Culture method for obtaining high-quality hemsley rockvine root callus
By disinfecting and ZT soaking the young and tender stems of the 3-leaf green green, combining zein and light quality regulation, and daily temperature gradient management, the problem of slow growth of callus and uncertain root height in the culture of 3-leaf green green, is solved, and the induction and succession of high-quality callus is achieved, providing high-quality materials for molecular biology research.
Patent Information
- Application Number
- CN202411829065.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-03-14
AI Technical Summary
In the tissue culture of Trichophyllum, there are problems of difficulty in establishing a regeneration system, including slow growth of callus, high incidence of uncertain roots, premature callus aging, severe browning, and high mortality after subculture, which affects the development of its molecular biology research.
After disinfecting the tender stems of the three-leaf green leaves, it is soaked in sterile water containing ZT to balance the auxin content, reduce the occurrence of uncertain roots, and promote callus differentiation. During the process of inheritance, the content of zein is increased, the light quality ratio is adjusted, and the circulation management method of gradual daily temperature is adopted.
The induction and succession of high-quality callus in Trichophyllum has been achieved, the health and succession of callus have been improved, uncertain roots and browning have been reduced, and high-quality research materials have been provided.
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Figure CN119605648A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of tissue culture, and in particular relates to a culture method for obtaining high-quality callus of Tripterygium wilfordii. Background Art
[0002] The scientific name of Sanyeqing is Ligusticum ternatum, which is a perennial climbing vine of the genus Ligusticum in the family Vitaceae. Sanyeqing is a rare medicinal plant unique to China. The root is its main medicinal and economic organ. Its root extract has anti-inflammatory, analgesic, detoxifying and antipyretic effects. Its root extract can effectively inhibit the migration and proliferation of tumor cells and promote the apoptosis of tumor cells, showing great potential for anti-tumor clinical applications. With the in-depth exploration of the medicinal value of Sanyeqing, it is gradually becoming an emerging bulk Chinese medicinal material.
[0003] At present, cutting propagation is the main propagation method of Tripterygium wilfordii. Although the survival rate of cutting propagation is high, the reproduction rate of sexual reproduction is extremely low, and molecular biology research cannot be carried out through sexual reproduction, so it is particularly important to establish an efficient Tripterygium wilfordii tissue culture system. In recent years, with the rapid development of molecular biology research on Tripterygium wilfordii, the establishment of a regeneration system has become increasingly critical. There has always been a problem of difficulty in establishing a regeneration system in the tissue culture of Tripterygium wilfordii. During the growth of callus tissue, there are shortcomings such as slow growth, high incidence of adventitious roots, premature aging of callus, severe browning, and high mortality after subculture. In recent years, due to the continuous development of the medicinal value of Tripterygium wilfordii, Tripterygium wilfordii callus is an important material for its molecular biology research, which can be used for research such as transgenics and enrichment of secondary metabolites. Therefore, cultivating healthy and subcultured callus is very important for the development of molecular biology research on Tripterygium wilfordii. Summary of the invention
[0004] In order to solve the above-mentioned problems of the prior art, the present invention provides a culture method for obtaining high-quality callus of Tripterygium wilfordii. The obtained young stem segments of Tripterygium wilfordii are first disinfected, and then soaked in sterile water containing ZT to balance the high auxin content in the stems of Tripterygium wilfordii, reduce the occurrence of adventitious roots after inoculation, and promote callus differentiation. It was found that the use of ZT can have a high induction rate of Tripterygium wilfordii in young stem segments, which can reach 100%. In the process of callus induction, a relatively ideal primary generation callus of Tripterygium wilfordii can be obtained by combining with a low level of NAA. In the process of subculture, the content of zeatin is increased, the light quality ratio is adjusted, and a cyclic management method with gradual daily temperature changes is adopted to obtain high-quality subculture callus of Tripterygium wilfordii.
[0005] The technical solution adopted by the present invention is:
[0006] The present invention protects a culture method for obtaining high-quality callus of Tripterygium wilfordii, comprising the following steps:
[0007] Step 1: Pretreatment of explants;
[0008] Step 2: Callus induction culture: Place the treated explants in a callus induction medium and place them in an environment with a day-night temperature difference for callus induction culture;
[0009] Step 3: Subculture and expansion of callus tissue: After the callus produced on the stem segment in step 2 is peeled off, it is transferred to the subculture proliferation medium, the light quality ratio of red light and blue light is adjusted, and subculture is carried out in an environment with gradual temperature change during the day to obtain high-quality callus of Sanyeqing.
