Tissue culture method of artemisia williamsii

Abstract

The invention discloses a tissue culture method of Artemisia glandulifera, and relates to the technical field of plant tissue culture, and the tissue culture method comprises the following steps: S1, selecting a tender stem segment of Artemisia glandulifera as an explant, and carrying out disinfection treatment on the explant; s2, inoculating the tender stems treated in the step S1 into an induced proliferation culture medium for proliferation culture until lateral buds grow to 5-6 cm; s3, cutting off the lateral buds grown in the S2, and inoculating the lateral buds into a rooting culture medium for rooting culture; the rooting culture medium contains activated carbon; s4, after rooting culture in the step S3, performing seedling hardening under natural illumination, cleaning a root culture medium, and transplanting the seedlings into a matrix for survival; according to the method, technical reference is provided for efficient utilization, large-area propagation, large-scale planting and uniform seedling quality of artemisia williamsii resources; a large batch of high-quality seedlings with uniform quality can be obtained in a short time, and a technical guarantee is provided for germplasm resource protection of Artemisia latifolia.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a tissue culture method of Artemisia latifolia. Background Art

[0002] Artemisia stolonifera, also known as Jiuniucao, is a perennial herbaceous plant of the genus Artemisia in the family Compositae. It is a close species of Artemisia argyi Lévl. et Van., and its dried leaves are used as wormwood in some areas. It blooms in autumn and grows on branches. The whole grass is collected in summer and autumn, and the seedlings are collected in spring and early summer. They are sieved or dried for use. It is bitter, warm, and slightly toxic. It has the effects of warming the meridians to stop bleeding, dispersing cold and relieving pain. It is mainly used for hematemesis, epistaxis, metrorrhagia, cold menstruation, and uterine cold infertility. As a high-quality moxibustion material resource, Artemisia stolonifera has high economic value and research prospects. Due to its difficulty in seed germination, wild germplasm resources are currently scarce throughout the country. 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. At present, broad-leaved Artemisia is mainly propagated through rhizomes. Since the offspring of rhizome propagation have trait separation, it is not conducive to the protection of high-quality germplasm resources. Therefore, a broad-leaved Artemisia tissue rapid propagation system has been established to provide guarantee and technical support for germplasm resource protection and breeding of improved varieties.

[0003] At present, due to the scarcity of maternal resources of Artemisia latifolia, seed germination is difficult, rhizome propagation takes a long time, offspring traits are severely separated, and the number of reproduction is limited, which is far from meeting the market promotion needs. Tissue culture rapid propagation technology is urgently needed for seedling expansion. Literature search found that there are no relevant reports on tissue culture rapid propagation technology of Artemisia latifolia. Experiments show that the currently published tissue rapid propagation methods for Artemisia plants are not suitable for Artemisia latifolia, and are prone to high explant contamination rate, explant browning, yellowing of culture medium, slow bud growth, long rooting time, and poor seedling quality.

[0004] In order to overcome the lack of tissue culture rapid propagation technology of Artemisia latifolia and solve the problem of seed germination difficulties, the purpose of this study is to construct an efficient in vitro rapid propagation technology system of Artemisia latifolia, effectively shorten the breeding cycle, reduce production and planting costs, and provide technical guarantees for the introduction and domestication, seedling breeding, germplasm resource protection and industrial development of Artemisia latifolia. Summary of the invention

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

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

[0007] A tissue culture method of Artemisia latifolia comprises the following steps:

[0008] S1: Select young stem segments of Artemisia latifolia as explants and sterilize the explants;

[0009] S2: The young stem segments treated by S1 are inoculated into the induction proliferation medium for proliferation culture until the lateral buds grow to 5-6 cm;

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

[0011] The rooting medium contains activated carbon;

[0012] S4: After rooting culture in S3, place the seedlings under natural light for hardening, wash the root culture medium, and transplant them into the substrate for survival.

[0013] As a preferred embodiment of the present invention, in step S1, the disinfection treatment is specifically:

[0014] Soak the explants in a detergent solution, rinse repeatedly with clean water, wipe dry, and 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 bacterial disinfectant and rinse repeatedly with sterile water.

[0015] As a preferred embodiment of the present invention, in step S2, the proliferation culture is specifically:

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

[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, and the pH value is 5.7-5.9.

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

[0019] As a preferred embodiment of the present invention, in step S3, the rooting medium is: 1 / 2MS, 0.1 mg / L NAA, 3 g / L activated carbon, 4 g / L agar, and the pH value is 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 the present invention are as follows: the present invention has the advantages of fast induction speed, short time consumption, high induction rate, robust bud growth, high rooting rate, developed root system, high transplant survival rate and low production cost. In addition, the hormone level in the rooting culture process of broad-leaved Artemisia and the addition of a small amount of activated carbon can significantly improve the survival rate of broad-leaved Artemisia transplanting and hardening, keep the seedlings strong and uniform in quality. The present invention only takes about 60 days from explant induction and proliferation to transplanting and hardening survival, solves the difficulty of seed germination, effectively shortens the breeding cycle, reduces the production and planting cost, and provides technical guarantee for the introduction and domestication, seedling breeding, germplasm resource protection and industrial development of broad-leaved Artemisia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Inoculate the stem segments to induce axillary bud formation;

