A method for efficient tissue culture and rapid propagation of Artemisia selengensis

Abstract

The invention discloses a method for efficient tissue culture and rapid propagation of Artemisia selengensis, comprising: using current year stem segments of Artemisia selengensis as explants, disinfecting the surface of the segments, cutting the segments and sequentially inoculating the segments into an axillary bud germination induction medium, an adventitious bud induction medium, and an adventitious bud elongation and rooting synchronization medium to obtain regenerated plants, and then disinfecting the surfaces and transplanting the plants; the axillary bud germination induction medium is WPM+0.2-1.0 mg / L ZT+3.0% (w / v) sucrose+0.6% (w / v) agar, with a pH of 5.8; the adventitious bud induction medium is WPM+0.5-2.0 mg / L ZT+0.1-0.5 mg / L 6-BA+3.0% (w / v) sucrose+0.6% (w / v) agar, with a pH of 5.8; the adventitious bud elongation and rooting synchronization medium is WPM+0.5-2.0 mg / L ZT+0.1-0.5 mg / L 6-BA+3.0% (w / v) sucrose+0.6% (w / v) agar, with a pH of 5.8; and the adventitious bud elongation and rooting synchronization medium is WPM+0.5-2.0 mg / L KT + 2.0% (w / v) sucrose + 0.8% (w / v) agar, pH = 5.8.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and in particular to a method for efficient tissue culture and rapid propagation of Artemisia argyi. Background Art

[0002] Mugwort (Artemisia argyi), a typical representative of Traditional Chinese Medicine (TCM), is a perennial herb of the genus Artemisia in the Asteraceae family. It is a major TCM ingredient that combines medicine, food, and health products. Modern research shows that mugwort leaves are rich in mineral elements, flavonoids, and volatile oils, and have the effects of regulating qi and blood, dispelling cold and dampness, warming the meridians and activating the collaterals, and having antibacterial and anti-inflammatory, anti-cancer, and immunomodulatory effects. In my country, mugwort is primarily produced in Henan, Hubei, Hunan, Anhui, Shandong, and Hebei. In recent years, with the rapid development of the TCM health service industry, market demand for the mugwort industry has been increasing. The unique role of mugwort and its products in preventing disease, beautifying skin, and other aspects has been widely recognized by all sectors of society, and is particularly favored by merchants from Japan and South Korea. The international market has a large demand and a promising future.

[0003] Nanyang City, Henan Province, is one of China's major production areas for Wan Ai (Chinese mugwort) and the nation's largest supplier of raw mugwort. Nanyang City, known as "Wan," is the name for the mugwort produced there. Wan Ai has a long history and has garnered significant attention for its high medicinal value, diverse varieties, rich ingredients, and high yield. In recent years, my country has implemented a series of strategies to support the development of the Traditional Chinese Medicine (TCM) industry, integrating traditional Chinese and Western medicine and preserving TCM's syndrome-based treatment principles. This has provided significant opportunities for the development of the Wan Ai industry chain. However, due to the abundance of wild Wan Ai resources and numerous cultivated varieties, some currently promoted varieties exhibit inconsistent plant morphology, yield, and quality. These characteristics gradually disappear after long-term cultivation. Therefore, there is an urgent need to select and breed high-quality Wan Ai varieties and develop a supporting breeding technology system. Plant tissue culture rapid propagation technology allows for the rapid production of large quantities of high-quality, genetically consistent Wan Ai seedlings in a short period of time. This technology can, to a certain extent, meet the demand for high-quality Wan Ai seedlings for large-scale cultivation. Furthermore, this technology can enable the genetic improvement of these high-quality Wan Ai varieties and the large-scale production of their secondary metabolites. Currently, research on Wan Ai focuses primarily on its chemical composition, biological activity, and cultivation techniques. While there have been reports on using tissue culture techniques to induce adventitious buds from mugwort, there is still a lack of research on in vitro rapid propagation and cultivar cultivation of Wan Ai using tissue culture.

