naturally to obtain an in vitro stem tip after running - water cleaning.

[0060] (2) The in vitro stem tips after running water washing were operated in a laminar flow hood; they were soaked and disinfected with 0.2 wt% mercuric chloride aqueous solution for 8 min, and shaken well during this process to ensure the sterilization effect. Then they were rinsed 4 times with sterile water, and then the excess water was blotted dry with sterile filter paper under sterile conditions. About 0.1 cm of the wound at both ends of the disinfected stem segments was excised, and then they were cut into 1.5 cm small segments, ensuring that there was at least one axillary bud on each stem segment. The lower morphological end was inoculated on a solid induction medium (MS + 30 g / L sucrose + 7 g / L agar + 4.0 mg / L 6-BA) and cultured at 25 ± 2 °C. The culture conditions were as follows: temperature: 25 ± 2 °C, light intensity 1500 - 2000 Lx, light duration 12 h / d, see Figure 1 . Taking the day of inoculation as day 0, when cultured for 8 days, cluster bud points or callus appeared on the stem segments; (3) After the bud points appeared, continue the culture. Taking the day when cluster bud points appeared as day 0, when cultured for 2 - 5 days, cluster buds with a bud height of 0.5 to 1 cm appeared, see Figure 2 . In this stage, the culture time with 4 mg / L 6-BA should not be too long, and vitrification will occur in 8 - 10 days; (4) The cluster buds were cut from the base into square tissues with 1 to 2 cluster buds and transferred to a cluster bud proliferation medium for cluster bud proliferation culture. 5 pieces of cluster buds were inoculated in each petri dish. The culture conditions were as follows: temperature: 25 ± 2 °C, light intensity 1200 Lx, light cycle: 14 h of light and 10 h of darkness. Taking the day of transfer to the cluster bud proliferation medium as day 0, after culturing for 10 days, proliferated unrooted seedlings appeared, see Figure 3 .

[0061] For the cluster bud proliferation medium, the concentration of 6-BA can be continuously adjusted according to the actual demand for the number of seedlings: If proliferation and propagation are required, 3 mg / L 6-BA can be used for continuous culture, and after 10 - 12 days of culture, it can be replaced with 2 mg / L 6-BA for subculture, or directly replaced with 1 mg / L 6-BA for subculture; If a large amount of proliferation is not required, 2 mg / L 6-BA can be directly used for culture for 15 - 20 days, and then 1 mg / L 6-BA can be used for subculture for 10 days to start rooting. Except for hormones, other components of the cluster bud proliferation medium are the same as those of the induction medium. The types and dosages of hormones can be referred to Table 3.

[0062] (5) Cut the proliferated unrooted seedlings into single seedlings from the base, transfer them to test-tube plantlet bottles containing 45 ml of adventitious root rooting medium (1 / 2 MS + 7 g / L agar + 30 g / L sucrose + 1 mg / L NAA, pH value 5.8 - 6.0) for cultivation. The cultivation conditions are as follows: temperature: 25 ± 2 °C, light intensity 1200 Lx, light cycle: 14 h of light and 10 h of darkness. Take the day when transferred to the adventitious root rooting medium as day 0, and cultivate for 10 - 12 days to obtain rooted seedlings, see Figure 4 .

[0063] Example 2 Test the influence of induction media with different hormone ratios on the induction effect of clustered buds Prepare the clustered bud induction media according to the method and Table 1 of Example 1, where the difference in each numbered experiment lies only in the different hormone combination ratios.

[0064] Among them, according to Guo Xinrong et al., Yang Zhengxiu et al., and Yu Feifei et al., it has been reported that the combination of 6-BA and NAA is very beneficial for inducing clustered buds from Artemisia annua explants and then for rapid rooting and propagation. Therefore, this combination is selected.

[0065]

[0066] For each hormone combination, there are 3 parallel groups, and each group treats 15 explants. Then, respectively, count the callus formation rate (induction rate), clustered bud induction rate, and the time to form bud points.

[0067] The results are shown in Table 2.

[0068]

[0069] As shown in Table 2, the combination of No. 13 has the best cultivation effect, with the advantages of high bud induction rate, short number of days to form bud points, low callus induction, and can directly develop into seedlings, greatly reducing the time for early differentiation to form clustered buds, and directly generating clustered buds during the cultivation process. Although individual explants will produce callus, after the callus is formed, it can quickly differentiate into clustered buds, and the overall differentiation time of the clustered buds does not exceed 12 days. It can be differentiated and completed in the bud induction medium without changing the medium, with fast speed and high efficiency.