[0010] Preferably, in step 1, the explant is pretreated by cutting the young stem segments of Huaiyushan clover, rinsing them under running water for 30-40 minutes, cutting them, disinfecting them with 0.1% mercuric chloride for 8-12 minutes, rinsing them with sterile water for 3-5 times, disinfecting them with 70% alcohol for 10-20 seconds, rinsing them with sterile water for 3-5 times, soaking them in sterile water containing ZT, and absorbing the water with sterile filter paper to obtain the pretreated stem segments.
[0011] More preferably, the length of the young stem segment of Huaiyushan clover is 1 cm, and the stem segment close to the top does not have buds.
[0012] Young and tender stem segments can more easily form callus, and inoculation in callus induction medium can more effectively increase the primary callus induction rate, with an induction rate of 100%. The callus is light green in color, loose in texture, strong in growth potential, easy to separate, with a high survival rate in succession, and the rate of adventitious root production is extremely low during callus formation.
[0013] More preferably, the concentration of ZT in sterile water is 4 mg / L, the soaking time is 10-20 min, and then the water is dried with sterile dry filter paper.
[0014] Since the surface of the young stem segments of the three-leaf green will absorb dust and other impurities, rinsing under running water for 30-40 minutes can effectively remove the surface dust and microbial attachments. 0.1% mercuric chloride has a strong disinfection effect, but it is easy to kill the explants if the time is too long, so about 8-12 minutes is appropriate. 75% alcohol has strong penetration and can kill microorganisms under the epidermis; subsequent rinsing with sterile water can eliminate disinfectant residues and promote hydration of the explants. Then soaking in sterile water with a ZT concentration of 4 mg / L for 10-20 minutes can balance the higher auxin content in the stems of the three-leaf green, reduce the occurrence of adventitious roots after inoculation, and promote callus differentiation.
[0015] More preferably, the callus induction medium in step 1 comprises 4-5 mg / L ZT, 0.25 mg / L NAA and 6 g / L sucrose.
[0016] More preferably, the callus induction culture time is 18-20 days, the photoperiod is 12h light / 12h dark, the daytime temperature is 26-28°C, and the dark temperature is 20-22°C.
[0017] The callus induction effect of ZT in the tissue culture of Tripterygium wilfordii is now better than other cytokinins such as 6-BA and KT. The induction rate of 4-5 mg / L ZT (zeatin) for young stem segments can reach 100%. At the same time, the callus browning rate is low, the color is light green and the growth potential is good, and the incidence of adventitious roots is extremely low (2%). The next step of subculture can be carried out. At the same time, there is a certain temperature difference between day and night, which can significantly increase the growth of callus tissue and reduce the browning of callus tissue.
[0018] More preferably, in step 2, the subculture medium comprises 6 mg / L ZT, 0.25 mg / L NAA and 6 g / L sucrose.
[0019] Continuing to use zeatin in the subculture medium and increasing the hormone concentration to 6 mg / L had a good stabilizing effect on the subculture of Tripterygium wilfordii. After three subcultures, the cultured callus would produce loose and translucent callus tissue, which was conducive to further inducing differentiation.
[0020] More preferably, the subculture time is 40 days, during which the culture medium is replaced every 20 days, the photoperiod is 12 hours light / 12 hours dark, and the light intensity is 100 μ.mol.m -2 s -1 .
[0021] More preferably, the light quality ratio of red light (660-665nm) to blue light (435-445) is 3:1, and the optical density is 80-100μ.mol.m -2 s -1 .
[0022] More preferably, in step 2, the daily temperature change is adjusted by a step temperature change method, from 0 (24 o'clock) to 23 o'clock, the temperature changes at 1°C / every 1h, the maximum temperature is 28°C, and the minimum temperature is 16°C. The temperature gradient changes are: 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C and 16°C.
[0023] Simulating the daylight cycle and daily temperature changes of the Huaiyushan Tripterygium wilfordii production area, and appropriately increasing the proportion of blue light, has a good promoting effect on the callus growth of Huaiyushan Tripterygium wilfordii. The callus grows rapidly, the browning rate is low, the dry matter accumulation is increased, and the subculture cycle is shortened.
[0024] Beneficial effects:
[0025] (1) The present invention selects young stem segments as explants and adds ZT (zeatin) to the culture medium during callus induction culture, which can better promote the occurrence of callus.