[0004] Chinese patent application publication number CN116114598A discloses a method for rapid tissue culture propagation of mugwort and its application. The method comprises cultivating young shoots from mugwort explants in a bud induction medium; cultivating clustered shoots from the budding explants in a proliferation and expansion medium; cultivating rooting clusters from the clustered shoots in a rooting medium; and hardening the rooted, virus-free seedlings in floating trays. Aiming to increase the proliferation rate of mugwort clustered shoots and obtain virus-free mugwort seedlings, the method optimizes the hormone combination in the mugwort tissue culture rapid propagation medium, resulting in a proliferation rate exceeding 10 times per cycle and over 90% of the shoots growing into virus-free seedlings suitable for transplanting, allowing for the rapid production of large numbers of mugwort seedlings in a short period of time. However, the method disclosed in Chinese patent application publication number CN116114598A is not universally applicable due to factors such as the origin, variety, explant material, and collection period, which directly affect the plant's regeneration ability. Therefore, in view of the many problems existing in the production of Wan Ai mentioned above, it is urgent to develop a method for efficient tissue culture and rapid propagation of Wan Ai to meet the needs of rapid propagation, variety improvement and secondary metabolite production of excellent Wan Ai strains. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for efficient tissue culture and rapid propagation of Artemisia selengensis by using plant tissue culture technology.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] A method for efficient tissue culture and rapid propagation of Artemisia selengensis, comprising the following steps:

[0008] (1) Explant collection and surface disinfection: Use the stem segments of Artemisia selengensis that were grown in the current year as explants, and cut them into segments of appropriate size after surface disinfection.

[0009] (2) Axillary bud germination induction culture: The above-mentioned segments were inoculated into an axillary bud germination induction medium for axillary bud germination induction culture; wherein the axillary bud germination induction medium is: WPM + 0.2-1.0 mg / L ZT + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8;

[0010] (3) Adventitious bud induction culture: Axillary buds were separated and transferred to adventitious bud induction medium for adventitious bud induction culture; wherein the adventitious bud induction medium was: WPM + 0.5-2.0 mg / L ZT + 0.1-0.5 mg / L 6-BA + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8;

[0011] (4) Synchronous culture of adventitious bud elongation and rooting: The induced adventitious buds are separated and transferred to a synchronous culture medium for adventitious bud elongation and rooting to obtain complete regenerated plants of Ai wan; wherein the synchronous culture medium for adventitious bud elongation and rooting is: WPM + 0.5-2.0 mg / L KT + 2.0% (w / v) sucrose

[0012] + 0.8% (w / v) agar, pH = 5.8;

[0013] (5) Acclimation and transplantation of regenerated plants: After the surface of the obtained regenerated plants is disinfected, they are directly transplanted for seedling cultivation to obtain robust Wan Ai tissue culture regenerated plants.

[0014] Preferably, the current year's stem segment is a non-lignified current year's stem segment or a semi-lignified current year's stem segment.

[0015] Preferably, the appropriately sized segments are segments with one axillary bud.

[0016] Preferably, in (1), the surface disinfection comprises the following steps: the stem segments are divided into beakers and rinsed under running water for 10 to 20 minutes, during which 1 to 2 ml of hand soap is added 1 to 2 times; thereafter, they are wiped with 70% to 75% (v / v) ethanol, and then placed on a sterile operating table, rinsed with sterile water 4 to 6 times, and then surface disinfected with 70% to 75% (v / v) ethanol for 30 to 45 seconds, 0.1% (w / v) mercuric chloride solution for 1 to 4 minutes, and then rinsed with sterile water 6 to 8 times; and the surface moisture of the stem segments is absorbed with sterile filter paper.

[0017] Preferably, the axillary bud germination induction medium is: WPM+0.5mg / L ZT+3.0% (w / v) sucrose

[0018] + 0.6% (w / v) agar, pH = 5.8.

[0019] Preferably, the germination induction culture time of axillary buds is 15 to 20 days; the induction culture time of adventitious buds is 20 to 25 days; and the synchronous culture time of elongation and rooting of adventitious buds is 15 to 20 days.

[0020] Preferably, the adventitious bud induction medium is: WPM+1.0 mg / L ZT+0.3 mg / L 6-BA+3.0% (w / v) sucrose+0.6% (w / v) agar, pH=5.8.

[0021] Preferably, the adventitious bud elongation and rooting synchronization medium is: WPM+1.0 mg / L KT+2.0% (w / v) sucrose+0.8% (w / v) agar, pH=5.8.

