Tissue culture breeding method of Mongolian medicine plant Artemisia stelleriana

Abstract

The invention belongs to the technical field of plant asexual propagation, and particularly relates to a Mongolian medicine plant Artemisia stelleriana tissue culture propagation method. The tissue culture breeding method comprises the following steps: explant selection: selecting an artemisia stelleriana aseptic seedling plant, and cutting stems and leaves as an explant material; callus induction culture: segmenting the explant, inoculating the explant into a primary culture medium, and culturing to obtain callus; differentiated bud induction culture: inoculating the callus into a differentiated bud induction culture medium, and inducing to generate differentiated buds; rooting induction culture: when the differentiated buds are transferred into a rooting culture medium, rooting seedlings are obtained; and rooting seedling culture: performing domestication and seedling hardening on the rooting seedlings, and transplanting and culturing to obtain survived artemisia fruticosa tissue culture seedlings. According to the method, key parameters such as explant selection and culture medium formula are optimized, standardized production is realized, and industrial popularization is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of plant asexual reproduction, and particularly relates to a tissue culture and breeding method for the Mongolian medicinal plant Artemisia annua. Background Art

[0002] Artemisia frigida, the aerial part of the Artemisia frigida plant in the Asteraceae family, has been a popular traditional Mongolian medicine for generations. Currently, its production relies primarily on wild collection, with seed propagation employed in some areas. Because Artemisia frigida is a water and soil conservation plant, its growth pattern is closely tied to its habitat. Harvesting it from the wild can easily damage the environment, leading to soil erosion and increased desertification of grasslands.

[0003] Propagating Artemisia annua from seeds often results in low seedling emergence rates, weak stress resistance during the seedling stage, and an excessively slow growth cycle. Furthermore, problems such as low germination rates, uneven germination, and unstable seedling quality severely restrict the standardized cultivation and production of Artemisia annua. Tissue culture technology can effectively and rapidly improve the propagation speed and quality of Artemisia annua seedlings, enabling factory-scale production of high-quality Artemisia annua seedlings to meet production needs. Summary of the Invention

[0004] In response to the shortcomings of existing technologies, the present invention provides a method for tissue culture and propagation of the Mongolian medicinal plant Artemisia annua. By leveraging multiple advantages, including efficient propagation, quality assurance, resource conservation, and cost control, the method promotes the modernization and sustainable development of Chinese herbal medicine production, achieving both economic and social benefits.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: A first aspect of the present invention provides a method for tissue culture and propagation of a Mongolian medicinal plant Artemisia annua, the method comprising the following steps: Selection of explants: Select sterile seedlings of Artemisia annua and cut the stems and leaves as explant materials; Callus induction culture: The explants were segmented and inoculated into primary culture medium to obtain callus tissue. The culture temperature was 25±2°C, the light duration was 12 h / d, and the light intensity was 1800-2200 lux. Differentiation bud induction culture: inoculate the callus tissue into differentiation bud induction medium to induce differentiation buds. The culture temperature is 25±2℃, the light duration is 10-12 h / d, and the light intensity is 2000-3000 lux. Rooting induction culture: When the differentiated buds are transferred to the rooting medium to obtain rooted shoots, they are cultured in the dark at a temperature of 25±2℃; Rooted seedling cultivation: the rooted seedlings are acclimated and transplanted to obtain surviving Artemisia annua tissue culture seedlings.

[0006] As a preferred embodiment, the culturing of the sterile seedlings specifically includes: removing the white mucus on the surface of the Artemisia annua seeds, then sterilizing the seeds with 75% ethanol, rinsing with sterile water, and then sterilizing with 2% NaClO for 5 minutes, 10 minutes, 15 minutes, and 20 minutes respectively, rinsing with sterile water, and then drying the surface of the seeds, and inoculating them into MS culture medium for culturing; As a preferred embodiment, the method for removing the white mucus on the surface of Artemisia annua seeds includes physical scrubbing, warm water soaking, sand friction or detergent solution cleaning.

