A tissue culture method and a seedling hardening and transplanting method for coptis deltoidea
By using tissue culture and hardening-off transplanting methods for tender shoots of Coptis chinensis rhizomes, the problems of small seeds and long dormancy period of Coptis chinensis have been solved, achieving efficient propagation and resource preservation of Coptis chinensis, improving the growth cycle and survival rate, and supporting the sustainable utilization of Coptis chinensis.
Patent Information
- Application Number
- CN202511316006.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-15
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2045-09-15
AI Technical Summary
Coptis chinensis seeds are small, have a long dormancy period, and produce uneven seedlings with slow growth. This results in a large market demand but a scarcity of wild resources, low efficiency in artificial cultivation, and difficulty in achieving sustainable utilization of the resources.
Tender shoots from the rhizomes of Coptis chinensis were used as explants. Adventitious shoots were induced, proliferated, and rooted using tissue culture methods with specific ratios of culture medium and hormones. Finally, the seedlings were hardened off in a light incubator and transplanted into an optimized seedling substrate for further cultivation.
It improved the propagation efficiency of Coptis chinensis, shortened the growth cycle, achieved a proliferation coefficient of 3.5, a rooting rate of 95.4%, and a seedling survival rate of 92.7%, thus supporting the rapid propagation and sustainable utilization of Coptis chinensis resources.
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Figure CN120858878B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for tissue culture and seedling hardening and transplanting of Coptis chinensis. Background Technology
[0002] Coptis chinensis Franch., belonging to the genus Coptis in the family Ranunculaceae, is also known as Wei Lian, Chuan Lian, and Ji Zhua Lian. It possesses various pharmacological effects, including reducing internal heat and dampness, antibacterial properties, and relieving dysentery. Coptis chinensis prefers cool, moist, and shady conditions, and dislikes high temperatures and drought. It is generally distributed in high-altitude areas, requiring a low-temperature, high-humidity natural environment. It cannot withstand strong sunlight and prefers low light, thus requiring shade. Coptis chinensis has shallow roots and thrives in loose, fertile topsoil rich in humus. It commonly grows in the cool, damp shade of high-altitude forests. Coptis chinensis is mainly propagated by seeds, but division is also possible.
[0003] The market demand for Coptis chinensis is high, but long-term over-harvesting has led to a scarcity of wild resources. In artificial cultivation, the small size of the seeds, long dormancy period, uneven emergence, and slow growth of Coptis chinensis, requiring 6-7 years before harvest, thus limiting its production. Plant tissue culture technology can enrich and optimize Coptis chinensis germplasm resources, addressing its resource scarcity problem. Establishing an effective tissue culture regeneration system can be used for the preservation of endangered Coptis chinensis germplasm, achieving its sustainable utilization. Summary of the Invention
[0004] The purpose of this invention is to provide a tissue culture method and a seedling hardening and transplanting method for Coptis chinensis, providing technical support for the enrichment, optimization and preservation of Coptis chinensis germplasm resources.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for tissue culture of Coptis chinensis, comprising the following steps:
[0007] (1) Using the tender shoots of Coptis chinensis rhizome as explants, sterile seedlings of Coptis chinensis were obtained by inoculating them into a basic culture medium after disinfection.
[0008] (2) The aseptic seedlings of Coptis chinensis were inoculated into an adventitious bud differentiation medium and cultured to obtain adventitious buds of Coptis chinensis; the adventitious bud differentiation medium contained 1.3-1.7 mg / L 6-BA and 0.25-0.35 mg / L NAA;
[0009] (3) The adventitious buds of Coptis chinensis were inoculated into a proliferation medium and cultured to obtain Coptis chinensis proliferating seedlings; the proliferation medium contained 1.8-2.2 mg / L TDZ and 0.45-0.55 mg / L IAA;
[0010] (4) The Coptis chinensis seedlings were inoculated into a rooting medium and cultured to obtain Coptis chinensis test tube seedlings.
[0011] Preferably, the disinfection method in step (1) is as follows: disinfect with 70-80% alcohol for 30-60 seconds, rinse with sterile water 2-3 times, then disinfect with 0.08-0.12% mercuric chloride for 8-14 minutes, and rinse with sterile water 3-5 times.
[0012] Preferably, the basic culture medium in step (1) is MS culture medium, and the culture time is 8 to 12 days.