[0022] Preferably, the acclimation and transplanting of the regenerated plants comprises the following steps: separating the obtained regenerated plants from the adventitious bud elongation and rooting synchronization culture medium, rinsing with running water and disinfecting with potassium permanganate solution, and then directly transplanting them into a seedling tray filled with a nutrient matrix for seedling cultivation; wherein the nutrient matrix is ​​garden soil or a mixture of garden soil, vermiculite and nutrient soil, and the nutrient matrix is ​​thoroughly watered and sprayed with a 0.75% by mass fraction of carbendazim solution and WPM macroelements one day before use.

[0023] Preferably, the culture conditions for the axillary bud germination induction culture, adventitious bud induction culture, and adventitious bud elongation and rooting synchronous culture are: temperature of 25±2°C, light intensity of 50-60 μmol m -2 s -1 The photoperiod was 14 / 10 h (light / dark); the conditions for the seedling culture were: temperature 22-28 ° C, light intensity 40-50 μmol m -2 s -1 , humidity 70-85%, water every 5-7 days, and spray 0.75% carbendazim solution every 7-10 days during the first 20 days of planting.

[0024] The advantages of the present invention are:

[0025] The present invention provides a method for efficient tissue culture and rapid propagation of Artemisia argyi. Specifically, the method uses young stem segments of the current year's fine Artemisia argyi strain as explants. Based on the induction of axillary bud germination, the method directly induces clustered bud regeneration to achieve rapid propagation of the fine Artemisia argyi strain. The method has the following outstanding advantages: First, the method uses young stem segments of the current year's fine Artemisia argyi strain with axillary buds as explants. The axillary buds are used to directly induce adventitious buds during the propagation process, thereby maintaining the germplasm characteristics of the mother plant. Second, the method achieves simultaneous cultivation of adventitious bud elongation and rooting, greatly reducing the complexity of propagation and shortening the propagation cycle. Rapid and efficient propagation of fine Artemisia argyi individual plants can be achieved within two months. Third, the method has a high regeneration efficiency, with a clustered bud induction rate of up to 93.7%, an average of up to 3.9 adventitious buds per explant, a rooting rate of up to 98.6%, and a transplant survival rate of regenerated plants of up to 100%. The method provides important technical support for the rapid propagation and genetic improvement of fine Artemisia argyi strains.

[0026] The present invention is of great significance to the research related to large-scale reproduction, variety improvement and secondary metabolite production of excellent Wan Ai strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The axillary buds of Artemisia wanensis germinated and inoculated on the adventitious bud induction medium after being separated in Example 2 of the present invention;

[0028] Figure 2The induction of adventitious buds of Artemisia selengensis in Example 2 of the present invention;

[0029] Figure 3 The complete regenerated plant obtained after synchronous cultivation of elongation and rooting of the adventitious buds of Artemisia annua in Example 2 of the present invention;

[0030] Figure 4 This is a rooting diagram of the complete regenerated plant obtained in Example 2 of the present invention;

[0031] Figure 5 This is the complete regenerated plant of Artemisia selengensis transplanted into the cultivation medium in Example 2 of the present invention. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0034] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0035] Example 1

[0036] A method for efficient tissue culture and rapid propagation of Wan Ai, the specific steps are as follows:

[0037] (1) Select a well-growing strain of Artemisia selengensis and cut the current year's unlignified branches as explants; remove the leaves and cut into 4-5 cm stem segments; divide the segments into 500 ml beakers and rinse under running water for 10 minutes, adding 1 ml of hand sanitizer twice during the process. Then wipe the stem segments with 70% ethanol; then place the stem segments on a sterile operating table, rinse with sterile water 4 times, and then disinfect the surface with 70% ethanol for 45 seconds, 0.1% (w / v) mercuric chloride solution for 1 minute, and then rinse with sterile water 6 times. Use sterile filter paper to absorb the surface moisture of the stem segments and cut them into segments of appropriate size with one axillary bud for later use.