[0026] (2) The present invention uses a gradual temperature change during the subculture stage of the three-leaf green callus to promote the growth of the three-leaf green callus. The gradual temperature simulates the daily temperature changes in the high mountains in September, October and November where the three-leaf green grows best. This high-altitude gradual temperature plays an important role in the accumulation of dry matter and can significantly inhibit the browning phenomenon of the three-leaf green callus in the subculture culture. The callus remains light green and slightly loose. This three-leaf green callus tissue can be used as an ideal material for molecular biology research.
[0027] (3) The present invention has found in the research process that the use of zeatin can have a high induction rate of 100% for young stem segments of Tripterygium wilfordii. In the callus induction process, low levels of NAA can be used to obtain ideal primary callus of Tripterygium wilfordii. In the subculture process, by increasing the content of zeatin, adjusting the light quality ratio, and using a cyclic management method with gradual daily temperature changes, high-quality Tripterygium wilfordii callus can be obtained for factory production or scientific research of secondary metabolites. The method of the present invention is simple and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0029] Figure 1 This is the callus induction and subculture growth of Tripterygium wilfordii in Example 3.
[0030] Figure 2 This is the callus proliferation in Comparative Example 1.
[0031] Figure 3 This is the callus proliferation in Comparative Example 2.
[0032] Figure 4 This is the callus proliferation in Comparative Example 3.
[0033] Figure 5 This is the callus proliferation in Comparative Example 4.
[0034] Figure 6 This is the callus proliferation in Comparative Example 5.
[0035] Figure 7 This is the callus proliferation in Comparative Example 6.
[0036] Figure 8 This is the callus proliferation in Comparative Example 7. DETAILED DESCRIPTION
[0037] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.
[0038] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0042] The raw materials used in the following examples were purchased from commercial sources.
[0043] Example 1
[0044] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, comprising the following steps:
[0045] Step 1: Pretreatment of explants: Cut the tender stem segments of Huaiyushan clover, rinse them under running water for 30 minutes, cut them into 1 cm stem segments, disinfect them with 1% mercuric chloride for 8 minutes, rinse them with sterile water for 3 times, and then disinfect them with 75% alcohol for 10 seconds, finally rinse them with sterile water for 3 times, and then soak them in sterile water containing ZT (4 mg / L) for 10 minutes, and use sterile filter paper to absorb the moisture to obtain the pre-treated stem segments.
[0046] Step 2: Place the young stem segments treated as above in a culture medium containing 4 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The primary callus induction culture time is 18 days. The photoperiod is 12 hours (light) / 12 hours (darkness), the temperature is 26°C during the day and 20°C in the dark.
[0047] Step 3: Subculture and expansion of callus tissue: The callus tissue with callus stem segments was peeled off and transferred to the subculture proliferation medium. The concentration of the subculture proliferation medium was 6 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The culture time was 40 days, during which the culture medium was replaced every 20 days. The photoperiod was 12 hours (light) / 12 hours (darkness). The light quality ratio was red light (660-665nm) / blue light (435-445nm): 3:1, and the optical density was 80μ.mol.m -2 s -1 ; The daily temperature was adjusted by step temperature change, the temperature changed by 1℃ every 1h, the highest temperature was 26℃, the lowest temperature was 14℃, and the temperature from 0 (24 hours) to 23 hours was 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 26℃, 25℃, 24℃, 23℃, 22℃, 21℃, 20℃, 19℃, 18℃, 17℃, 16℃. Finally, the contamination rate of the stem segments of Huaiyushan Sanyeqing callus was 15%, the callus induction rate was 100%, the rooting rate was 0%, and the value-added multiple was 3.2.
[0048] Example 2
[0049] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, comprising the following steps:
[0050] Step 1: Pretreatment of explants: Cut the tender stem segments of Huaiyushan clover, rinse them under running water for 30 minutes, cut them into 1 cm stem segments, disinfect them with 1% mercuric chloride for 9 minutes, rinse them with sterile water for 4 times, and then disinfect them with 75% alcohol for 15 seconds, finally rinse them with sterile water for 4 times, and then soak them in sterile water containing ZT (4 mg / L) for 15 minutes, and use sterile filter paper to absorb the moisture to obtain the pre-treated stem segments.
[0051] Step 2: Induction culture of callus tissue: The young stem segments after the above treatment are placed in a culture medium containing 4.5 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The initial callus induction culture time is 19 days, the photoperiod is 12 hours (light) / 12 hours (darkness), the temperature is 27°C during the day and 21°C in the dark.