[0013] Preferably, the adventitious bud differentiation medium in step (2) is: MS + 1.3-1.7 mg / L 6-BA + 0.25-0.35 mg / L NAA + 25-35 g / L sucrose + 7.0-8.0 g / L agar; the culture time is 25-35 days.
[0014] Preferably, the proliferation medium in step (3) is: MS + 1.8-2.2 mg / L TDZ + 0.45-0.55 mg / L IIA + 25-35 g / L sucrose + 7.0-8.0 g / L agar; the culture time is 30-40 days.
[0015] Preferably, the rooting medium in step (4) is: 1 / 2 MS + 0.45-0.55 mg / L NAA + 0.08-0.12 mg / L IBA + 0.8-1.2 g / L activated carbon + 25-35 g / L sucrose + 7.0-8.0 g / L agar; the culture time is 18-25 days.
[0016] Preferably, the pH of the adventitious bud differentiation medium, the proliferation medium and the rooting medium are all 5.8 to 6.0; the light intensity during the culture in steps (1) to (4) is 2000 to 3000 lx, the temperature is 25 ± 2℃, the humidity is 65% to 75%, and the light-dark cycle is 10 to 14 hours of light and 10 to 14 hours of dark alternating culture.
[0017] This invention provides a method for hardening and transplanting Coptis chinensis seedlings. When the roots of the Coptis chinensis test-tube seedlings reach 3-5 cm in length and the seedling height reaches 4-6 cm, the bottle cap is removed and the seedlings are hardened in a light incubator for 4-7 days. After hardening, the Coptis chinensis test-tube seedlings are taken out, sterilized, and then transplanted into a seedling substrate for cultivation.
[0018] Preferably, the temperature during seedling hardening is 25±2℃, and the sterilization method is to soak the Coptis chinensis test tube seedlings in a carbendazim solution for 20-30 minutes, wherein the concentration of the carbendazim solution is 1:800-1:1200.
[0019] Preferably, the seedling substrate is peat moss, vermiculite and river sand in a mass ratio of 1.5-2.5:0.8-1.2:0.8-1.2.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] This invention provides a method for cultivating tissue culture seedlings and hardening-off transplanting of Coptis chinensis using tender shoots from its rhizomes as explants. The induction rate of adventitious buds can reach 90.3%, the proliferation coefficient reaches 3.5, the rooting rate reaches 95.4%, and the survival rate of hardened-off transplanted seedlings reaches 92.7%. The tissue culture and hardening-off transplanting method provided by this invention can improve the propagation efficiency of Coptis chinensis, significantly shorten its growth cycle, and provide technical support for the rapid propagation and sustainable utilization of Coptis chinensis resources, showing broad application prospects. Attached Figure Description
[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0023] Figure 1 The mother plant of Coptis chinensis used in Example 1;
[0024] Figure 2 The aseptic seedlings of Coptis chinensis obtained in Example 1;
[0025] Figure 3 The image shows the growth status of adventitious buds in Coptis chinensis after 7 days of differentiation culture in Example 1.
[0026] Figure 4 The growth status of adventitious buds in Coptis chinensis after 10 days of differentiation culture in Example 1;
[0027] Figure 5 The growth status of adventitious buds in Coptis chinensis after 20 days of differentiation culture in Example 1;
[0028] Figure 6 The growth status of adventitious buds in Coptis chinensis after 30 days of differentiation culture in Example 1;
[0029] Figure 7 The Coptis chinensis propagation seedlings obtained in Example 1;
[0030] Figure 8 The rooted seedlings of Coptis chinensis obtained in Example 1;
[0031] Figure 9 The growth status of the Coptis chinensis seedlings transplanted after hardening in Example 1;
[0032] Figure 10 This shows the growth status of Coptis chinensis seedlings that have been transplanted and cultured for 30 days in Example 1. Detailed Implementation
[0033] This invention provides a method for tissue culture of Coptis chinensis, comprising the following steps:
[0034] (1) Using the tender shoots of Coptis chinensis rhizome as explants, sterile seedlings of Coptis chinensis were obtained by inoculating them into a basic culture medium after disinfection.
[0035] (2) The aseptic seedlings of Coptis chinensis were inoculated into an adventitious bud differentiation medium and cultured to obtain adventitious buds of Coptis chinensis; the adventitious bud differentiation medium contained 1.3-1.7 mg / L 6-BA and 0.25-0.35 mg / L NAA;
[0036] (3) The adventitious buds of Coptis chinensis were inoculated into a proliferation medium and cultured to obtain Coptis chinensis proliferating seedlings; the proliferation medium contained 1.8-2.2 mg / L TDZ and 0.45-0.55 mg / L IAA;
[0037] (4) The Coptis chinensis seedlings were inoculated into a rooting medium and cultured to obtain Coptis chinensis test tube seedlings.