[0038] (2) The stem segments cut in step (1) were planted vertically in axillary bud germination induction medium at a temperature of 25 ± 2 °C and a light intensity of 50 to 60 μmol m -2 s -1Axillary bud germination was induced in a constant temperature incubator with a 14 / 10 h light / dark photoperiod. Axillary buds began to germinate after 6 days of light culture. After 20 days of culture, the stem contamination rate was 16.3% and the axillary bud germination rate was 83.4%. The axillary bud germination induction medium consisted of WPM + 0.2 mg / L ZT + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0039] (3) The axillary buds germinated in step (2) were separated and transferred to adventitious bud induction medium at a temperature of 25 ± 2 °C and a light intensity of 50 to 60 μmol m -2 s -1 Adventitious bud induction was performed in a constant temperature incubator with a 14 / 10 h light / dark photoperiod. After 25 days of culture, the induction rate of axillary buds was 81.5%, with an average of 2.5 buds produced per explant. The adventitious bud induction medium consisted of WPM + 0.5 mg / L ZT + 0.1 mg / L 6-BA + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0040] (4) The adventitious buds induced in step (3) were separated and transferred to the adventitious bud elongation and rooting synchronization medium at a temperature of 25±2°C and a light intensity of 50-60 μmol m -2 s -1 Adventitious bud elongation and rooting were carried out in a constant temperature culture room with a photoperiod of 14 / 10 h (light / dark). After 20 days of light culture, regenerated plants with an average plant height of 3.2 cm, a strong root system and 3 to 5 complete leaves were obtained. The induction rate of adventitious roots was 77.4%, with an average of 2.3 adventitious roots per explant and an average adventitious root length of 3.2 cm. The synchronous culture medium for adventitious bud elongation and rooting was: WPM + 0.5 mg / L KT + 2.0% (w / v) sucrose + 0.8% (w / v) agar, pH = 5.8.

[0041] (5) After the regenerated plants were separated from the adventitious bud elongation and rooting synchronization medium, they were rinsed with running water and disinfected with 0.2% potassium permanganate solution, and then directly transplanted into a plastic seedling tray filled with nutrient medium at a temperature of 22-28°C and a light intensity of 40-50 μmol m -2 s -1, and seedling cultivation was carried out in a greenhouse with a humidity of 70-85%. The nutrient matrix was composed of garden soil, and the nutrient matrix was watered thoroughly and sprayed with a 0.75% carbendazim solution and WPM macroelements the day before use. During the cultivation process, water was applied every 5-7 days, and a 0.75% carbendazim solution was sprayed every 7-10 days for the first 20 days after transplanting. After 30 days of cultivation, the survival rate of Wan Ai plants after transplantation was as high as 81.2%.

[0042] Example 2

[0043] A method for efficient tissue culture and rapid propagation of Wan Ai, the specific steps are as follows:

[0044] (1) Select a well-growing strain of Artemisia selengensis and cut the semi-lignified branches of the current year as explants; after removing the leaves, cut into 4-5 cm stem segments; divide the segments into 500 ml beakers and rinse under running water for 20 minutes, adding 2 ml of hand sanitizer once during the process. Then wipe the stem segments with 75% ethanol; then place the stem segments on a sterile operating table, rinse with sterile water 4 times, and then disinfect the surface with 75% ethanol for 30 seconds, 0.1% (w / v) mercuric chloride solution for 2 minutes, and then rinse with sterile water 6 times. Use sterile filter paper to absorb the surface moisture of the stem segments, cut into segments of appropriate size with one axillary bud, and set aside.

[0045] (2) The stem segments cut in step (1) were planted vertically in axillary bud germination induction medium at a temperature of 25 ± 2 °C and a light intensity of 50 to 60 μmol m -2 s -1 Axillary bud germination was induced in a constant temperature incubator with a 14 / 10 h light / dark photoperiod. Axillary buds began to germinate after 4 days of light culture. After 15 days of culture, the stem contamination rate was 0.5% and the axillary bud germination rate was 98.7%. The axillary bud germination induction medium consisted of WPM + 0.5 mg / L ZT + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0046] (3) Separate the axillary buds germinated in step (2) and transfer them to adventitious bud induction medium ( Figure 1 ), at a temperature of 25 ± 2 °C and a light intensity of 50–60 μmol m -2 s -1 Adventitious buds were induced in a constant temperature culture room with a photoperiod of 14 / 10 h (light / dark). After 25 days of culture, the induction rate of adventitious buds was 93.7%, and an average of 3.9 adventitious buds were produced per explant ( Figure 2 The adventitious bud induction medium was WPM + 1.0 mg / L ZT + 0.3 mg / L 6-BA + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0047] (4) The adventitious buds induced in step (3) were separated and transferred to the adventitious bud elongation and rooting synchronization medium at a temperature of 25±2°C and a light intensity of 50-60 μmol m -2 s -1 The elongation and rooting of adventitious buds were carried out in a constant temperature culture room with a photoperiod of 14 / 10h (light / dark). After 15 days of light culture, regenerated plants with an average plant height of 4.3cm, a strong root system and 3 to 5 complete leaves were obtained ( Figure 3 The adventitious root induction rate was as high as 98.6%, with an average of 4.1 adventitious roots per explant and an average adventitious root length of 4.8 cm ( Figure 4 The adventitious bud elongation and rooting synchronization medium is: WPM + 1.0 mg / L KT + 2.0% (w / v) sucrose + 0.8% (w / v) agar, pH = 5.8.