[0070] In contrast, for the same high-concentration 6-BA treatment, the combination of No. 14 adds NAA to the bud induction medium, and the callus induction rate is higher than that of No. 13. Although the callus can also differentiate into clustered buds during the cultivation process, the time to form clustered buds is prolonged. And due to the too long differentiation time of the callus, the plants are under the cultivation of high-concentration 6-BA cytokinin for a long time, and the vitrification phenomenon increases.

[0071] At the same time, for low-concentration 6-BA treatments, such as No. 1 - 3, a large amount of brown callus will appear (seeFigure 8 ), and it cannot be successfully differentiated using the same culture medium. It is necessary to change to other culture media for differentiation induction during the subsequent culture process. The overall time to form cluster buds is long, and the operation is much more cumbersome than that of No. 13.

[0072] Moreover, by comparing the combinations of No. 1, 5, 9, and 13, it can be found that the higher the 6-BA concentration, the shorter the time to form cluster buds, which is beneficial to directly forming cluster buds in the early stage of the tissue culture process.

[0073] Example 3. Test and analyze the influence of proliferation culture media with different concentrations of 6-BA on the proliferation effect According to the results of Example 2, select the cluster bud seedlings of the best induction culture medium (i.e., the 4 mg / L 6-BA group of No. 13 culture medium), and according to Table 3 below, test and analyze the influence of proliferation culture media with different concentrations of 6-BA on the proliferation effect.

[0074]

[0075] It is found from the above table that the proliferation efficiency of using high-concentration 6-BA alone is higher than that of combining 6-BA with the same concentration of NAA. Therefore, 6-BA is added alone for culture during the differentiation and proliferation process.

[0076] It can be found from No. 1 that when the germination culture medium is cultured with 4 mg / L 6-BA, it is necessary to change to a culture medium with a lower concentration as soon as possible. Continuous culture with 4 mg / L is very likely to cause vitrification; when changing to a lower concentration of 6-BA, such as No. 4, No. 7, and No. 10, the vitrification phenomenon is significantly alleviated, and the culture time for the vitrification phenomenon is continuously extended. Therefore, after successful induction of buds, it is necessary to change to a culture medium with a lower concentration of 6-BA for proliferation culture as soon as possible.

[0077] During the proliferation culture process without adding NAA for culture, the proliferation coefficient continuously decreases from No. 4 with a higher concentration, and No. 10 is the lowest. However, the vitrification phenomenon continuously decreases, and no vitrification phenomenon is found when directly using 1 mg / L 6-BA for proliferation culture after successful induction of buds.

[0078] Therefore, considering the need for large-scale cultivation of seedlings, after successful induction of buds, continue to proliferate and culture with No. 4 at a reduced concentration. The culture time for this process is 10 - 12 days. For the second subculture, select No. 7 to continue the culture, which takes about 20 - 25 days. Then use No. 10 for strengthening the seedlings, and finally perform rooting treatment. If large-scale seedlings are not needed, then No. 7 can be directly used for culture in the first subculture, which takes about 10 - 15 days. For the second subculture, use No. 10 for culture for 10 - 15 days, and finally perform rooting and acclimatization.

[0079] Example 4. Test and analyze the rooting efficiency of the present invention According to the method of Example 1 and Table 4, a cluster root culture medium was prepared, and the difference in the experiments with each serial number was only in the hormone combination ratio.

[0080]

[0081] When NAA was added alone for rooting, according to the experimental results of different concentrations in Table 4, it was found that the rooting effect was the best with 1 mg / L NAA. As the concentration of NAA increased, the rooting rate continuously decreased. The average rooting rate and the number of roots with 2 mg / L NAA were significantly lower than those with 1 mg / L NAA. Therefore, it was the best to use 1 mg / L NAA for rooting treatment.

[0082] 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 Serial No. 1, Serial No. 4, and Serial No. 7. Moreover, using NAA alone could also reduce the cost loss in the early stage. Therefore, for subsequent large-scale seedling hardening, the culture medium 1 / 2 MS + 7 g / L agar + 30 g / L sucrose + 1 mg / L NAA was used, and its rooting induction effect was the best, with an average rooting rate of 97.78 ± 3.85.

[0083] For the cluster buds obtained from the explants of Artemisia annua at different parts and used for rooting treatment, there was no essential difference, and the rooting rate mainly depended on the type and ratio of hormones. Therefore, the pre-proliferation method used in the present invention had extremely strong adaptability and could be combined with different rooting culture media for rooting induction in the later stage.