[0038] In this invention, the disinfection method described in step (1) is as follows: disinfect with 70-80% alcohol for 30-60 seconds, rinse with sterile water 2-3 times, then disinfect with 0.08-0.12% mercuric chloride for 8-14 minutes, and rinse with sterile water 3-5 times.
[0039] In this invention, the disinfection method described in step (1) is preferably: disinfecting with 72-78% alcohol for 40-50 seconds, rinsing with sterile water 2-3 times, then disinfecting with 0.09-0.11% mercuric chloride for 11-13 minutes, and rinsing with sterile water 3-5 times.
[0040] In this invention, the disinfection method described in step (1) is preferably: disinfecting with 75% alcohol for 45 seconds, rinsing twice with sterile water, then disinfecting with 0.1% mercuric chloride for 12 minutes, and rinsing four times with sterile water.
[0041] In this invention, the basic culture medium in step (1) is MS culture medium, and the culture time is 8 to 12 days, preferably 9 to 11 days, and more preferably 10 days.
[0042] In this invention, the adventitious bud differentiation culture medium in step (2) is: MS + 1.3-1.7 mg / L 6-BA + 0.25-0.35 mg / L NAA + 25-35 g / L sucrose + 7.0-8.0 g / L agar, preferably MS + 1.4-1.6 mg / L 6-BA + 0.28-0.32 mg / L NAA + 28-32 g / L sucrose + 7.2-7.8 g / L agar, and more preferably MS + 1.5 mg / L 6-BA + 0.30 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar; the culture time is 25-35 days, preferably 28-32 days, and more preferably 30 days.
[0043] In this invention, the proliferation medium in step (3) is: MS + 1.8-2.2 mg / L TDZ + 0.45-0.55 mg / L IAA + 25-35 g / L sucrose + 7.0-8.0 g / L agar, preferably MS + 1.9-2.1 mg / L TDZ + 0.48-0.52 mg / L IAA + 28-32 g / L sucrose + 7.2-7.8 g / L agar, and more preferably MS + 2.0 mg / L TDZ + 0.5 mg / L IAA + 30 g / L sucrose + 7.5 g / L agar; the culture time is 30-40 days, preferably 32-38 days, and more preferably 35 days.
[0044] In this invention, the rooting medium in step (4) is: 1 / 2 MS + 0.45-0.55 mg / L NAA + 0.08-0.12 mg / L IBA + 0.8-1.2 g / L activated carbon + 25-35 g / L sucrose + 7.0-8.0 g / L agar, preferably 1 / 2 MS + 0.48-0.52 mg / L NAA + 0.09-0.11 mg / L IBA + 0.9-1.1 g / L activated carbon + 28-32 g / L sucrose + 7.2-7.8 g / L agar, and more preferably 1 / 2 MS + 0.5 mg / L NAA + 0.1 mg / L IBA + 1.0 g / L activated carbon + 30 g / L sucrose + 7.5 g / L agar; the culture time is 18-25 days, preferably 20-23 days, and more preferably 21 days.
[0045] In this invention, the pH of the adventitious bud differentiation medium, the proliferation medium and the rooting medium are all 5.8 to 6.0, preferably 5.9.
[0046] In this invention, the light intensity during cultivation in steps (1) to (4) is 2000-3000 lx, preferably 2200-2800 lx, more preferably 2400-2600 lx, and even more preferably 2500 lx; the temperature is 25±2℃, preferably 25±1℃, and even more preferably 25℃; the humidity is 65%-75%, preferably 67%-73%, more preferably 69%-71%, and even more preferably 70%; the light-dark cycle is 10-14h light and 10-14h dark alternating cultivation, preferably 11-13h light and 11-13h dark alternating cultivation, and even more preferably 12h light and 12h dark alternating cultivation.
[0047] This invention provides a method for hardening and transplanting Coptis chinensis seedlings. When the roots of the Coptis chinensis test-tube seedlings reach 3-5 cm in length and the seedling height reaches 4-6 cm, the bottle cap is removed and the seedlings are hardened in a light incubator for 4-7 days. After hardening, the Coptis chinensis test-tube seedlings are taken out, sterilized, and then transplanted into a seedling substrate for cultivation.