[0007] As a preferred embodiment, the detergent solution cleaning method has a better cleaning effect, a low seed contamination rate, and a high germination rate. Therefore, the detergent solution cleaning method is preferably used as the Artemisia annua seed cleaning method.

[0008] As a preferred embodiment, removing the white mucus on the surface of the Artemisia annua seeds includes: soaking the Artemisia annua seeds in tap water for 1 hour, rubbing and cleaning the soaked seeds with detergent, and rinsing with tap water until the detergent is completely removed.

[0009] As a preferred embodiment, the explant is segmented into 0.5 to 1 cm segments.

[0010] As a preferred embodiment, the primary culture medium consists of MS + IBA + NAA + sucrose + agar; As a preferred embodiment, the mass concentration of IBA is 1-5 mg / L, the mass concentration of NAA is 1-5 mg / L, the mass concentration of sucrose is 30 g / L, the mass concentration of agar is 3-10 g / L; As a preferred embodiment, the pH value of the primary culture medium is 5.8-6.0.

[0011] In the present invention, in the differentiation bud induction medium, the mass concentration of IBA can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L or 5 mg / L, etc., the mass concentration of NAA can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L or 5 mg / L, etc., the mass concentration of agar can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, etc., and the pH value can be 5.8, 5.9 or 6.0, etc., but is not limited to the values listed above, and other values not listed within the above numerical range are equally applicable.

[0012] As a preferred embodiment, the differentiation bud induction medium consists of MS + 6-BA + IBA + sucrose + agar.

[0013] As a preferred embodiment, the mass concentration of 6-BA is 1-5 mg / L, the mass concentration of IBA is 1-5 mg / L, the mass concentration of sucrose is 30 g / L, and the mass concentration of agar is 3-10 g / L.

[0014] As a preferred embodiment, the pH value of the differentiation bud induction medium is 5.8-6.0.

[0015] In the present invention, in the differentiation bud induction medium, the mass concentration of 6-BA can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L or 5 mg / L, etc., the mass concentration of IBA can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L or 5 mg / L, etc., the mass concentration of agar can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, etc., and the pH value can be 5.8, 5.9 or 6.0, etc., but is not limited to the values listed above, and other values not listed within the above numerical range are equally applicable.

[0016] As a preferred embodiment, the rooting medium consists of 1 / 2MS + GA3 + IBA + sucrose + agar.

[0017] As a preferred embodiment, the mass concentration of GA3 is 0.5-3 mg / L, the mass concentration of IBA is 0.5-3 mg / L, the mass concentration of sucrose is 30 g / L, and the mass concentration of agar is 3-10 g / L.

[0018] As a preferred embodiment, the pH value of the rooting medium is 5.8-6.0.

[0019] In the present invention, in the rooting medium, the mass concentration of GA3 can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L or 3 mg / L, and the mass concentration of IBA can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 The mass concentration of agar can be 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, and the pH value can be 5.8, 5.9 or 6.0, but is not limited to the values listed above. Other values not listed within the above numerical range are also applicable.

[0020] As a preferred embodiment, the period of acclimation and transplanting is late spring or early autumn.

[0021] As a preferred embodiment, in the culture of the rooted seedlings, acclimation is performed when the rooted seedlings grow to 3-4 cm in height, have 3-5 roots, and 3-4 leaves.

[0022] As a preferred embodiment, the acclimation includes: hardening the rooted seedlings in the culture bottle under natural light for 3-8 days, then opening the sealing film of the culture bottle to harden the seedlings for 1-3 days, during which time water is sprayed on the leaves; removing the seedlings from the culture bottle, cleaning the root culture medium, and then transplanting the seedlings into the substrate for planting.

[0023] As a preferred embodiment, the matrix formula is humus soil: vermiculite = 1:3.

[0024] As a preferred embodiment, the time for transplanting the seedlings into the substrate for planting is 40 days.

[0025] As a preferred embodiment, the seedlings are transplanted into the substrate and planted for 14 days, and then transplanted outdoors for further planting and cultivation for 26 days.

[0026] As a preferred embodiment, the plants are moved outdoors for planting and covered.