[0084] Example 5. Field cultivation of rapid-propagated tissue culture seedlings Seedling hardening preparation: When the roots of the tissue culture seedlings were about 1 - 1.5 cm long, seedling hardening preparation was carried out. Open the bottle cap of the test-tube seedlings, adapt in the tissue culture room for 1 day, take out the rooted seedlings, and wash the residual culture medium attached to the roots of the rooted seedlings. Place the tissue culture seedlings under outdoor conditions to adapt to room temperature conditions, and pay attention to avoiding direct sunlight for about 2 - 3 days; Seedling hardening of tissue culture seedlings: Gently clamp the tissue culture seedlings out of the culture medium, wash the culture medium on the roots with clean tap water, and transplant them into loose soil. About 8 - 9 seedlings are transplanted into a flower pot with a diameter of 20 cm. After transplantation, water the soil thoroughly for seedling hardening of the tissue culture seedlings; Film covering for seedling hardening: To ensure the water demand, the just-transplanted tissue culture seedlings are sealed with a transparent plastic wrap or plastic bag with good light transmittance, and the placement should avoid direct sunlight, see Figure 5 ; Open-air seedling hardening: Remove the plastic wrap or plastic bag 5 days after film covering for seedling hardening, and carry out seedling hardening in place for 3 days, and then move it to the sun for seedling hardening, see Figure 6 ; Field transplanting: After acclimatizing the tissue culture seedlings under direct sunlight for more than 20 days, they grow to about 10 cm. At this time, pay attention to fertilization. When they reach 15 cm, they can be transplanted to the field, and then managed according to normal seedlings. See Figure 7 .

[0085] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Therefore, any simple modifications, equivalent changes, and decorations made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A tissue culture method of Artemisia annua, characterized in that, It includes the following steps: (1) Cut the Artemisia annua stem segments with axillary buds as explants, and perform sterilization and pre-inoculation treatment; (2) Inoculate the explants on the induction medium for culture to induce the formation of cluster buds; The induction medium consists of MS medium, sucrose, agar and 6-BA, where the concentration of 6-BA is 3.5 - 4.5 mg / L; (3) Transfer the germinated cluster buds to the proliferation medium for cluster bud proliferation culture; Except for the concentration of 6-BA, other components and contents of the proliferation medium are the same as those of the induction medium, where the initial concentration of 6-BA is not higher than 3 mg / L and gradually decreases; (4) Cut the proliferated unrooted seedlings and transfer them to the rooting medium for rooting culture.

2. The artemisia annua tissue culture method according to claim 1, wherein The sterilization in step (1) is to immerse the explants in mercuric chloride solution and shake continuously to make the solution and the explants fully contact.

3. The artemisia annua tissue culture method according to claim 1, characterized in that, In step (2), culture on the induction medium for 9 - 10 days.

4. The artemisia annua tissue culture method according to claim 1 or 3, characterized in that, The concentration of 6-BA in the induction medium in step (2) is 4 mg / L.

5. The artemisia annua tissue culture method according to claim 1 or 3, characterized in that, The sucrose concentration in the induction medium in step (2) is 20 - 40 g / L, and the agar concentration is 6 - 8 mg / L.

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

7. The artemisia annua tissue culture method according to claim 1 or 3, characterized in that The rooting medium in step (4) consists of 1 / 2MS medium, sucrose, agar, and NAA, and the concentration of NAA is 0.8 - 1.2 mg / L.

8. The tissue culture method of Artemisia annua according to claim 7, characterized in that, The sucrose concentration in the rooting medium in step (4) is 25 - 35 g / L, and the agar concentration is 6 - 8 mg / L.

9. A method for rapid propagation of Artemisia annua with one-step seedling formation, characterized in that, It includes the following steps: (1) Perform tissue culture on Artemisia annua explants according to the method of any one of claims 1 - 8 to generate tissue culture seedlings; (2) Acclimatization pretreatment: Prepare for acclimatization of the tissue culture seedlings; (3) Acclimatization of tissue culture seedlings: Transplant the tissue culture seedlings into loose soil, pour the soil thoroughly for acclimatization of the tissue culture seedlings; (4) Film-covered acclimatization: Seal the tissue culture seedlings with a transparent plastic wrap or plastic bag and place them. The placement location should avoid direct sunlight; (5) Open-air acclimatization: Remove the plastic wrap or plastic bag, acclimatize in place and then move to the sun for acclimatization; (6) Field transplantation: Perform field transplantation and planting after the acclimatization is completed.

10. The method for directly growing seedlings of Artemisia annua for rapid propagation according to claim 9, wherein, Remove the plastic wrap or plastic bag 5 days after film-covered acclimatization, acclimatize in place for 3 days, then move to the sun for acclimatization for 20 days. When it grows to 15 cm, perform field transplantation.

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