[0048] In this invention, it is preferable to open the bottle cap when the root length of the Coptis chinensis test-tube seedlings reaches 3.5-4.5 cm and the seedling height reaches 4.5-5.5 cm, and harden the seedlings in a light incubator for 5-7 days; more preferably, it is preferable to open the bottle cap when the root length of the Coptis chinensis test-tube seedlings reaches 4 cm and the seedling height reaches 5 cm, and harden the seedlings in a light incubator for 6 days.
[0049] In this invention, the temperature during seedling hardening is 25±2℃, preferably 25±1℃, and more preferably 25℃; the sterilization method is to soak the Coptis chinensis test tube seedlings in a carbendazim solution for 20-30 minutes, preferably 22-28 minutes, and more preferably 25 minutes; the concentration of the carbendazim solution is 1:800-1:1200, preferably 1:900-1:1100, and more preferably 1:1000.
[0050] In this invention, the seedling substrate is peat moss, vermiculite and river sand, with a mass ratio of 1.5-2.5:0.8-1.2:0.8-1.2, preferably 1.8-2.2:0.9-1.1:0.9-1.1, and more preferably 2:1:1.
[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The Coptis chinensis material used in the following embodiments comes from Juhe Village, Daxi Town, Dafang County, Bijie City, Guizhou Province, and the variety is Coptis chinensis Franch.
[0052] Example 1
[0053] A method for tissue culture and hardening-off transplanting of Coptis chinensis, the steps of which are as follows:
[0054] (1) Obtaining sterile Coptis chinensis seedlings
[0055] Collect Coptis chinensis ( Figure 1 The tender shoots of the rhizome were used as explants for tissue culture. After cleaning and disinfection, they were inoculated into MS basal medium and cultured for 10 days to obtain sterile Coptis chinensis seedlings. Figure 2 The disinfection method is as follows: disinfect with 75% alcohol for 45 seconds, rinse twice with sterile water, then disinfect with 0.1% mercuric chloride (HgCl2) for 12 minutes, and rinse four times with sterile water.
[0056] (2) Culture of adventitious shoots differentiated from Coptis chinensis
[0057] The obtained sterile Coptis chinensis seedlings were inoculated into adventitious bud differentiation medium and cultured for 30 days to obtain adventitious buds of Coptis chinensis (growth status at 7, 10, 20 and 30 days of culture is shown in the figure). Figures 3-6 (as shown); the adventitious bud differentiation medium is: MS + 1.5 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar, pH 5.9.
[0058] (3) Proliferation culture of adventitious buds of Coptis chinensis
[0059] The obtained adventitious buds of Coptis chinensis were inoculated into a proliferation medium for proliferation culture. After 35 days of culture, Coptis chinensis proliferated seedlings were obtained. Figure 7 The proliferation medium was: MS + 2.0 mg / L TDZ + 0.5 mg / L IAA + 30 g / L sucrose + 7.5 g / L agar, with a pH of 5.9.
[0060] (4) Rooting culture of Coptis chinensis
[0061] The obtained Coptis chinensis propagation seedlings were transferred to rooting medium and cultured for 21 days to obtain Coptis chinensis test-tube seedlings. Figure 8 The rooting medium is: 1 / 2 MS + 0.5 mg / L NAA + 0.1 mg / L IBA + 1.0 g / L activated carbon + 30 g / L sucrose + 7.5 g / L agar, with a pH of 5.9.
[0062] (5) Hardening-off and transplanting of Coptis chinensis test-tube seedlings
[0063] When the roots of the Coptis chinensis plantlets reached 4cm and the seedlings were 5cm tall, the tissue culture bottle was opened, and the plantlets were hardened off at 25℃ in a light incubator for 6 days. The plantlets were then removed, the agar at the base was washed off, and they were soaked in a 1:1000 carbendazim solution for 25 minutes. Finally, they were transplanted into seedling trays filled with a seedling substrate (peat:vermiculite:river sand = 2:1:1), thoroughly watered, covered with a thin film to retain moisture, and ensured ventilation and a suitable temperature (25℃). The film was removed after one week. The growth status of the transplanted Coptis chinensis seedlings after hardening off is as follows. Figure 9 As shown, the growth status of Coptis chinensis seedlings after 30 days of hardening-off and transplanting is as follows. Figure 10 As shown.