[0027] As a preferred embodiment, the covering treatment includes pine needle covering and Mongolian oak fallen leaf covering, preferably Mongolian oak fallen leaf covering.

[0028] The beneficial effects of one or more of the above technical solutions are as follows: (1) There is no tissue culture method for Artemisia annua in the prior art. The present invention has conducted in-depth research on the entire process of obtaining sterile Artemisia annua seedlings, the ratio of the culture medium in the culture stage, acclimation and hardening of the seedlings, and transplanting, and has developed a dedicated tissue culture system suitable for the Mongolian medicine Artemisia annua. This system can effectively and quickly improve the reproduction speed and quality of Artemisia annua seedlings, and realize the factory-based cultivation of high-quality Artemisia annua seedlings.

[0029] (2) The tissue culture method of the present invention can realize the rapid in vitro propagation of Artemisia annua tissue culture seedlings under sterile conditions, which is not restricted by season, climate and region, greatly shortens the seedling raising period, and realizes standardized production by optimizing key parameters such as explant selection, hormone ratio and culture conditions, which is convenient for industrial promotion.

[0030] (3) Tissue culture can avoid genetic recombination during sexual reproduction through meristem culture, retain the excellent traits of the mother plant, and ensure the uniform quality of the medicinal materials. Combined with heat treatment or virus detection technology, virus-free seedlings can be obtained, improving the yield and quality of medicinal plants. At the same time, it can reduce the pressure of wild collection and promote ecological balance. At the same time, through tissue culture combined with low-temperature preservation, wild plant genetic resources can be stored for a long time.

[0031] (4) By adjusting the composition of the culture medium, the accumulation of secondary metabolites can be stimulated, thereby increasing the medicinal value. Some plants can directly produce active ingredients through cell culture, reducing dependence on the whole plant.

[0032] (5) Tissue culture can be carried out in a three-dimensional manner in the laboratory, which takes up little space, has a high degree of automation, and has lower long-term costs than traditional field seedling cultivation. The sterile environment reduces the use of pesticides and is in line with the trend of green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 This is a schematic diagram of sterile Artemisia annua seedlings in Example 1 of the present invention; Figure 2 This is a schematic diagram of callus induction of Artemisia annua in Example 1 of the present invention; Figure 3 This is a schematic diagram of differentiation induction of Artemisia annua in Example 1 of the present invention; Figure 4 This is a schematic diagram of rooting induction of Artemisia annua in Example 1 of the present invention; Figure 5 This is a schematic diagram of the regenerated Artemisia annua seedlings in Example 1 of the present invention; Figure 6 This is a schematic diagram of the indoor acclimation of Artemisia annua tissue culture seedlings in Example 1 of the present invention; Figure 7 This is a schematic diagram of outdoor acclimation of Artemisia argyi in Example 1 of the present invention; Figure 8 Schematic diagram of using Mongolian oak fallen leaves and pine needles to cover Artemisia annua in an embodiment of the present invention; Figure 9 This is a schematic diagram of the tissue culture seedlings after acclimation and transplantation for 2 years. The red part is the acclimated seedlings covered with pine needles, and the yellow part is the acclimated seedlings covered with Mongolian oak. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] The present invention will now be further described with reference to specific examples. The following examples are intended only to explain the present invention and are not intended to limit its contents. If the specific experimental conditions are not specified in the examples, they are generally in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, are conventional reagents, methods, and equipment used in the present invention in the art and are all commercially available.

[0038] Example 1 Currently, there is no technical process for tissue culture of Artemisia annua. The purpose of this embodiment is to explore a technical solution suitable for tissue culture of Artemisia annua.