[0052] Step 3: Subculture and expansion of callus tissue: The callus tissue with callus stem segments was peeled off and transferred to the subculture proliferation medium. The concentration of the subculture proliferation medium was 6 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The culture time was 40 days, during which the culture medium was replaced every 20 days. The photoperiod was 12 hours (light) / 12 hours (darkness). The light quality ratio was red light (660-665nm) / blue light (435-445nm): 3:1, and the optical density was 90μ.mol.m -2 s -1 . ; The daily temperature was adjusted by step temperature change, the temperature changed by 1℃ every 1h, the highest temperature was 27℃, the lowest temperature was 15℃, and the temperature from 0 (24 hours) to 23 hours was 117℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 27℃, 26℃, 25℃, 24℃, 23℃, 22℃, 21℃, 20℃, 19℃, 18℃, 17℃, 16℃,. Finally, the contamination rate of the stem segments of Huaiyushan Sanyeqing callus was 8.3%, the callus induction rate was 100%, the rooting rate was 0%, and the value-added multiple was 3.4.
[0053] Example 3
[0054] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, comprising the following steps:
[0055] Step 1: Pretreatment of explants: Cut the tender stem segments of Huaiyushan clover, rinse them under running water for 30 minutes, cut them into 1 cm stem segments, disinfect them with 0.1% mercuric chloride for 12 minutes, rinse them with sterile water for 5 times, and then disinfect them with 70% alcohol for 20 seconds, finally rinse them with sterile water for 5 times, and then soak them in sterile water containing ZT (4 mg / L) for 20 minutes, and use sterile filter paper to absorb the moisture to obtain the pre-treated stem segments.
[0056] Step 2: Induction culture of callus tissue: The young stem segments after the above treatment are placed in a culture medium containing 5 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The initial callus induction culture time is 20 days, the photoperiod is 12 hours (light) / 12 hours (darkness), the temperature is 28°C during the day and 22°C in the dark.
[0057] Step 3: Subculture and expansion of callus tissue: The callus tissue with callus stem segments was peeled off and transferred to the subculture proliferation medium. The concentration of the subculture proliferation medium was 6 mg / L ZT (zeatin), 0.25 mg / L NAA and 6 g / L sucrose. The culture time was 40 days, during which the culture medium was replaced every 20 days. The photoperiod was 12 hours (light) / 12 hours (darkness). The light quality ratio was red light (660-665nm) / blue light (435-445nm): 3:1, and the optical density was 100μ.mol.m -2 s -1 The daily temperature was adjusted by step temperature change, the temperature changed by 1°C every 1h, the highest temperature was 28°C, the lowest temperature was 16°C, and the temperatures from 0 (24 hours) to 23 hours were 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, and 15°C. The callus induction and subculture growth of Tripterygium wilfordii in this example are shown in the figure. Figure 1 As shown, the contamination rate of the stem segments of Huaiyushan Tripterygium wilfordii callus was 5%, the callus induction rate was 100%, the rooting rate was 0%, and the multiplication factor was 3.9.
[0058] Comparative Example 1
[0059] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0060] like Figure 2 As shown, in step (1), without soaking in ZT (4 mg / L), the rooting rate of Tripterygium wilfordii was higher, callus formation was delayed, callus induction rate was 100%, rooting rate was 0%, but callus tissue showed browning.
[0061] Comparative Example 2
[0062] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0063] In step (2), 6BA (3 mg / L) was added to the callus induction medium to replace ZT, the concentration of NAA was 0.4 mg / L, and the remaining steps were the same as in Example 3.
[0064] like Figure 3 As shown, the callus induction rate of Tripterygium wilfordii was low, and the adventitious roots of the explants grew vigorously, with a callus induction rate of 10% and a rooting rate of 100%.
[0065] Comparative Example 3
[0066] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0067] Add KT 4 mg / ml and NAA 0.25 mg / L to the callus induction medium, and the remaining steps are the same as Example 3.
[0068] like Figure 4 As shown, the callus induction rate of Tripterygium wilfordii was low, and the adventitious roots of the explants grew vigorously, with a callus induction rate of 30% and a rooting rate of 100%.
[0069] Comparative Example 4
[0070] A method for obtaining high-quality callus of Tripterygium wilfordii, which differs from Example 3 only in that:
[0071] ZT 4 mg / ml and NAA 0.3 mg / L were added to the callus induction medium to replace the concentrations, and the remaining steps were the same as in Example 3.
