A tissue culture method for Artemisia broadleaf

Abstract

This invention discloses a tissue culture method for Artemisia broadleaf, belonging to the field of plant tissue culture technology, including the following steps: S1: Selecting tender stem segments of Artemisia broadleaf as explants and disinfecting the explants; S2: Inoculating the tender stem segments treated in S1 into an induction proliferation medium for proliferation culture until lateral buds grow to 5-6 cm; S3: Cutting off the lateral buds grown in S2 and inoculating them into a rooting medium for rooting culture; the rooting medium contains activated carbon; S4: After rooting culture in S3, placing the seedlings under natural light to harden them off, washing the culture medium from the roots, and transplanting them into a substrate to survive; This invention provides a technical reference for the efficient utilization of Artemisia broadleaf resources, large-scale propagation, large-scale planting, and uniform seedling quality; it enables the acquisition of a large number of high-quality seedlings with uniform quality in a short period of time, providing technical support for the protection of Artemisia broadleaf germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and specifically to a method for tissue culture of broadleaf Artemisia. Background Technology

[0002] *Artemisia stolonifera*, also known as Jiuniu grass, is a perennial herb belonging to the genus *Artemisia* in the family Compositae. It is a closely related species to *Artemisia argyi* Lévl. et Van., and its dried leaves are used as mugwort in some regions. It flowers in autumn, growing on the tips of branches. The whole plant is harvested in summer and autumn, while seedlings are harvested in early spring and summer, sifted for fluffing or dried for use. It is bitter and warm in nature, and slightly toxic. It has the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain, and is mainly used for hematemesis, epistaxis, metrorrhagia, menstrual disorders due to cold, and infertility due to uterine cold. As a high-quality moxibustion material, *Artemisia stolonifera* has high economic value and research prospects. However, due to the difficulty of seed germination, wild germplasm resources are currently scarce nationwide. Therefore, it is necessary to protect its resources. In recent years, the development of *Artemisia stolonifera* resources has reached a certain scale. A certain scale of *Artemisia stolonifera* GAP planting base has been established in Jiangxi Province, my country, which has played a certain role in promoting the protection of wild *Artemisia stolonifera* resources. Currently, broadleaf artemisia is mainly propagated through rhizomes. However, since the offspring of rhizome propagation exhibit phenotypic segregation, it is not conducive to the protection of high-quality germplasm resources. Therefore, a rapid tissue propagation system for broadleaf artemisia has been established to provide guarantees and technical support for germplasm resource protection and the breeding of superior varieties.

[0003] Currently, due to the scarcity of *Artemisia latifolia* parent plants, difficulties in seed germination, and the time-consuming nature of rhizome propagation, resulting in severe phenotypic segregation in offspring and limited propagation numbers, the demand for seedlings is far from being met. Therefore, tissue culture rapid propagation technology is urgently needed for seedling expansion. Literature review revealed no reports on tissue culture rapid propagation technology for *Artemisia latifolia*. Experiments show that currently published methods for rapid tissue propagation of *Artemisia* species are not suitable for *Artemisia latifolia*, easily leading to drawbacks such as high explant contamination rates, explant browning, yellowing of the culture medium, slow bud growth, long rooting time, and poor seedling quality.

[0004] To overcome the lack of tissue culture technology for rapid propagation of broadleaf artemisia and to solve the problem of difficult seed germination, this study aims to construct an efficient in vitro rapid propagation technology system for broadleaf artemisia, effectively shortening the propagation cycle, reducing production and planting costs, and providing technical support for the introduction, domestication, seedling propagation, germplasm resource protection, and industrial development of broadleaf artemisia. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art and to provide a tissue culture method for Artemisia argyi.

[0006] The technical solution of the present invention is as follows:

[0007] A method for tissue culture of Artemisia broadleaf includes the following steps:

[0008] S1: Select tender stem segments of broadleaf Artemisia as explants and disinfect the explants;

[0009] S2: The tender stem segments treated with S1 were inoculated into the induction and proliferation medium for proliferation culture until lateral buds grew to 5-6 cm.

[0010] S3: Cut off the lateral buds that grow from S2 and inoculate them into the rooting medium for rooting culture;

[0011] The rooting medium contains activated carbon;

[0012] S4: After rooting culture in S3, the seedlings are hardened off under natural light, the culture medium is washed off the roots, and they are transplanted into the substrate to survive.