[0039] (1) Cultivation of sterile seedlings White mucus will soon appear on the surface of the Artemisia annua seeds after they come into contact with water. The mucus on the Artemisia annua seeds is not conducive to the penetration of disinfectants, and the disinfection effect is very poor. Therefore, soak the Artemisia annua seeds in tap water for 1 hour. Rub the soaked seeds with detergent and rinse with tap water several times until the detergent is completely removed, and wash away the white mucus on the surface of the seeds to the greatest extent. Then transfer the seeds with the outer seed coat peeled off to a clean workbench for disinfection. The seeds are first disinfected with 75% ethanol for 1 min, rinsed with sterile water 3 times, and then disinfected with 2% NaClO for 5 min, 10 min, 15 min and 20 min respectively, and rinsed with sterile water 3 times. Place the disinfected seeds on sterile filter paper to absorb the moisture on the surface, and then inoculate them on MS culture medium for culture to obtain sterile seedlings, such as Figure 1 shown.

[0040] (2) Effects of different hormones on callus induction of Artemisia annua explants Different concentrations of 6-BA, NAA, IAA, and IBA were added to the MS basal medium. Each hormone was set at 0.5 mg / L, 1.5 mg / L, 3 mg / L, and 5 mg / L in four gradients, for a total of 16 callus induction medium formulas. Sucrose 30 g / L and agar 8 g / L were added. The pH was adjusted to 5.8 and sterilized at 121°C for 20 min.

[0041] Cut the sterilized leaves into 0.5 cm-1 cm pieces and inoculate them into sterilized callus induction medium. Inoculate 10 explants into each bottle. Make 6 bottles in parallel for each medium formula, repeat 3 times, and culture in an incubator at 25℃±2℃. Count the number of calli after 21 days. The calli obtained by culture are as follows: Figure 2 After 40 days, the callus mortality rate was calculated: callus rate = number of explants forming callus / total number of inoculated explants × 100%, and callus mortality = number of dead callus / number of explants forming callus × 100%. The results are shown in Table 1.

[0042] Table 1 Effects of different auxin treatments on callus induction of Artemisia annua explants

[0043] As shown in Table 1, the induction rates in the 6-BA treatment group were generally low at all concentrations, reaching a maximum of only 17.34% (at 3 mg / L). This indicates that 6-BA was not effective in inducing callus in Artemisia explants within the tested concentration range. In the NAA treatment group, the induction rate increased with increasing concentration. At 1.5 mg / L, the induction rate reached 38.42%, while at 3 mg / L and 5 mg / L, the induction rates reached 78.45% and 77.31%, respectively. Callus mortality was zero at all three concentrations. This indicates that NAA is highly effective in inducing callus in Artemisia explants within the tested concentration range. In the IAA treatment group, the induction rates at 0.5 mg / L and 3 mg / L were zero, while at 1.5 mg / L, the induction rate was 11.7%, but callus mortality was relatively high at 50.05%. In the IBA treatment group, the induction rates reached 56.67% and 77.34% at concentrations of 1.5 mg / L and 3 mg / L, respectively, and the callus mortality rate at these two concentrations was 0. This indicates that IBA has a good effect on callus induction in Artemisia annua explants within the concentration range of 1.5 mg / L to 3 mg / L.

[0044] (3) Callus induction of explants from different parts of Artemisia annua MS basal medium was supplemented with 1.5 mg / L NAA, 30 g / L sucrose, and 8 g / L agar. The pH was adjusted to 5.8 and sterilized at 121°C for 20 min. Disinfected leaves, stems, and roots were cut into 0.5-1 cm pieces and inoculated onto sterilized callus induction medium. Ten explants were seeded into each bottle. Six bottles were replicated for each medium formulation, with three replicates. Cultures were maintained in an incubator at 25°C ± 2°C. Callus rates were calculated after 21 days, and callus mortality was calculated after 40 days. The results are shown in Table 2.

[0045] Table 2 Effects of different explants of Artemisia annua on callus induction

[0046] Note: a, b, and c in the table refer to the significant differences between a and b, and between b and c in the variance analysis. That is, the same letters represent no significant differences, and different letters represent significant differences.

[0047] As shown in Table 2, the callus induction rate for leaf explants of Artemisia annua was 50.32%, with a relatively low callus mortality rate of 3.31%. For root explants of Artemisia annua, the callus induction rate was 56.21%, but the callus mortality rate was as high as 47.7%. The callus induction efficiency of stem explants of Artemisia annua was even higher, with an induction rate of 67.15% and a callus mortality rate of only 4.12%. The induction efficiency of leaf tissue was slightly lower than that of stem tissue, and it can also be used as material for callus induction experiments.

