Atractylodes macrocephala hairy root induction method and application
Through the Agrobacterium-mediated induction method of atractylodesia, the problems of reduced atractylodesia resources and high transplantation thresholds were solved, and the effective induction and cultivation of atractylodesia-like hairy roots were achieved, laying the foundation for the sustainable use of atractylodesia resources.
Patent Information
- Application Number
- CN202510182499.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art lacks effective methods to induce and cultivate hairy roots of Atractylodes, resulting in a decrease in Atractylodes resources and a high threshold for planting.
By subculturing the uncertain buds of Atractylodes tissue culture seedlings, and infecting Atractylodes root seedlings with Agrobacterium infestation solution, and then culturing them in proliferation medium in the light, the hairy roots of Atractylodes were successfully induced and cultivated.
The induction and culture of hairy roots of Atractylodesia is realized, providing the basis for the large-scale production of active ingredients such as Atractylodesia ketone and Atractylodesia lactone, and solving the problem of reduced Atractylodesia resources.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant cultivation and transgenic technology, and in particular relates to a method for inducing hairy roots of Atractylodes macrocephala and its application. Background Art
[0002] Atractylodes macrocephala Koidz is a perennial herbaceous plant of the genus Atractylodes in the Asteraceae family. Its dried rhizome is used as medicine, which has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. It is mainly used to treat spleen deficiency, poor appetite, abdominal distension and diarrhea, phlegm and fluid retention, dizziness and palpitations, edema, spontaneous sweating, and fetal movement disorders. Modern pharmacological studies have shown that Atractylodes has anti-tumor, gastric mucosal protection, antioxidant, anti-inflammatory, antibacterial, and lipid metabolism regulation. The main chemical components of Atractylodes include volatile oils, among which atractylone is one of the main components, with a content of up to 60%-70%, and together with other terpenes and other substances, it constitutes the various effects of Atractylodes. The problem of continuous cropping obstacles in the production and planting of Atractylodes is relatively serious. The soil that has been planted with Atractylodes usually needs to be replanted with Atractylodes after an interval of 3 to 5 years. The high planting threshold, reduced planting area, the impact of natural disasters, and increased market demand have led to a year-on-year reduction in Atractylodes resources.
[0003] The application of modern biotechnology to produce natural medicinal ingredients can effectively alleviate the pressure on medicinal plant resources and provide guarantees for the safety, effectiveness, stability and controllability of traditional Chinese medicine. As a bioreactor, hairy roots have the characteristics of short production cycle, easy scalability, no interference from the external environment, strong physiological metabolic activity, and good genetic stability. They have been widely used in in vitro culture of medicinal plants and research on effective ingredient extraction. In addition, the Agrobacterium-mediated genetic transformation and plant regeneration system of hairy roots of medicinal plants provides an important platform for studying the gene function and synthetic pathway analysis of Chinese medicinal materials. At present, genetic transformation of hairy roots has been applied to the cultivation process of many plants, but the cultivation of Atractylodes macrocephala itself is relatively difficult. There is no existing technology that provides a technical solution for the induction and cultivation of hairy roots of Atractylodes macrocephala mediated by Agrobacterium. In addition, its genetic transformation system still needs further research. Summary of the invention
[0004] In view of this, the object of the present invention is to provide a method and application of inducing hairy roots of Atractylodes macrocephala.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for inducing hairy roots of Atractylodes macrocephala, comprising the following steps:
[0007] S1. After subculturing the adventitious buds in the Atractylodes macrocephala tissue culture seedlings for 20 to 25 days, the rooting medium was used for 5 to 9 weeks to obtain the rooted Atractylodes macrocephala seedlings;
[0008] S2. After infecting the rooted Atractylodes macrocephala seedlings with the Agrobacterium infection solution, culturing the infected rooted Atractylodes macrocephala seedlings;
[0009] S3. placing the newly grown hairy roots of the infected Atractylodes macrocephala rooting seedlings in a proliferation medium and culturing them at 23-27°C in the dark;
[0010] The Agrobacterium is K599 Agrobacterium.
[0011] Preferably, the culture medium for subculture of the Atractylodes macrocephala tissue culture seedlings in S1 is MS+6-BA 2.0-3.0 mg / L+NAA 0.3-0.8 mg / L+sucrose 28-32 g / L+agar 5-10 g / L; the rooting medium is MS+0.3-0.8 mg / L NAA.