[0023] Figure 2 For axillary bud proliferation;

[0024] Figure 3 To induce adventitious roots to form in young shoots;

[0025] Figure 4 To add activated carbon culture medium for root proliferation;

[0026] Figure 5 To multiply and root the culture medium without activated carbon;

[0027] Figure 6 To refine the seedlings for half-opened bottles;

[0028] Figure 7 This shows the growth status of Artemisia latifolia tissue culture seedlings after transplanting. DETAILED DESCRIPTION

[0029] The present invention discloses a tissue culture technology of Artemisia latifolia, comprising the following steps:

[0030] S1. Explant disinfection: Select young stem segments of Artemisia latifolia as explants, soak them in a detergent-water solution, rinse them repeatedly with clean water, wipe them dry, and disinfect them in a clean bench: disinfect with ethanol for 30 seconds, rinse them twice with sterile water; disinfect with HgCl2, rinse them with sterile water; disinfect with NaClO, rinse them with sterile water; disinfect with bacterial culture, rinse them repeatedly with sterile water, wipe them dry, cut them into 2.0 cm long stem segments, and inoculate them on the induction proliferation medium (reference Figure 1).

[0031] S2, induction proliferation culture: the young stem segments obtained in step S1 are inoculated into the induction proliferation culture medium for proliferation culture, first cultured in full darkness at 25-28°C for 3 days, then cultured under light conditions of 12 hours per day, with a light intensity of 1900 lx and a culture temperature of 25°C, and induced to form lateral buds in about 2 weeks, and the proliferation culture can be continued in the culture medium until the lateral buds grow to about 5-6 cm (reference Figure 2 ). The induction proliferation medium was: MS, 0.05 mg / L 6-BA, 0.05 mg / L KT, 30 g / L sucrose, 4 g / L agar, pH 5.9;

[0032] S3, rooting culture: cut off the lateral buds grown to 5-6 cm in step S2, and inoculate them on the rooting medium for rooting culture. After transferring to the culture medium, culture them in the dark at 25°C for 2 days, and then culture them under the conditions of 12 hours of light per day, 3000 lx of light intensity, and 25°C of culture temperature until roots are formed (refer to Figure 4 ); rooting medium: 1 / 2MS, 0.1mg / L NAA, 3g / L activated carbon, 4g / L agar, pH 5.9.

[0033] S4. Hardening and transplanting: After Artemisia latifolia has taken root in the bottle for 30 days, place it under natural light for 5 days to harden the seedlings (refer to Figure 6 ), wash the root culture medium, and transplant it to a matrix composed of nutrient soil and perlite (mass ratio 9:1) for survival (reference Figure 7 ).

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0035] Example 1

[0036] On June 21, 2023, we collected young seedlings of Artemisia latifolia from the Artemisia latifolia planting base in Zhangshu, Jiangxi Province, and carried out a tissue culture and rapid propagation experiment. The main steps are as follows:

[0037] From the robust Artemisia latifolia mother plant, branches with full terminal buds or lateral buds are collected as tissue culture explants; generally, a sunny morning after 5 days of clear weather is selected to collect the explants;

[0038] Bring the explants back to the laboratory, rinse them with running water to clean the debris on the surface of the explants, continue to rinse with running water for 1 hour, soak them in detergent water for 30 minutes, and then rinse them with running water;

[0039] The rinsed explants were placed in a clean bench for subsequent disinfection steps: soaked in 75% alcohol for 1 minute, rinsed with sterile water 3 times, taken out and immersed in 8% NaClO for 5 minutes, rinsed with sterile water 3 times, immersed in 0.1% HgCl2 for 8 minutes, rinsed with sterile water 4 times, and then placed on sterilized filter paper to absorb surface moisture. Use a scalpel to cut the stem explants with buds into stem segments of about 2 cm in a sterile culture dish for later use;

[0040] Induced proliferation culture: The cut stem segments with buds were inoculated into an induction medium, which contained MS, 1.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, 4 g / L agar, and a pH of 5.9. After inoculation into the induction medium, the buds were cultured in the dark at 26°C for 3 days, and then switched to light conditions for 16 hours per day, with a light intensity of 1200 lx and a culture temperature of 26°C. After 21 days of culture, the lateral buds could elongate to 5 cm, and the proliferation rate was 1.84.

[0041] Rooting culture: Cut the lateral buds of about 5 cm obtained in the previous step and inoculate them on the rooting medium for rooting culture. After inoculation, first culture them in the dark at 26°C for 3 days, then place them in the light for 15 hours a day, the light intensity is 2200lx, and the culture temperature is 26°C. Culture for 7 days to root, and the rooting rate is 100%. The rooting medium is: 1 / 2MS, 1.0mg / L NAA, 3g / L activated carbon, 4g / L agar, pH value is 5.9;

[0042] Hardening and transplanting: After the broad-leaved Artemisia has taken root in the bottle for 14 days, place it under natural light for 5 days, wash the root culture medium, and transplant it into a matrix mixed with peat soil and perlite in a ratio of 8:2. The survival rate of transplanting is 90%.