[0064] In steps (1) to (4), the light intensity during cultivation is 2500 lx, the temperature is 25℃, the humidity is 70%, and the light-dark cycle is 12h light and 12h dark alternating cultivation.
[0065] Example 2
[0066] The inventors previously studied the effects of the time for mercuric chloride disinfection of explants in Example 1, the hormone ratios in the adventitious bud differentiation medium, proliferation medium, and rooting medium, and the composition of the seedling substrate on the survival rate of Coptis chinensis plant tissue culture and transplanting. The results are shown in Tables 1 to 5.
[0067] Table 1. Effects of different disinfection times on the growth of Coptis chinensis explants.
[0068]
[0069] As shown in Table 1, the disinfection time of 8–12 min had little effect on the mortality of Coptis chinensis explants. Among them, the sterility rate and mortality rate of explants were low when the disinfection time was 12 min, while the sterility rate and mortality rate of Coptis chinensis explants increased when the disinfection time was too long.
[0070] Table 2. Effects of different hormone ratios on the induction of adventitious shoots from Coptis chinensis.
[0071] Types of culture media 6-BA (mg / L) NAA (mg / L) Induction rate (%) MS 0.5 0.1 75.1 MS 0.5 0.3 79.5 MS 0.5 0.5 78.9 MS 1.0 0.1 81.5 MS 1.0 0.3 80.7 MS 1.0 0.5 78.6 MS 1.5 0.1 84.9 MS 1.5 0.3 90.3 MS 1.5 0.5 88.4
[0072] As shown in Table 2, the concentrations of 6-BA and NAA in the adventitious shoot differentiation medium have a significant impact on the induction effect. The adventitious shoot induction rate is the highest, reaching 90.3%, when the concentration of 6-BA is 1.5 mg / L and the concentration of NAA is 0.3 mg / L.
[0073] Table 3 Effects of different hormone ratios on the proliferation culture of Coptis chinensis
[0074] Types of culture media TDZ (mg / L) IAA (mg / L) Proliferation coefficient MS 1.0 0.1 2.3 MS 1.0 0.5 2.7 MS 1.0 1.0 2.5 MS 2.0 0.1 3.1 MS 2.0 0.5 3.5 MS 2.0 1.0 2.8 MS 3.0 0.1 3.0 MS 3.0 0.5 2.6 MS 3.0 1.0 2.5
[0075] As shown in Table 3, the concentrations of TDZ and IAA in the proliferation medium also have a significant effect on the proliferation of Coptis chinensis. The proliferation coefficient is the largest when the concentration of TDZ is 2.0 mg / L and the concentration of IAA is 0.5 mg / L, reaching 3.5.
[0076] Table 4. Effects of different hormone ratios on the rooting culture of Coptis chinensis.
[0077] Types of culture media NAA (mg / L) IBA (mg / L) Rooting rate (%) MS 0.1 0.1 80.5 MS 0.3 0.2 85.2 MS 0.5 0.3 86.4 1 / 2MS 0.1 0.2 87.1 1 / 2MS 0.3 0.3 91.3 1 / 2MS 0.5 0.1 95.4 1 / 4MS 0.1 0.3 89.2 1 / 4MS 0.3 0.1 83.7 1 / 4MS 0.5 0.2 78.5
[0078] As shown in Table 4, the concentrations of NAA and IBA in the rooting medium also have a significant impact on the rooting rate. The rooting effect is best when the concentration of NAA is 0.5 mg / L and the concentration of IBA is 0.1 mg / L, with a rooting rate of up to 95.4%.
[0079] Table 5. Effects of different substrate ratios on the survival rate of Coptis chinensis seedlings after transplanting.
[0080] Serial Number Seedling substrate Proportion Survival rate (%) 1 Nutrient soil: vermiculite 1：1 79.8 2 Nutrient soil: vermiculite 2:1 85.7 3 Nutrient soil: vermiculite 3:1 82.4 4 Nutrient soil: vermiculite: river sand 1:1:1 88.9 5 Nutrient soil: vermiculite: river sand 2:1:1 90.8 6 peat moss: vermiculite 1:1 85.1 7 peat moss: vermiculite 2:1 89.6 8 peat moss: vermiculite 3:1 88.3 9 Peat moss: Vermiculite: River sand 1:1:1 90.5 10 Peat moss: Vermiculite: River sand 2:1:1 92.7
[0081] As shown in Table 5, the composition and ratio of the seedling substrate have a significant impact on the survival rate of Coptis chinensis seedlings after transplanting. The highest survival rate, 92.7%, is achieved when the composition of the seedling substrate is peat moss:vermiculite:river sand = 2:1:1.