[0072] like Figure 5 As shown, the callus induction rate of Tripterygium wilfordii was low, and the adventitious roots of the explants grew vigorously, with a callus induction rate of 100% and a rooting rate of 100%.
[0073] Comparative Example 5
[0074] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0075] In step (3), the concentration of the subculture proliferation medium is 4 mg / L ZT (zeatin) and the concentration of NAA is 0.4 mg / L. The remaining steps are the same as Example 5.
[0076] like Figure 6 As shown, the callus proliferation rate of Tripterygium wilfordii is low, and the explant proliferation and growth are slow. The callus proliferation rate of Tripterygium wilfordii is 1.5 times, and the rooting rate is 50%.
[0077] Comparative Example 6
[0078] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0079] The concentration of the subculture proliferation medium is 5 mg / L ZT (zeatin) and the concentration of NAA is 0.6 mg / L. The remaining steps are the same as Example 3.
[0080] like Figure 7 As shown, the callus proliferation rate of Tripterygium wilfordii is low, the explant proliferation grows slowly, the callus proliferation rate of Tripterygium wilfordii is 2 times, the rooting rate is 0%, and the browning phenomenon is serious.
[0081] Comparative Example 7
[0082] A method for obtaining high-quality callus of Huaiyushan Tripterygium wilfordii, which differs from Example 3 only in that:
[0083] Use ordinary fluorescent lamps, the temperature is 28°C during the day and 22°C in the dark, and the remaining steps are the same as Example 3.
[0084] like Figure 8 As shown, the callus proliferation rate of Tripterygium wilfordii was low, and the explant proliferation and growth were slow. The callus proliferation rate of Tripterygium wilfordii was 3 times, the rooting rate was 0%, and some calli turned brown.
[0085] The embodiments described above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belong to the protection scope of the present invention.
Claims
1. A method for obtaining high-quality callus of Tripterygium wilfordii, characterized in that: The following steps are involved: Step 1: Pretreatment of explants; Step 2: Callus induction culture: Place the treated explants in a callus induction medium and place them in an environment with a day-night temperature difference for callus induction culture; Step 3: Subculture and expansion of callus tissue: After the callus produced on the stem segment in step 2 is peeled off, it is transferred to the subculture proliferation medium, the light quality ratio of red light and blue light is adjusted, and subculture is carried out in an environment with gradual temperature change during the day to obtain high-quality callus of Sanyeqing.
2. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 1, characterized in that: In step 1, pre-treatment of explants: Cut the tender stem segments of Huaiyushan clover, rinse them under running water for 30-40 minutes, cut them, disinfect them with 0.1% mercuric chloride for 8-12 minutes, rinse them with sterile water for 3-5 times, disinfect them with 70% alcohol for 10-20 seconds, rinse them with sterile water for 3-5 times, soak them in sterile water containing ZT, and absorb the moisture with sterile filter paper to obtain the pre-treated stem segments.
3. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 2, characterized in that: During the pre-treatment of the stem segments, they were cut into 1 cm stem segments.
4. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 2, characterized in that: In step 1, the concentration of ZT in sterile water is 4 mg / L, and the immersion time is 10-20 min.
5. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 1, characterized in that: The callus induction medium in step 2 includes 4-5 mg / L ZT, 0.25 mg / L NAA and 6 g / L sucrose.
6. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 5, characterized in that: The callus induction culture time is 18-20 days, the photoperiod is 12h light / 12h dark, the daytime temperature is 26-28℃, and the dark temperature is 20-22℃.
7. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 5, characterized in that: The subculture medium in step 3 included 6 mg / L ZT, 0.25 mg / L NAA, and 6 g / L sucrose.
8. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 7, characterized in that: The subculture time was 40 days, during which the culture medium was replaced every 20 days, and the photoperiod was 12 h light / 12 h dark.
9. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 7, characterized in that: The light quality ratio of red light to blue light is 3:1, and the optical density is 80-100μ.mol.m -2 s -1 .
10. The method for obtaining high-quality callus of Tripterygium wilfordii according to claim 8, characterized in that: In step 2, the daily temperature gradient is adjusted by a step temperature change method. From 0:00 to 23:00, the temperature changes at 1°C / hour, with the highest temperature being 27-29°C and the lowest temperature being 14-16°C.