[0048] (4) Effects of different hormones on callus differentiation induction of Artemisia annua Add 1.5 mg / L NAA to MS basal medium, along with 30 g / L sucrose and 8 g / L agar. Adjust the pH to 5.8 and sterilize at 121°C for 20 minutes. Cut the sterilized leaves, stems, and roots into 0.5-1 cm pieces and inoculate them onto sterilized callus induction medium for induction. Induce callus at a temperature of 25 ± 2°C, with a light intensity of 2000-3000 lux and a photoperiod of 10-12 hours per day.

[0049] The callus tissue obtained from the primary culture was inoculated into the differentiation bud induction medium. The differentiation bud induction medium was based on the MS basal medium, with different concentrations (1mg / L, 3mg / L, 4mg / L, 5mg / L) of IAA, IBA, NAA, and 6-BA added. Add 30g / L sucrose and 8g / L agar, adjust the pH to 5.8, and sterilize at 121°C for 20 min. Cut the sterilized explants into 0.5cm pieces and inoculate them into the sterilized callus induction medium. Inoculate 10 explants into each bottle, make 6 bottles in parallel for each culture medium formula, repeat 3 times, and culture in a tissue culture incubator at 25±2°C. Continue to culture under the conditions of light intensity of 2000lx and light of 12h / d. After 30 days, the differentiation rate was counted, and the differentiated callus tissue was as follows. Figure 3 The results are shown in Table 3, where differentiation rate (%) = number of differentiated calli / total number of inoculated calli × 100%, and mortality rate of differentiated seedlings (%) = number of dead differentiated seedlings / number of differentiated calli × 100%.

[0050] Table 3 Effects of different hormones on callus differentiation induction of Artemisia annua

[0051] Table 3 shows that for the 6-BA treatment groups, the callus differentiation induction rate initially increased and then stabilized with increasing concentration. Specifically, at a concentration of 1 mg / L, the induction rate was 24.02%. When the concentration increased to 3 mg / L, the induction rate significantly increased to 76.11%, reaching a peak. Subsequently, at concentrations of 4 mg / L and 5 mg / L, the induction rate decreased slightly but remained at high levels of 68.01% and 69.35%, respectively. The mortality rate was zero in all IBA-treated groups, demonstrating the high safety and efficacy of IBA in inducing callus differentiation in Artemisia annua. Within the IBA-treated groups, the induction rate showed significant fluctuations with concentration. The induction rate was 34.12% at 1 mg / L and rose to 57.03% at 3 mg / L, reaching its highest value for this treatment group. However, as the concentration increased further to 4 mg / L and 5 mg / L, the induction rate dropped sharply to 16.14% and 21.34%, respectively. The mortality rates of differentiated seedlings at 1 mg / L and 3 mg / L concentrations were 1.18% and 3.19%, respectively. While mortality was 0% at 4 mg / L and 5 mg / L, the significant decrease in induction rate suggests that high concentrations of 6-BA may have a negative impact on callus differentiation in Artemisia annua. The results were less favorable for the NAA treatment group. Within the concentration range of 1 mg / L to 5 mg / L, the induction rate was generally low, reaching a maximum of only 2.21%. This suggests that NAA is ineffective in inducing callus differentiation in Artemisia annua within the tested concentration range, and that high-concentration treatment may result in higher mortality. In summary, this study found that 6-BA, within the 3 mg / L to 5 mg / L concentration range, was significantly effective in inducing callus differentiation in Artemisia annua and was highly safe, whereas NAA treatment was relatively ineffective.