[0082] As can be seen from the above, this invention provides a method for cultivating Coptis chinensis seedlings using tender shoots from the rhizomes of Coptis chinensis as explants. The optimal disinfection time for the explants is 12 minutes; the optimal induction medium for adventitious shoot differentiation is MS + 1.5 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar, with an induction rate of 90.3%; the optimal medium for proliferation culture is MS + 2.0 mg / L LTDZ + 0.5 mg / L IAA + 30 g / L sucrose + 7.5 g / L agar, with a proliferation coefficient of 3.5; the optimal medium for rooting culture is 1 / 2 MS + 0.5 mg / L NAA + 0.1 mg / L IBA + 1.0 g / L activated carbon + 30 g / L sucrose + 7.5 g / L agar, with a rooting rate of 95.4%; the optimal composition of the seedling substrate is peat moss:vermiculite:river sand = 2:1:1, with a seedling survival rate of 92.7% after hardening and transplanting.
[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for tissue culture of Coptis chinensis, characterized in that, Includes the following steps: (1) Using tender shoots of Coptis chinensis rhizomes as explants, sterilized plants were inoculated into a basic culture medium to obtain sterile Coptis chinensis seedlings; the basic culture medium was MS medium. (2) The aseptic seedlings of Coptis chinensis were inoculated into an adventitious bud differentiation medium and cultured to obtain adventitious buds of Coptis chinensis; the adventitious bud differentiation medium was: MS + 1.3~1.7mg / L 6-BA + 0.25~0.35mg / L NAA + 25~35g / L sucrose + 7.0~8.0g / L agar; (3) The adventitious buds of Coptis chinensis were inoculated into a proliferation medium and cultured to obtain Coptis chinensis proliferating seedlings; the proliferation medium was: MS + 1.8~2.2 mg / L TDZ + 0.45~0.55 mg / L IAA + 25~35 g / L sucrose + 7.0~8.0 g / L agar; (4) The Coptis chinensis seedlings were inoculated into a rooting medium and cultured to obtain Coptis chinensis test-tube seedlings; the rooting medium was: 1 / 2 MS + 0.45~0.55 mg / L NAA + 0.08~0.12 mg / L IBA + 0.8~1.2 g / L activated carbon + 25~35 g / L sucrose + 7.0~8.0 g / L agar.
2. The method as described in claim 1, characterized in that, The disinfection method described in step (1) is as follows: disinfect with 70-80% alcohol for 30-60 seconds, rinse with sterile water 2-3 times, then disinfect with 0.08-0.12% mercuric chloride for 8-14 minutes, and rinse with sterile water 3-5 times.
3. The method as described in claim 1, characterized in that, The culture time described in step (1) is 8~12 days.
4. The method as described in claim 1, characterized in that, The culture time described in step (2) is 25~35 days.
5. The method as described in claim 1, characterized in that, The culture time described in step (3) is 30~40 days.
6. The method as described in claim 1, characterized in that, The culture time described in step (4) is 18~25 days.
7. The method according to any one of claims 1 to 6, characterized in that, The pH of the adventitious bud differentiation medium, the proliferation medium and the rooting medium are all 5.8~6.0; the light intensity during the culture in steps (1)~(4) is 2000~3000lx, the temperature is 25±2℃, the humidity is 65%~75%, and the light-dark cycle is 10~14h light and 10~14h dark alternating culture.
8. The method as described in claim 1, characterized in that, It also includes the step of hardening off and transplanting seedlings; the method of hardening off and transplanting seedlings is as follows: when the roots of the Coptis chinensis test tube seedlings grow to 3-5cm and the seedling height is 4-6cm, the bottle cap is opened and the seedlings are hardened off in a light incubator for 4-7 days; after hardening off, the Coptis chinensis test tube seedlings are taken out, sterilized and then transplanted into the seedling substrate for cultivation.
9. The method as described in claim 8, characterized in that, The temperature during seedling hardening is 25±2℃, and the sterilization method is to soak the Coptis chinensis test tube seedlings in a carbendazim solution for 20~30 minutes, wherein the concentration of the carbendazim solution is 1:800~1:1200.
10. The method as described in claim 8, characterized in that, The seedling substrate consists of peat moss, vermiculite, and river sand in a mass ratio of 1.5~2.5:0.8~1.2:0.8~1.2.