[0012] Preferably, the culture conditions in S2 are a temperature of 23-27° C., a light exposure time of 11-13 h / d, and a light intensity of 2000-3000 Lx.
[0013] Preferably, the method for preparing the Agrobacterium infection solution comprises the following steps:
[0014] The Agrobacterium was expanded and cultured to OD600 = 0.6-0.8, and then centrifuged at 3000-5000 rpm and resuspended in infection solution. The culture was allowed to stand at 24-28° C. in the dark for 2-3 hours to obtain the Agrobacterium infection solution.
[0015] Preferably, Agrobacterium is cultured in YEB liquid medium;
[0016] The YEB liquid culture medium contains 25-75 μg / mL of streptomycin.
[0017] Preferably, the infection solution is prepared by adding the following components to MS liquid culture medium: 8-12 mM MES, 8-12 mM MgCl2, 80-120 μM acetosyringone, pH = 5.8-6.0.
[0018] Preferably, the infection method of the Agrobacterium infection solution is to directly create wounds on the Atractylodes macrocephala tissue culture seedlings, and cover the wounded Atractylodes macrocephala tissue culture seedlings with the Agrobacterium infection solution;
[0019] The part of the Atractylodes macrocephala tissue culture seedlings infected with the Agrobacterium infection solution includes the stem segment or leaf axil of the tissue culture seedlings.
[0020] Preferably, 4 to 7 wounds are made on each part of the Atractylodes macrocephala tissue culture seedling using a syringe needle, and the amount of Agrobacterium infection solution used each time is 3-8 μl.
[0021] Preferably, the Atractylodes macrocephala hairy root proliferation culture medium is MS+sucrose 28-32 g / L+agar 5-10 g / L.
[0022] The present invention provides application of the Atractylodes macrocephala hairy root induction method in Atractylodes macrocephala hairy root induction, genetic transformation, gene function research and / or Atractylodes macrocephala metabolite production.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention provides a method for inducing hairy roots in a sterile system of Atractylodes macrocephala. The hairy roots of Atractylodes macrocephala are successfully cultivated by optimizing the steps of culturing and diluting Agrobacterium bacterial liquid, infection of explants, etc., laying a foundation for the large-scale production of active ingredients such as atractylodesone and atractylodes lactone using the hairy roots of Atractylodes macrocephala grown autonomously, and solving the problem of limited wild resources of Atractylodes macrocephala. The culturing method of the Atractylodes macrocephala tissue culture seedlings of the present invention can enable Atractylodes macrocephala to grow, differentiate and root rapidly, and the induction and cultivation of the hairy roots thereof have the advantages of convenient material collection, no seasonal restrictions, short culturing time, genetic stability, etc., and has a positive effect on the improvement of the transgenic technology of Atractylodes macrocephala, the mass cultivation and utilization of the hairy roots of Atractylodes macrocephala, and the synthesis and production of secondary metabolites. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 To grow strong and healthy Atractylodes macrocephala sterile seedlings;
[0026] Figure 2 These are suspected hairy roots induced from the stem segments of sterile seedlings of Atractylodes macrocephala;
[0027] Figure 3 This is the PCR validation diagram of Atractylodes macrocephala hairy roots;
[0028] Figure 4 This is a growth status diagram of Atractylodes macrocephala hairy roots after one month of proliferation culture in solid culture medium;
[0029] Figure 5 This is the successful induction of hairy roots of Atractylodes macrocephala. DETAILED DESCRIPTION
[0030] The present invention provides a method for inducing hairy roots of Atractylodes macrocephala, comprising the following steps:
[0031] S1. After subculturing the adventitious buds in the Atractylodes macrocephala tissue culture seedlings for 20 to 25 days, the rooting medium was used for 5 to 8 weeks to obtain the rooted Atractylodes macrocephala seedlings;
[0032] S2. After infecting the rooted Atractylodes macrocephala seedlings with the Agrobacterium infection solution, culturing the infected rooted Atractylodes macrocephala seedlings;
[0033] S3. placing the newly grown hairy roots of the infected Atractylodes macrocephala rooting seedlings in a proliferation medium and culturing them at 23-27°C in the dark;
[0034] The Agrobacterium is K599 Agrobacterium.