[0052] (4) Effects of different formulas on the rooting culture of Artemisia annua On the basis of MS basic medium, different concentrations of IAA, IBA, NAA, and 6-BA were added to design a callus induction medium formula, see Table 4. Differentiated cluster seedlings were inoculated on this medium, and rooting was observed after 7 days. The rooting rate was calculated after 45 days. Figure 4 As shown, regenerated seedlings were obtained by dark culture at a culture temperature of 25±2°C. The schematic diagram of regenerated seedlings is shown in Figure 5 The rooting rate (%) = number of rooted buds / total number of inoculated differentiated seedlings × 100%, and the mortality rate (%) = number of dead buds / total number of inoculated differentiated seedlings × 100%. The results are shown in Table 4.

[0053] Table 4 Effects of different hormones on the rooting of Artemisia annua callus

[0054] As shown in Table 4, when treated with 6-BA, the rooting rate was 2.33% at a test concentration of 1 mg / L; NAA treatment failed to induce rooting, and the rooting rate was 0. As the IBA concentration increased, the rooting rate of Artemisia explants showed an increasing and then decreasing trend. At all tested concentrations, as the concentration increased, the rooting rate under GA3 treatment showed a trend of first increasing and then decreasing, and remained at a high level overall. Among the four hormones, GA3 treatment had the shortest rooting time, and 6-BA treatment had the longest rooting time. This study shows that there are significant differences in the rooting induction effects of different hormones on Artemisia callus tissue. Among them, GA3 showed a good rooting induction effect at a lower concentration, while NAA failed to effectively induce rooting. The rooting induction effect of IBA fluctuated with changes in concentration. (5) Determination of the acclimatization method for Artemisia annua tissue culture seedlings Because the temperature difference between indoor and outdoor temperatures in winter and summer significantly affects the acclimation of Artemisia scabra, the acclimation and transplanting process is carried out in late spring (mid-April to early May) or early autumn (late August to early September). During the acclimation of Artemisia scabra seedlings, the use of different mulches (such as pine needles and Mongolian oak leaves) significantly affects the microenvironment, soil physical and chemical properties, and seedling adaptability and growth performance. The following is an analysis of the differences after mulching: Pretreatment: Pine needles are prone to harboring nematodes, so they need to be decomposed until they are dark brown and have no pine oil smell; diseased oak leaves need to be removed to avoid the spread of anthrax; pretreatment should be sprayed with 5% potassium permanganate solution.

[0055] When the rooted seedlings grow to 3-4 cm in height, have 3-5 roots, and 3-4 leaves, they can be hardened. Place the rooted seedlings under natural light for 5 days, then remove the sealing film on the bottle cap and harden them for 2 days. During this period, use a spray bottle to spray the leaves to ensure that the leaves do not lose water to enhance the adaptability of the test tube seedlings to the outdoor environment. Then remove the seedlings from the culture bottle, wash the root culture medium, and then plant them in the substrate. The substrate formula is humus soil: vermiculite = 1:3. The schematic diagram of the acclimation of Artemisia argyi tissue culture seedlings is shown below. Figure 6 As shown in Figure 2, after 14 days of planting and cultivation, the seedling trays were moved to the natural environment. Figure 7 As shown, keep the soil moist and place it in a cool and ventilated place. The light is from weak to strong in the early stage. The above substrate seedlings are divided into 3 groups, which are covered with pine needles, covered with Mongolian oak fallen leaves, and not covered. The covering diagram is shown in Figure 8 The acclimatization was continued for 26 days and transplanted after acclimatization was completed. The effects of different coverings on the survival rate of tissue culture seedlings after transplanting are shown in Table 5.

[0056] Table 5 Effects of different covering materials on the survival rate of tissue culture seedlings

[0057] Among them, the plants after acclimation and transplantation of tissue culture seedlings for 2 years are as follows Figure 9 As shown, the red portion represents acclimated seedlings covered with pine needles, while the yellow portion represents acclimated seedlings covered with Mongolian oak. Plants covered with Mongolian oak leaf litter are generally stronger, primarily because Mongolian oak leaf litter provides moderate thermal insulation and improved air permeability (preventing high temperatures from suffocating the roots), promoting root growth, while also providing high safety and a stable survival rate. Pine needle mulch, on the other hand, is known for its excellent thermal insulation, which may have inhibited root growth, and thus plant growth.