[0013] In a preferred embodiment of the present invention, the disinfection process in step S1 specifically includes:

[0014] Soak the explants in a dish soap solution, rinse them repeatedly with clean water, and then dry them. Then, disinfect them in a clean bench: first, disinfect with ethanol and rinse with sterile water; then disinfect with HgCl2 and rinse with sterile water; continue with NaClO disinfection and rinse with sterile water; finally, disinfect with plant growth regulator and rinse repeatedly with sterile water.

[0015] In a preferred embodiment of the present invention, step S2, the proliferation culture specifically includes:

[0016] First, culture in complete darkness at 25-28℃ for 3 days, then place in a light intensity of 1900-2300 lx and a culture temperature of 25-27℃, with 8-12 hours of light per day. After 2 weeks of culture, lateral buds can be induced. Continue to proliferate and culture in the same medium until the lateral buds grow to 5-6 cm.

[0017] As a preferred embodiment of the present invention, in step S2, the induction proliferation medium is: MS, 0.05-0.2 mg / L 6-BA, 0.05-0.1 mg / L KT, 30 g / L sucrose, 4 g / L agar, with a pH value of 5.7-5.9.

[0018] As a preferred embodiment of the present invention, in step S3, the rooting culture specifically involves: first, culturing in complete darkness at 25°C for 2 days, and then culturing under conditions of light intensity of 2500-3000 lx and culture temperature of 25°C until rooting occurs, with 12 hours of light per day.

[0019] As a preferred embodiment of the present invention, in step S3, the rooting medium is: 1 / 2 MS, 0.1 mg / L NAA, 3 g / L activated carbon, 4 g / L agar, with a pH value of 5.7-5.9.

[0020] As a preferred embodiment of the present invention, in step S4, the matrix includes nutrient soil and perlite in a mass ratio of 9:1.

[0021] The beneficial effects of this invention are: rapid induction speed, short time consumption, high induction rate, robust shoot growth, high rooting rate, well-developed root system, high transplant survival rate, and low production cost. Furthermore, appropriate hormone levels and the addition of a small amount of activated carbon during the rooting culture of *Artemisia argyi* can significantly improve the survival rate of transplanted seedlings, maintaining robust seedling growth and uniform quality. This invention requires only about 60 days from explant induction and proliferation to transplant survival, solving the problem of seed germination difficulties, effectively shortening the propagation cycle, and reducing production and planting costs. It provides technical support for the introduction and domestication of *Artemisia argyi*, seedling propagation, germplasm resource protection, and industrial development. Attached Figure Description

[0022] Figure 1 Inducing axillary bud formation by inoculating stem segments;

[0023] Figure 2 For axillary bud proliferation;

[0024] Figure 3 To induce the formation of adventitious roots in young shoots;

[0025] Figure 4 To promote root growth in culture medium supplemented with activated carbon;

[0026] Figure 5 Propagation and rooting in culture medium without activated carbon;

[0027] Figure 6 To prepare seedlings by partially opening the bottle;

[0028] Figure 7 This shows the growth of broadleaf Artemisia seedlings after transplanting. Detailed Implementation

[0029] This invention discloses a tissue culture technique for Artemisia broadleaf, comprising the following steps:

[0030] S1. Explant disinfection: Select tender stem segments of Artemisia argyi as explants, soak them in a solution of detergent and water, rinse repeatedly with clean water, dry them, and then disinfect them in a clean bench: ethanol disinfection for 30 seconds, rinse twice with sterile water; HgCl2 disinfection, rinse with sterile water; NaClO disinfection, rinse with sterile water; plant growth regulator disinfection, rinse repeatedly with sterile water, dry them, cut them into 2.0 cm long stem segments, and inoculate them onto the induction proliferation medium (refer to...). Figure 1).

[0031] S2. Induction and Proliferation Culture: The young stem segments obtained in step S1 are inoculated into the induction and proliferation culture medium for proliferation culture. First, they are cultured in complete darkness at 25-28℃ for 3 days. Then, they are placed under 12 hours of light per day, with a light intensity of 1900 lx, and a culture temperature of 25℃. Lateral buds can be induced in about 2 weeks. Proliferation culture can continue in this medium until the lateral buds grow to about 5-6 cm (reference). Figure 2 The induction proliferation medium consisted of MS, 0.05 mg / L 6-BA, 0.05 mg / L KT, 30 g / L sucrose, and 4 g / L agar, with a pH of 5.9.