[0048] (5) After the regenerated plants were separated from the adventitious bud elongation and rooting synchronization medium, they were rinsed with running water and disinfected with a 0.2% potassium permanganate solution. They were then directly transplanted into a plastic seedling tray containing a nutrient medium at a temperature of 22-28°C and a light intensity of 40-50 μmol m -2 s -1 , the seedlings were cultured in a greenhouse with a humidity of 70-85% (such as Figure 5 The composition and volume ratio of the nutrient medium is garden soil: vermiculite: Pin's nutrient soil = 2:1:2. The nutrient medium should be thoroughly watered and sprayed with a 0.75% carbendazim solution and WPM macronutrients the day before use. During the cultivation process, water should be applied every 5-7 days. For the first 20 days after planting, spray with a 0.75% carbendazim solution every 7-10 days. After 30 days of cultivation, the survival rate of Wan Ai plants after transplantation reached 100%.

[0049] Example 3

[0050] A method for efficient tissue culture and rapid propagation of Wan Ai, the specific steps are as follows:

[0051] (1) Select a well-growing strain of Artemisia selengensis and cut the semi-lignified branches of the current year as explants; remove the leaves and cut into 4-5 cm stem segments; divide the segments into 500 ml beakers and rinse under running water for 20 minutes, adding 2 ml of hand sanitizer once during the process. Then wipe the stem segments with 75% ethanol; then place the stem segments on a sterile operating table, rinse with sterile water 6 times, and then disinfect the surface with 75% ethanol for 30 seconds, 0.1% (w / v) mercuric chloride solution for 4 minutes, and then rinse with sterile water 8 times. Use sterile filter paper to absorb the surface moisture of the stem segments and cut them into segments of appropriate size with one axillary bud for later use.

[0052] (2) The stem segments cut in step (1) were planted vertically in axillary bud germination induction medium at a temperature of 25 ± 2 °C and a light intensity of 50 to 60 μmol m -2 s -1 Axillary bud germination was induced in a constant temperature incubator with a 14 / 10 h light / dark photoperiod. Axillary buds began to germinate after 8 days of light culture. After 20 days of culture, the stem contamination rate was 0.0% and the axillary bud germination rate was 91.1%. The axillary bud germination induction medium consisted of WPM + 1.0 mg / L ZT + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0053] (3) The axillary buds germinated in step (2) were separated and transferred to adventitious bud induction medium at a temperature of 25 ± 2 °C and a light intensity of 50 to 60 μmol m -2 s -1 Adventitious bud induction was performed in a constant temperature incubator with a 14 / 10 h light / dark photoperiod. After 25 days of culture, the induction rate of adventitious buds was 95.3%, with an average of 3.2 buds produced per explant. The adventitious bud induction medium consisted of WPM + 2.0 mg / L ZT + 0.5 mg / L 6-BA + 3.0% (w / v) sucrose + 0.6% (w / v) agar, pH = 5.8.

[0054] (4) The adventitious buds induced in step (3) were separated and transferred to the adventitious bud elongation and rooting synchronization medium at a temperature of 25±2°C and a light intensity of 50-60 μmol m -2 s -1 Adventitious bud elongation and rooting were carried out in a constant temperature culture room with a photoperiod of 14 / 10 h (light / dark). After 15 days of light culture, regenerated plants with an average plant height of 4.1 cm, a strong root system and 3 to 5 complete leaves were obtained. The adventitious root induction rate was as high as 94.4%, with an average of 3.8 adventitious roots per explant and an average adventitious root length of 3.9 cm. The synchronous culture medium for adventitious bud elongation and rooting was: WPM + 2.0 mg / L KT + 2.0% (w / v) sucrose + 0.8% (w / v) agar, pH = 5.8.