[0058] Example 2 (1) Cultivation of sterile seedlings Same as Example 1.

[0059] (2) Preparation of culture medium: ① Callus culture medium: MS + 1.5 mg / L IBA + 5 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; ② Differentiation bud induction medium: MS + 4 mg / L 6-BA + 3 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; ③Rooting medium: 1 / 2 MS + 2 mg / L GA3 + 2 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; (3) Material processing: Material collection method: Select sterile seedlings of Artemisia annua, cut the stems and leaves as explant materials; peel off stem segments of about 0.5 to 1 cm, and inoculate them into primary culture medium for cultivation.

[0060] (4) Callus induction culture: Cut stems or leaves of about 0.5 cm-1 cm in size and inoculate them into primary culture medium. The culture temperature is 25±2℃, the light duration is 12 h / d, and the light intensity is 2000 lux to induce callus tissue.

[0061] (5) Differentiation induction culture: The primary cultured callus was inoculated into differentiation medium at 25±2°C, 10-12 hours per day, and 2000-3000 lux to induce the formation of differentiated shoots.

[0062] (6) Rooting induction culture: The differentiated buds were transferred to a rooting medium and cultured in the dark at a temperature of 25±2°C to induce rooted shoots.

[0063] (7) Rooted seedlings acclimatization and transplanting: Acclimation and transplanting: In late spring, when rooted seedlings reach 3-4 cm in height, have 3-5 roots, and 3-4 leaves, they are hardened under natural light for 5 days. Afterward, the bottle cap is sealed and hardened for 2 days. During this time, the leaves are sprayed with a watering can to ensure they retain moisture and enhance their adaptability to the outdoor environment. The seedlings are then removed from the culture bottle, the root culture medium is cleaned, and they are planted in a medium composed of humus soil and vermiculite in a ratio of 1:3. After 14 days of incubation, the seedling trays are transferred to a natural environment, where soil moisture is maintained and the seedlings are placed in a cool, well-ventilated area. Light levels are increased from low to high, and the seedlings are covered with fallen Mongolian oak leaves. Acclimation continues for 26 days, and transplanting is performed after acclimation is complete. The survival rate can exceed 90%.

[0064] Example 3 (1) Cultivation of sterile seedlings Same as Example 2.

[0065] (2) Preparation of culture medium: ① Callus culture medium: MS + 5 mg / L IBA + 1.5 mg / L NAA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; ② Differentiation bud induction medium: MS + 5 mg / L 6-BA + 3 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; ③Rooting medium: 1 / 2 MS + 0.5 mg / L GA3 + 0.5 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH adjusted to between 5.8-6.0; (3) Material processing: Material collection method: Select sterile seedlings of Artemisia annua, cut the stems and leaves as explant materials; peel off about 0.5 to 1 cm of stems or leaves, and inoculate them into primary culture medium for cultivation.

[0066] (4) Callus induction culture: Cut stems or leaves of about 0.5 cm to 1 cm in size and inoculate them into primary culture medium. The culture temperature is 25±2°C, the light duration is 12 h / d, and the light intensity is 2000 lux to induce callus tissue.

[0067] (5) Differentiation induction culture: The primary cultured callus was inoculated into differentiation medium at 25±2°C, 10-12 hours per day, and 2000-3000 lux to induce the formation of differentiated shoots.

[0068] (6) Rooting induction culture: The differentiated buds were transferred to a rooting medium and cultured in the dark at a temperature of 25±2°C to induce rooted shoots.

[0069] (7) Rooted seedlings acclimatization and transplanting: The acclimatization and transplanting process begins in late spring, when rooted seedlings reach 3-4 cm in height, have 3-5 roots, and 3-4 leaves. Harden the seedlings under natural light for 5 days, then remove the film from the bottle cap and harden them for 2 days. During this time, spray the leaves with a spray bottle to ensure they retain moisture and enhance their adaptability to the outdoor environment. The seedlings are then removed from the culture bottle, the root culture medium cleaned, and planted in a medium composed of humus soil and vermiculite in a ratio of 1:3. After 14 days of planting, the seedling trays are transferred to a natural environment, where soil moisture is maintained and placed in a cool, well-ventilated area. Light levels are increased from low to high, and the seedlings are covered with fallen Mongolian oak leaves. Acclimatization continues for 26 days. Transplantation is complete, and the survival rate can exceed 90%.