[0032] S3. Rooting Culture: Cut off the lateral buds that have grown to 5-6 cm in step S2 and inoculate them onto rooting medium for rooting culture. After transferring to the medium, first incubate in complete darkness at 25℃ for 2 days, then place them under conditions of 12 hours of light per day, light intensity of 3000 lx, and culture temperature of 25℃ until rooting occurs (reference). Figure 4 The rooting medium consisted of 1 / 2 MS, 0.1 mg / L NAA, 3 g / L activated carbon, and 4 g / L agar, with a pH of 5.9.

[0033] S4. Hardening off and transplanting: After the broadleaf artemisia has rooted in the bottle for 30 days, place it under natural light for 5 days to harden off (reference). Figure 6 After washing away the culture medium from the roots, transplant the seedlings into a substrate composed of potting soil and perlite (9:1 by weight) to ensure survival (see reference). Figure 7 ).

[0034] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0035] Example 1

[0036] On June 21, 2023, tender seedlings of *Artemisia broadleaf* were collected from the *Artemisia broadleaf* planting base in Zhangshu, Jiangxi Province, and their rapid propagation through tissue culture was carried out. The main steps are as follows:

[0037] Take plump terminal buds or lateral buds from healthy broadleaf Artemisia mother plants as explants for tissue culture; generally, take explants on a sunny morning 5 days after the weather has cleared.

[0038] The explants were brought back to the laboratory, rinsed with running water to remove debris from the surface of the explants, rinsed with running water for 1 hour, then soaked in dish soap water for 30 minutes and rinsed clean with running water.

[0039] After rinsing, the explants were placed in a laminar flow hood for further sterilization: They were soaked in 75% alcohol for 1 minute, rinsed three times with sterile water, then immersed in 8% NaClO for 5 minutes, rinsed three times with sterile water, and finally submerged in 0.1% HgCl2 for 8 minutes, rinsed four times with sterile water. The explants were then placed on sterilized filter paper to absorb surface moisture. Using a scalpel, the explants with buds were cut into approximately 2cm segments in a sterile culture dish for later use.

[0040] Induction and proliferation culture: Cut stem segments with buds were inoculated into an induction medium consisting of MS, 1.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 4 g / L agar, with a pH of 5.9. After inoculation into the induction medium, the cells were cultured in complete darkness at 26°C for 3 days. Then, they were transferred to light conditions with 16 hours of light per day at a light intensity of 1200 lx and a culture temperature of 26°C for 21 days. At this time, the lateral buds elongated to 5 cm, and the proliferation rate was 1.84.

[0041] Rooting culture: The lateral buds, approximately 5 cm long, obtained in the previous step were cut and inoculated onto rooting medium for rooting culture. After inoculation, they were first cultured in complete darkness at 26℃ for 3 days, and then placed under conditions of 15 hours of light per day, a light intensity of 2200 lx, and a culture temperature of 26℃ for 7 days until rooting occurred, with a rooting rate of 100%. The rooting medium consisted of 1 / 2 MS, 1.0 mg / L NAA, 3 g / L activated carbon, and 4 g / L agar, with a pH of 5.9.

[0042] Hardening off and transplanting: After the broadleaf wormwood has been rooted in the bottle for 14 days, it is placed under natural light for 5 days to harden off. After washing off the culture medium from the roots, it is transplanted into a substrate made of peat moss and perlite in a ratio of 8:2. The survival rate of transplanting is 90%.

[0043] Example 2

[0044] On May 15, 2024, healthy, disease-free lateral branches with buds from the current year's growth were selected from the Jiuniu Grass planting base of Jiangxi Shennong Herbal Planting Garden. These explants were soaked in a dish soap solution for 30 minutes, then rinsed with tap water for 1 hour, and dried before use. In a clean bench, the explants were soaked in 75% ethanol for 30 seconds, rinsed three times with sterile water, disinfected with 8% NaClO for 5 minutes, rinsed twice with sterile water, disinfected with 0.1% HgCl2 solution for 10 minutes, rinsed three times with sterile water, disinfected with 0.6% plant growth regulator for 10 minutes, rinsed eight times with sterile water, dried, and cut into 1.5cm long stem segments before inoculating onto the induction medium.