[0043] Example 2

[0044] On May 15, 2024, disease-free and healthy plants with buds and side branches were selected from the Jiuniucao planting base of Shennong Herbal Plantation in Jiangxi Province as explants, soaked in a detergent solution for 30 minutes, then rinsed with tap water for 1 hour, and dried for use. Soaked in 75% ethanol by volume for 30 seconds in a clean bench, rinsed with sterile water three times, disinfected with 8% NaClO by volume for 5 minutes, rinsed with sterile water twice, disinfected with 0.1% HgCl2 solution by volume for 10 minutes, rinsed with sterile water three times, and then disinfected with 0.6% plant bacteria for 10 minutes, and then washed with sterile water 8 times, dried and cut into stem segments with a length of 1.5 cm and inoculated on the induction medium.

[0045] Induced proliferation culture: The clustered buds obtained in the above steps are transferred to a proliferation culture medium for proliferation culture. After inoculation, they are first cultured in total darkness at 26°C until a lateral bud sprouts from the leaf axil. Then, they are cultured under the conditions of 16 hours of light per day, 1200 lx of light intensity, and 26°C of culture temperature. After 21 days of culture, the lateral buds can be extended to 5 cm, and the proliferation rate is 1.77. The proliferation culture medium is: MS, 0.05 mg / L KT, 0.1 mg / L 6-BA, 30 g / L sucrose, 4 g / L agar, and the pH value is 5.9.

[0046] Rooting culture: The obtained buds with a height of about 6 cm are cut and inoculated on a rooting medium for rooting culture. After inoculation, they are first cultured in total darkness at 26°C for 6 days, and then placed under light for 15 hours a day, with a light intensity of 2200 lx and a culture temperature of 26°C for 7 days to root, and the rooting rate is 100%. The rooting medium is: MS, 1.0 mg / L NAA, 4 g / L activated carbon, 30 g / L sucrose, 4 g / L agar, and the pH value is 5.9.

[0047] Transplantation and seedling hardening: After the broad-leaved Artemisia tissue culture seedlings have taken root in the bottle for 3 weeks, they are placed in a half-open bottle under natural light for 7 days for seedling hardening. The roots are washed and transplanted to a matrix of peat soil and perlite (1:1) with added wood fiber. The survival rate is 100%.

[0048] Comparative Example 1

[0049] The difference from Example 2 is that no activated carbon is added to the rooting medium. The rest is the same as Example 2, and the survival rate is 88%.

[0050] In summary, it can be seen that the appropriate hormone level during the rooting culture of broad-leaved Artemisia and the addition of a small amount of activated carbon can significantly improve the survival rate of broad-leaved Artemisia transplanting and seedling hardening, keep the seedlings strong and uniform in quality. The present invention solves the difficulty of seed germination, effectively shortens the breeding cycle, reduces the production and planting costs, and provides technical guarantees for the introduction and domestication, seedling breeding, germplasm resource protection and industrial development of broad-leaved Artemisia.

[0051] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A tissue culture method for Artemisia latifolia, characterized in that: The following steps are involved: S1: Select young stem segments of Artemisia latifolia as explants and sterilize the explants; S2: The young stem segments treated by S1 are inoculated into the induction proliferation medium for proliferation culture until the lateral buds grow to 5-6 cm; S3: Cut off the lateral buds grown in S2 and inoculate them into rooting medium for rooting culture; The rooting medium contains activated carbon; S4: After rooting culture in S3, place the seedlings under natural light for hardening, wash the root culture medium, and transplant them into the substrate for survival.

2. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that: In step S1, the disinfection process is specifically as follows: Soak the explants in a detergent solution, rinse repeatedly with clean water, wipe dry, and 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 bacterial disinfectant and rinse repeatedly with sterile water.

3. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that In step S2, the proliferation culture is specifically as follows: First culture in total darkness at 25-28℃ for 2-4 days, then culture at a light intensity of 1900-2300lx, a culture temperature of 25℃-27℃, and 8-12h of light per day. After 2 weeks of culture, lateral buds can be induced to form. Continue to proliferate and culture in this culture medium until the lateral buds grow to 5-6cm.

4. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that: In step S2, the proliferation induction 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, and the pH value is 5.7-5.

9.

5. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that: In step S3, the rooting culture is specifically as follows: firstly culture in total darkness at 25°C for 2 days, then culture under conditions of a light intensity of 2500-3000lx and a culture temperature of 25°C until rooting, with light exposure of 10-12h per day.

6. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that: In step S3, the rooting medium is: 1 / 2MS, 0.1-1.0 mg / L NAA, 3 g / L activated carbon, 4 g / L agar, and the pH value is 5.

9.

7. The tissue culture method of Artemisia latifolia according to claim 1, characterized in that: In step S4, the matrix includes nutrient soil and perlite in a mass ratio of 1-9:1-3.

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