[0070] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for tissue culture and breeding of the Mongolian medicinal plant Artemisia annua, characterized in that: The tissue culture breeding method comprises the following steps: Selection of explants: Select sterile seedlings of Artemisia annua and cut the stems and leaves as explant materials; Callus induction culture: The explants were segmented and inoculated into primary culture medium to obtain callus tissue. The culture temperature was 25±2°C, the light duration was 12 hours / day, and the light intensity was 1800-2200 lux. Differentiation bud induction culture: inoculate the callus tissue into differentiation bud induction medium to induce differentiation buds. The culture temperature is 25±2℃, the light duration is 10-12h / d, and the light intensity is 2000-3000 lux. Rooting induction culture: When the differentiated buds are transferred to the rooting medium to obtain rooted shoots, they are cultured in the dark at a temperature of 25±2℃; Rooted seedling cultivation: the rooted seedlings are acclimated and transplanted to obtain surviving Artemisia annua tissue culture seedlings.

2. the Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, The sterile seedling cultivation comprises the following steps: removing white mucus from the surface of Artemisia annua seeds, sterilizing the seeds with 75% ethanol, rinsing with sterile water, and then sterilizing with 2% NaClO for 5 minutes, 10 minutes, 15 minutes, and 20 minutes, respectively; rinsing with sterile water, and then drying the surface of the seeds, and inoculating them into MS culture medium for cultivation; Preferably, removing the white mucus on the surface of the Artemisia annua seeds comprises: soaking the Artemisia annua seeds in tap water for 1 hour, rubbing and cleaning the soaked seeds with detergent, and rinsing with tap water until the detergent is completely removed.

3. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, The explant is segmented into 0.5-1 cm sections.

4. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, The primary culture medium consists of MS + IBA + NAA + sucrose + agar; Preferably, the mass concentration of IBA is 1-5 mg / L, the mass concentration of NAA is 1-5 mg / L, the mass concentration of sucrose is 30g / L, the mass concentration of agar is 3-10 g / L; Preferably, the pH value of the primary culture medium is 5.8-6.

0.

5. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, The differentiation bud induction medium consists of MS + 6-BA + IBA + sucrose + agar; Preferably, the mass concentration of 6-BA is 1-5 mg / L, the mass concentration of IBA is 1-5 mg / L, the mass concentration of sucrose is 30 g / L, and the mass concentration of agar is 3-10 g / L; Preferably, the pH value of the differentiation bud induction medium is 5.8-6.

0.

6. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, Rooting medium consisted of 1 / 2MS + GA3 + IBA + sucrose + agar; Preferably, the mass concentration of GA3 is 0.5-3 mg / L, the mass concentration of IBA is 0.5-3 mg / L, the mass concentration of sucrose is 30 g / L, and the mass concentration of agar is 3-10 g / L; Preferably, the pH value of the rooting medium is 5.8-6.

0.

7. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, In the rooted seedling cultivation, when the rooted seedling grows to 3 to 4 cm in height, has 3 to 5 roots and 3 to 4 leaves, it is acclimated.

8. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 1, is characterized in that, The acclimation comprises: hardening the rooted seedlings in the culture bottle under natural light for 3-8 days, then opening the sealing film of the culture bottle to harden the seedlings for 1-3 days, during which time water is sprayed on the leaves; taking out the seedlings from the culture bottle, cleaning the root culture medium, and then transplanting the seedlings into the substrate.

9. Mongolian medicine plant Artemisia annua tissue culture breeding method according to claim 8, is characterized in that, The matrix formula is humus soil: vermiculite = 1:

3.

10. The Mongolian medicine plant Artemisia annua tissue culture and breeding method according to claim 8, wherein The time for transplanting the seedlings into the substrate is 40 days.

Images