[0035] In the present invention, the conditions for the subculture in step S1 are to select the Atractylodes macrocephala tissue culture seedlings with good growth and no diseases and culture them in the culture medium.
[0036] The culture conditions are as follows: the temperature is preferably 23-27°C, more preferably 24-26°C, and further preferably 25°C; the light exposure time is preferably 11-13h / d, further preferably 11.5-12.5h / d, and further preferably 12h / d; the light intensity is preferably 2000-3000Lx, further preferably 2200-2800Lx, and further preferably 2500Lx; and the culture time is 20-25d, preferably 21-24d, and further preferably 23d.
[0037] The culture medium for subculture of the Atractylodes macrocephala tissue culture seedlings in step S1 is MS+6-BA+NAA+sucrose+agar;
[0038] The concentration of 6-BA in the culture medium is preferably 2.0 to 3.0 mg / L, more preferably 2.2 to 2.8 mg / L, and even more preferably 2.5 mg / L;
[0039] The concentration of NAA in the culture medium is preferably 0.3 to 0.8 mg / L, more preferably 0.4 to 0.7 mg / L, and even more preferably 0.5 mg / L;
[0040] The concentration of sucrose in the culture medium is preferably 28 to 32 g / L, more preferably 29 to 31 g / L, and even more preferably 30 g / L;
[0041] The concentration of agar in the culture medium is preferably 5 to 10 g / L, more preferably 6 to 9 g / L, and even more preferably 7.5 g / L.
[0042] The rooting medium is preferably MS+0.3-0.8 mg / L NAA, more preferably MS+0.4-0.7 mg / L NAA, and more preferably MS+0.5 mg / L NAA. The culture time of the rooting medium is 5 to 9 weeks, and more preferably 8 weeks. The culture conditions are: the temperature is preferably 23 to 27°C, more preferably 24 to 26°C, and more preferably 25°C. The light time is preferably 11 to 13 h / d, more preferably 11.5 to 12.5 h / d, and more preferably 12 h / d. The light intensity is preferably 2000 to 3000 Lx, more preferably 2200 to 2800 Lx, and more preferably 2500 Lx.
[0043] In the present invention, the method for preparing the Agrobacterium infection solution in step S2 preferably comprises the following steps:
[0044] Activate the strain → expand the culture → prepare Agrobacterium infection solution;
[0045] The activation of the strain preferably includes the following steps: inoculating the Agrobacterium strain on a YEB solid plate (containing 50 μg / mL of streptomycin), inverting and culturing to obtain a single Agrobacterium colony; picking a single Agrobacterium colony and preferably inoculating it in a YEB liquid culture medium, and adding streptomycin, the final concentration of streptomycin in the culture medium is preferably 25-75 μg / mL, more preferably 50 μg / mL, preferably culturing in a dark environment to obtain Agrobacterium; the temperature of the inverted culture is preferably 26-30°C, more preferably 27-29°C, and more preferably 28°C, the culture time is preferably 48-72h, more preferably 54-66h, and more preferably 60h; during the culture process, a rotary bed culture is preferably used, and the culture speed is preferably 180-220rpm, more preferably 190-210rpm, and more preferably 200rpm; the culture time is preferably 18-26h, more preferably 20-24h, and more preferably 22h to obtain an activated bacterial solution.
[0046] The expanded culture preferably includes the following steps: taking the activated bacterial solution to expand the culture in a liquid culture medium to obtain the activated bacterial solution. The volume of the activated bacterial solution is preferably 80 to 120 μL, more preferably 90 to 110 μL, and more preferably 100 μL; the volume of the liquid culture medium is 10 mL; the culture is preferably cultured to OD600 = 0.6 to 0.8, and more preferably OD600 = 0.8, to obtain the Agrobacterium bacterial solution.
[0047] The Agrobacterium infection solution is prepared by centrifuging the Agrobacterium culture obtained by expanded culture, resuspending it with the infection solution, and standing it in the dark at 24-28°C for 2-3 hours to obtain the Agrobacterium infection solution; the centrifugation speed is preferably 3000-5000rpm, more preferably 3500-4500rpm, and further preferably 4000rpm; the standing temperature is preferably 24-28°C, more preferably 24-26°C, and further preferably 25°C; the standing time is preferably 2-3h, more preferably 2.2-2.8h, and further preferably 2.5h.