[0055] (5) After the regenerated plants were separated from the adventitious bud elongation and rooting synchronization medium, they were rinsed with running water and disinfected with a 0.2% potassium permanganate solution. They were then directly transplanted into a plastic seedling tray containing a nutrient medium at a temperature of 22-28°C and a light intensity of 40-50 μmol m -2 s -1, and seedling cultivation was carried out in a greenhouse with a humidity of 70-85%. The composition and volume ratio of the nutrient matrix was garden soil: vermiculite: Pinshi nutrient soil = 2:1:1, and the nutrient matrix was watered thoroughly and sprayed with a 0.75% carbendazim solution and WPM macroelements the day before use. During the cultivation process, water was applied every 5-7 days, and a 0.75% carbendazim solution was sprayed every 7-10 days for the first 20 days after transplanting. After 30 days of cultivation, the survival rate of Wan Ai plants after transplanting was as high as 97.8%.

[0056] Comparative Example 1

[0057] In this comparative example, the in vitro regeneration of Artemisia vulgaris stem segments was studied using the mugwort regeneration method described in Chinese patent application publication number CN116114598A. A 4-cm-long stem segment bearing axillary buds closest to the meristematic zone of the Artemisia vulgaris was rinsed under tap water for 30 minutes, placed in a laminar flow hood, and disinfected with 75% ethanol for 1 minute, followed by 0.1% (w / v) mercuric chloride for 7 minutes, and then rinsed four times with sterile water to obtain a sterile explant. The sterile explants were cut into stem segments with axillary buds of 1 to 2 cm in length and inserted into a culture room containing MS+1.0 mg / L IAA+1.0 mg / L 6-BA+0.1 mg / L GA3 with a light intensity of 2000 to 3000 Lx, a temperature of 23±1°C, and a light intensity of 16 to 18 h / d. After 10 days of culture, it was found that 90% of the stem segment explants died due to the long disinfection time. For the stem segments that did not die, the buds were damaged due to the long disinfection time and no sprouting had occurred.

[0058] Comparative Example 2

[0059] In this comparative example, the surface-sterilized Artemisia selengensis stem segments in Example 2 were inoculated in the method mentioned in the Chinese patent application document with publication number CN116114598A to induce the culture of adventitious buds and adventitious roots of Artemisia selengensis stem segments. The stem segments obtained after disinfection in Example 2 were inoculated in the optimal culture medium (MS+1.5mg / L IAA+2.0mg / L 6-BA+0.1mg / L GA3) mentioned in the patent application document with publication number CN116114598A in a culture room with a light intensity of 2000-3000Lx, a temperature of 23±1°C, and a light intensity of 16-18h / d to induce the culture of adventitious buds. After 30 days of culture, axillary buds germinated from the stem segments of Artemisia selengensis. These stem segments were then inoculated with the proliferation and expansion medium MS + 1.0 mg / L 6-BA described in patent application publication number CN116114598A for bud induction. After 15 to 20 days of culture, each explant produced an average of only 2.1 adventitious buds, with an average bud height of 1.8 cm. This indicates that this medium is not ideal for bud proliferation and elongation in Artemisia selengensis.

[0060] The vigorous clusters of shoots growing in MS + 1.0 mg / L 6-BA were separated into individual plants and cultured in 1 / 2 MS + 0.3 mg / L IAA (the rooting medium described in patent application publication number CN116114598A) for rooting. After 10-15 days of culture, the adventitious root induction rate was only 73.4%, with an average of 2.2 adventitious roots per explant, and an average root length of 0.9 cm.