[0045] Induced proliferation culture: The clustered shoots obtained in the above steps were transferred to a proliferation medium for proliferation culture. After inoculation, they were first cultured in complete darkness at 26℃ until a lateral bud sprouted from the leaf axil. Then, they were placed under conditions of 16 hours of light per day, light intensity of 1200 lx, and culture temperature of 26℃ for 21 days. After that, the lateral buds could elongate to 5 cm, and the proliferation rate was 1.77. The proliferation medium consisted of MS, 0.05 mg / L KT, 0.1 mg / L 6-BA, 30 g / L sucrose, 4 g / L agar, and a pH of 5.9.

[0046] Rooting culture: The clustered shoots, approximately 6 cm in height, obtained above were cut and inoculated onto rooting medium for rooting culture. After inoculation, they were first cultured in complete darkness at 26℃ for 6 days, and then placed under conditions of 15 hours of light per day, a light intensity of 2200 lx, and a culture temperature of 26℃ for 7 days until rooting occurred, with a rooting rate of 100%. The rooting medium consisted of MS, 1.0 mg / L NAA, 4 g / L activated carbon, 30 g / L sucrose, 4 g / L agar, and a pH of 5.9.

[0047] Transplanting and hardening off: After the tissue culture seedlings of broadleaf Artemisia have rooted in the bottle for 3 weeks, they are placed under natural light for 7 days with the bottle half-open. After washing the roots, they are transplanted into a substrate of peat moss and perlite (1:1) with added wood fiber. The survival rate is 100%.

[0048] Comparative Example 1

[0049] Unlike Example 2, no activated carbon was added to the rooting medium. Otherwise, it was the same as Example 2, and the survival rate was 88%.

[0050] In summary, appropriate hormone levels and the addition of a small amount of activated carbon during the rooting culture of broadleaf artemisia can significantly improve the survival rate of transplanted seedlings, maintain robust seedling growth, and ensure uniform quality. This invention solves the problem of seed germination difficulties, effectively shortens the propagation cycle, reduces production and planting costs, and provides technical support for the introduction, domestication, seedling propagation, germplasm resource protection, and industrial development of broadleaf artemisia.

[0051] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for tissue culture of Artemisia broadleaf, characterized in that, Includes the following steps: S1: Select tender stem segments of broadleaf Artemisia as explants and disinfect the explants; S2: The tender stem segments treated with S1 were inoculated into the induction and proliferation medium for proliferation culture until lateral buds grew to 5-6 cm. S3: Cut off the lateral buds that grow from S2 and inoculate them into the rooting medium for rooting culture; The rooting medium contains activated carbon; S4: After rooting culture in S3, the seedlings are hardened off under natural light, the culture medium is washed off the roots, and they are transplanted into the substrate to survive. In step S2, the induction proliferation medium is: MS, 0.05-0.2 mg / L 6-BA, 0.05-0.1 mg / L KT, 30 g / L sucrose, 4 g / L agar, with a pH of 5.7-5.9; In step S3, the rooting medium consists of 1 / 2 MS, 0.1-1.0 mg / L NAA, 3 g / L activated carbon, 4 g / L agar, and a pH of 5.

9. In step S4, the substrate includes nutrient soil and perlite in a mass ratio of 1-9:1-3.

2. The method for tissue culture of Artemisia broadleaf according to claim 1, characterized in that, In step S1, the disinfection process specifically includes: Soak the explants in a dish soap solution, rinse them repeatedly with clean water, and then dry them. Then, disinfect them in a clean bench: first, disinfect with ethanol and rinse with sterile water; then disinfect with HgCl2 and rinse with sterile water; continue with NaClO disinfection and rinse with sterile water; finally, disinfect with plant growth regulator and rinse repeatedly with sterile water.

3. The method for tissue culture of Artemisia broadleaf according to claim 1, characterized in that... In step S2, the proliferation culture specifically includes: First, culture in complete darkness at 25-28℃ for 2-4 days, then place in a light intensity of 1900-2300 lx and a culture temperature of 25℃-27℃, with 8-12 hours of light per day. After 2 weeks of culture, lateral buds can be induced. Continue to culture in the same medium until the lateral buds grow to 5-6 cm.

4. The method for tissue culture of Artemisia broadleaf according to claim 1, characterized in that, In step S3, the rooting culture is specifically as follows: first, it is cultured in complete darkness at 25℃ for 2 days, and then placed under conditions of light intensity of 2500-3000 lx and culture temperature of 25℃ until rooting occurs, with light exposure of 10-12 h per day.

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