[0048] In the present invention, the infection solution is a component having the following concentrations added to MS culture medium (PH=5.8): the concentration of MES is preferably 8-12 mM, more preferably 9-11 mM, and even more preferably 10 mM;
[0049] The concentration of MgCl2 is preferably 8 to 12 mM, more preferably 9 to 11 mM, and even more preferably 10 mM;
[0050] The concentration of acetosyringone is preferably 80 to 120 μM, more preferably 90 to 110 μM, and even more preferably 100 μM.
[0051] In the present invention, the infection method of the Agrobacterium infection solution in step S2 is preferably to cover the wounded Atractylodes macrocephala tissue culture seedlings with the Agrobacterium infection solution; the infected Atractylodes macrocephala tissue culture seedling parts preferably include tissue culture seedling stem segments, leaves or leaf axils, more preferably stem segments, and more preferably the lower part of the stem segments. Each part preferably causes 4 to 7 wounds, more preferably 6, and the amount of Agrobacterium infection solution used each time is preferably 3-8 μl, more preferably 4-6 μl, and more preferably 5 μl. The amount of wounds and the Agrobacterium infection solution will affect the induction rate of hairy roots.
[0052] In the present invention, the culture conditions in step S2 are:
[0053] The temperature is preferably 23-27°C, more preferably 24-26°C, and even more preferably 25°C;
[0054] The illumination time is preferably 11 to 13 h / d, more preferably 11.5 to 12.5 h / d, and even more preferably 12 h / d;
[0055] The light intensity is preferably 2000 to 3000 Lx, more preferably 2200 to 2800 Lx, and even more preferably 2500 Lx.
[0056] In the present invention, the specific step of placing the newly grown hairy roots of the rooted Atractylodes macrocephala seedlings in a proliferation medium in step S3 is preferably to wait until the hairy roots grow to about 1 cm, remove the tip about 0.5-0.8 cm in a clean bench and place in a proliferation medium for culture.
[0057] In the present invention, the proliferation medium is MS+sucrose 28-32 g / L+agar 5-10 g / L;
[0058] The concentration of sucrose is preferably 28 to 32 g / L, more preferably 29 to 31 g / L, and even more preferably 30 g / L;
[0059] The concentration of agar is preferably 5 to 10 g / L, more preferably 6 to 9 g / L, and even more preferably 7.5 g / L.
[0060] In the present invention, the culture condition in step S3 is dark culture in an environment with a temperature of preferably 23 to 27°C, more preferably 24 to 26°C, and even more preferably 25°C.
[0061] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0062] Sources:
[0063] The tissue culture seedlings of Atractylodes macrocephala were obtained from the Tissue Culture Room of the Chinese Medicine Resource Center of the China Academy of Chinese Medical Sciences;
[0064] YEB liquid culture medium was purchased from Beijing Kezhi Biotechnology Co., Ltd. (catalog number: LZ-PYJ3423);
[0065] K599 was purchased from Weidi Company (item number: AC1080S);
[0066] Streptomycin solution was purchased from Coolaber (Cat. No.: SL3840);
[0067] Acetosyringone was purchased from Coolaber (Cat. No. SL9513).
[0068] Experimental Example 1 Hairy root induction in the aseptic system of Atractylodes macrocephala
[0069] The adventitious buds of the well-growing and disease-free Atractylodes macrocephala tissue culture seedlings were subcultured in MS+6-BA 3.0+0.5g / L NAA sucrose 30g / L+agar 7.5g / L (pH 5.8-6.0) medium for subculture and proliferation; the conditions were: temperature 25±2°C, light 12h / d, light intensity 2500Lx, and then cultured in rooting medium for 8 weeks to obtain Atractylodes macrocephala rooted seedlings, the rooting medium being MS+0.5mg / L NAA. Figure 1 .