[0061] In summary, the mugwort method mentioned in the patent application document with publication number CN116114598A is not suitable for the in vitro culture of Artemisia selengensis.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for rapid propagation of Artemisia selengensis tissue culture, characterized in that: The following steps are involved: (1) Explant collection and surface disinfection: The stem segments of Artemisia selengensis that were grown in the current year were used as explants, and after surface disinfection, they were cut into segments of appropriate size. The surface disinfection comprises the following steps: the stem segments were divided into beakers and rinsed under running water for 10 to 20 minutes, during which 1 to 2 ml of hand sanitizer was added 1 to 2 times; thereafter, they were wiped with 70% to 75% v / v ethanol, and then placed in a sterile operating table, rinsed with sterile water 4 to 6 times, and then surface disinfected with 70% to 75% v / v ethanol for 30 to 45 seconds, disinfected with 0.1% w / v mercuric chloride solution for 1 to 4 minutes, and then rinsed with sterile water 6 to 8 times; the surface moisture of the stem segments was dried with sterile filter paper; (2) Axillary bud germination induction culture: The above-mentioned segments were inoculated into an axillary bud germination induction medium for axillary bud germination induction culture; wherein the axillary bud germination induction medium is: WPM + 0.2-1.0 mg / L ZT + 3.0% w / v sucrose + 0.6% w / v agar, pH = 5.8; (3) Adventitious bud induction culture: Axillary buds were separated and transferred to adventitious bud induction medium for adventitious bud induction culture; wherein, the adventitious bud induction medium was: WPM + 0.5-2.0 mg / L ZT + 0.1-0.5 mg / L 6-BA + 3.0% w / v sucrose + 0.6% w / v agar, pH = 5.8; (4) Synchronous culture of adventitious bud elongation and rooting: The induced adventitious buds were separated and transferred to a synchronous culture medium for adventitious bud elongation and rooting to obtain complete regenerated plants of Artemisia wanfangensis; wherein the synchronous culture medium for adventitious bud elongation and rooting is: WPM + 0.5-2.0 mg / L KT + 2.0% w / v sucrose + 0.8% w / v agar, pH = 5.8; (5) Acclimation and transplantation of regenerated plants: After the surface of the obtained regenerated plants is disinfected, they are directly transplanted for seedling cultivation to obtain robust Wan Ai tissue culture regenerated plants.

2. The method for rapid propagation of Artemisia selengensis tissue culture according to claim 1, wherein: The current year stem segment is a current year non-lignified stem segment or a current year semi-lignified stem segment.

3. The method for rapid propagation of Artemisia selengensis tissue culture according to claim 1, wherein: The appropriate size of the cutting is the cutting with one axillary bud.

4. The method for rapid propagation of Artemisia selengensis tissue culture according to claim 1, wherein: The axillary bud germination induction medium is: WPM+0.5mg / L ZT+3.0% w / v sucrose+0.6% w / v agar, pH=5.

8.

5. The method for rapid propagation of Artemisia selengensis tissue culture according to claim 1, wherein: The germination induction culture time of axillary buds is 15 to 20 days; the induction culture time of adventitious buds is 20 to 25 days; the synchronous culture time of elongation and rooting of adventitious buds is 15 to 20 days.

6. The method for rapid propagation of Artemisia annua tissue culture according to claim 1, wherein: The adventitious bud induction medium is: WPM+1.0 mg / L ZT+0.3 mg / L 6-BA+3.0% w / v sucrose+0.6% w / v agar, pH=5.

8.

7. The method for rapid propagation of Artemisia annua tissue culture according to claim 1, wherein: The adventitious bud elongation and rooting synchronization culture medium is: WPM+1.0 mg / L KT+2.0% w / v sucrose+0.8% w / v agar, pH=5.

8.

8. The method for rapid propagation of Artemisia annua tissue culture according to claim 1, wherein: The acclimation and transplanting of the regenerated plants comprises the following steps: separating the obtained regenerated plants from the adventitious bud elongation and rooting synchronization culture medium, washing with running water and disinfecting with a potassium permanganate solution, and then directly transplanting them into a seedling tray filled with a nutrient matrix for seedling cultivation; wherein the nutrient matrix is ​​garden soil or a mixture of garden soil, vermiculite and nutrient soil, and the nutrient matrix is ​​irrigated with water and sprayed with a carbendazim solution with a mass fraction of 0.75% and WPM macroelements one day before use.

9. The method for rapid propagation of Artemisia annua tissue culture according to any one of claims 1 to 8, characterized in that: The culture conditions for the axillary bud germination induction culture, adventitious bud induction culture, adventitious bud elongation and rooting synchronous culture are: temperature 25±2°C, light intensity 50-60 μmol m -2 s -1 The photoperiod was 14 h light / 10 h dark; the conditions for the seedling culture were: temperature 22-28°C, light intensity 40-50 μmol m -2 s -1 , humidity 70-85%, water every 5-7 days, and spray 0.75% carbendazim solution every 7-10 days during the first 20 days of planting.

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