[0070] Agrobacterium rhizogenes K599 was inoculated on a YEB solid plate (containing 50 μg / mL of streptomycin), inverted and cultured at 28°C for 48 hours to obtain a single colony of Agrobacterium rhizogenes; a single colony of Agrobacterium rhizogenes was picked and inoculated into 2 mL YEB liquid culture medium, and streptomycin solution was added to a final concentration of 50 μg / mL, and cultured in the dark at 28°C and 200 rpm for 22 hours to obtain a culture solution; 100 μL of the culture solution was drawn into 10 mL YEB liquid culture medium for expansion culture until OD600 = 0.6-0.8 to obtain an Agrobacterium bacterial solution. The Agrobacterium bacterial solution was centrifuged at 3800 rpm and then resuspended with an infection solution [MES (final concentration of 10 mM), MgCl2 (final concentration of 10 mM), AS (acetosyringone) (final concentration of 100 μM), PH = 5.8-6.0 were added to 10 mL of MS liquid culture medium, and the solution was placed at 25-28°C in the dark for 2-3 hours to obtain an Agrobacterium infection solution.
[0071] In the clean bench, use a 1ml sterile syringe to select the stem, leaf or leaf axil of the rooting seedling of Atractylodes macrocephala, insert the needle to create a wound, and use the syringe to cover the wound with Agrobacterium infection solution. Each wound has about 5μl of Agrobacterium infection solution, so that the wound surface is in contact with the bacterial solution. Repeat the operation several times to create about 4-7 wounds on each stem. After infection, the culture conditions are as follows: temperature 25℃, light 12h / d, light intensity 2500Lx, and hairy roots will grow at the infected site after about 3-4 weeks. For details of the hairy roots, see Figure 2 .
[0072] When the hairy roots grow to about 1cm, remove the tip about 0.5cm in the clean bench and inoculate it into MS + sucrose 30g / L + agar 7.5g / L (pH5.8-6.0) culture medium. The culture conditions are: temperature 25±2℃, dark culture. After one week, it can grow to 2-3cm. Figure 4 This is a growth status diagram of Atractylodes macrocephala hairy roots after one month of proliferation culture in solid culture medium; Figure 5 This is the successful induction of hairy roots of Atractylodes macrocephala.
[0073] The identification of hairy roots of Atractylodes macrocephala can be carried out by identifying the rolB gene in the T-DNA region of the Ri plasmid of K599. The rolB gene is amplified by PCR on the extracted hairy root DNA. The positive control is the K599 bacterial solution; the negative control is the DNA of the roots of Atractylodes macrocephala tissue culture seedlings that have not been infected by K599 Agrobacterium. The PCR identification results show that the Agrobacterium T-DNA plasmid has been integrated into the hairy roots of Atractylodes macrocephala. For details, see Figure 3 .
[0074] Experimental Example 2 Effect of hormone combination on proliferation of Atractylodes macrocephala seedlings
[0075] Adventitious buds from the Atractylodes macrocephala tissue culture seedlings were inoculated in proliferation medium with different hormone ratios. After culturing for 4-6 weeks, the proliferation coefficient was calculated. The plant tissue culture proliferation coefficient = the number of effective seedlings formed by the bottle seedlings in one cycle / the number of inoculated seedlings. The results are shown in Table 1.
[0076] Table 1 Effects of different concentrations of hormone combinations on the proliferation of Atractylodes macrocephala tissue culture seedlings
[0077] Group Type of culture medium 6-BA(mg / L) NAA(mg / L) Proliferation coefficient A MS 2.0 0.5 2.1 B MS 2.0 0.8 2.8 C MS 3.0 0 2.5 D MS 3.0 0.5 3.4 E MS 3.0 0.8 3.1
[0078] From the results in Table 1, it can be seen that when the concentration of 6-BA is 3.0 mg / L and the concentration of NAA is 0.5 mg / L, the proliferation effect of Atractylodes macrocephala is the best. Therefore, a hormone ratio of 3.0 mg / L 6-BA and 0.5 mg / L NAA was selected and added to the MS culture medium.
[0079] Experimental Example 3 Effect of explants on the induction rate of hairy roots of Atractylodes macrocephala
[0080] In order to explore the effect of explant selection on the induction rate of hairy roots of Atractylodes macrocephala, leaves, stem segments and leaf axils of rooted Atractylodes macrocephala seedlings were selected as explants, and the OD prepared in Example 1 was used to 600 The explants were infected with Agrobacterium infection solution with a value of 0.6, and then the infected explants were induced to produce hairy roots under the conditions of 25°C and 12 hours of light. The induction rate of explants in different parts was studied, and the results are shown in Table 2.
[0081] Table 2 Effect of explants on the induction rate of hairy roots of Atractylodes macrocephala
[0082] Explant type Difficulty of sterilization Induction rate / % blade easy 0 stem segment Easier 30％ Leaf axil easy 10％
[0083] Table 2 shows that the induction rate of the explants at the stem segment position was the highest, at 30%, while the induction rate of the explants at the leaf axil position was lower, at 10%, and the explants at the leaf position were not successfully induced. Therefore, the stem segment was selected as the explant for subsequent experiments.
[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for inducing hairy roots of Atractylodes macrocephala, characterized in that: The steps include: S1. After subculturing the adventitious buds in the Atractylodes macrocephala tissue culture seedlings for 20 to 25 days, the rooting medium was used for 5 to 9 weeks to obtain the rooted Atractylodes macrocephala seedlings; S2. After infecting the rooted Atractylodes macrocephala seedlings with the Agrobacterium infection solution, culturing the infected rooted Atractylodes macrocephala seedlings; S3. Place the newly grown hairy roots of the infected Atractylodes macrocephala rooted seedlings in a proliferation medium and culture them in the dark at 23-27°C; The Agrobacterium is K599 Agrobacterium.
2. The method for inducing hairy roots of Atractylodes macrocephala according to claim 1, characterized in that: S1 The culture medium for subculture of the Atractylodes macrocephala tissue culture seedlings is MS+6-BA 2.0-3.0 mg / L+NAA 0.3-0.8 mg / L+sucrose 28-32 g / L+agar 5-10 g / L; the rooting medium is MS+0.3-0.8 mg / L NAA.
3. The method for inducing hairy roots of Atractylodes macrocephala according to claim 1, characterized in that: The culture conditions of S2 are as follows: temperature 23-27°C, light exposure time 11-13h / d, and light intensity 2000-3000Lx.
4. The method for inducing hairy roots of Atractylodes macrocephala according to claim 1, characterized in that: The preparation method of the Agrobacterium infection solution comprises the following steps: The Agrobacterium was expanded and cultured to OD600 = 0.6-0.8, and then centrifuged at 3000-5000 rpm and resuspended in infection solution. The culture was allowed to stand at 24-28° C. in the dark for 2-3 hours to obtain the Agrobacterium infection solution.
5. The method for inducing hairy roots of Atractylodes macrocephala according to claim 4, characterized in that: Agrobacterium was cultured in YEB liquid medium; The YEB liquid culture medium contains 25-75 μg / mL of streptomycin.
6. The method for inducing hairy roots of Atractylodes macrocephala according to claim 4, characterized in that: The infection solution is prepared by adding the following components in MS liquid culture medium: 8-12 mM MES, 8-12 mM MgCl2, 80-120 μM acetosyringone, and adjusting the pH to 5.8-6.
0.
7. The method for inducing hairy roots of Atractylodes macrocephala according to claim 1, characterized in that: The infection method of the Agrobacterium infection liquid is to directly make wounds on the Atractylodes macrocephala tissue culture seedlings, and then cover the wounded Atractylodes macrocephala tissue culture seedlings with the Agrobacterium infection liquid; The part of the Atractylodes macrocephala tissue culture seedlings infected by the Agrobacterium infection solution includes the stem segment or leaf axil of the tissue culture seedlings.
8. The method for inducing hairy roots of Atractylodes macrocephala according to claim 7, characterized in that: Each part of the Atractylodes macrocephala tissue culture seedling is infected 4 to 7 times, and the amount of Agrobacterium infection solution used each time is 3-8 μl.
9. The method for inducing hairy roots of Atractylodes macrocephala according to claim 1, characterized in that: The proliferation culture medium is MS+sucrose 28-32 g / L+agar 5-10 g / L.
10. Use of the Atractylodes macrocephala hairy root induction method according to claim 1 in Atractylodes macrocephala hairy root induction, genetic transformation, gene function research and / or Atractylodes macrocephala metabolite production.