Cultivation method and acclimatization and transplantation method for coptis chinensis test-tube plantlets

Abstract

The invention provides a culture method and an acclimatization and transplantation method for coptis chinensis test-tube plantlets, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) disinfecting a coptis chinensis explant, inoculating the disinfected coptis chinensis explant into an MS basal culture medium, and culturing to obtain a sterile seedling; (2) inoculating the aseptic seedlings into an adventitious bud culture medium for adventitious bud culture to obtain adventitious buds; (3) inoculating the adventitious buds into a proliferation culture medium for proliferation culture to obtain proliferation seedlings; and (4) inoculating the proliferation seedlings into a rooting culture medium for rooting culture to obtain the coptis chinensis test-tube plantlets. When the adventitious bud, proliferation and rooting culture medium is used for culturing the coptis chinensis test-tube plantlet, the proliferation coefficient of the test-tube plantlet reaches 3.0, the rooting rate reaches 96.3%, and the survival rate of acclimatization and transplantation reaches 91.5% through seedling acclimatization and transplantation. The culture method provided by the invention is used for preserving the germplasm resources of the coptis chinensis, and provides a basic guarantee for the germplasm improvement of the subsequent medicinal material coptis chinensis.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and in particular relates to a method for culturing and hardening off test-tube seedlings of Coptis chinensis from Huanjiang and a method for transplanting seedlings. Background Technology

[0002] Huanjiang Huanglian ( Coptis huanjiangensis LQ Huang, QJ Yuan & YH Wang) discovered a new species during the fourth national survey of Chinese medicinal resources in Huanjiang Maonan Autonomous County, a karst region in Guangxi. This species, previously unknown, possesses the basic morphological characteristics of Coptis species, but is significantly taller and has a thicker rhizome, making it a "giant" among Coptis species. Its value lies in its size. Firstly, it is a close relative of Coptis chinensis, currently the most widely used medicinal herb in the market. The rhizome of Coptis chinensis is used medicinally, and this closely related species is an important wild resource for the breeding and trait improvement of Coptis chinensis. In the future, with the help of this species, it may be possible to obtain Coptis chinensis with even thicker rhizomes, thus greatly increasing yield. Secondly, its tall stature and thick rhizome indicate that it is a new medicinal resource with enormous potential, which is of profound significance to the development of the Coptis chinensis industry. However, the resource of this new species is currently extremely scarce. Establishing an effective tissue culture regeneration system can be used for the preservation of this endangered germplasm, providing a fundamental guarantee for the subsequent germplasm improvement of Coptis chinensis. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for cultivating and hardening off test-tube seedlings of Coptis chinensis from Huanjiang and a method for transplanting the seedlings. The method of this invention can improve the rooting rate and transplant survival rate of test-tube seedlings of Coptis chinensis from Huanjiang, providing a basic guarantee for the subsequent preservation of Coptis chinensis from Huanjiang.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for culturing test-tube seedlings of Coptis chinensis from Huanjiang, comprising the following steps: (1) After disinfecting the explants of Coptis chinensis from Huanjiang, they were inoculated into MS medium for culture to obtain sterile seedlings; (2) Sterile seedlings were inoculated into an adventitious bud culture medium to obtain adventitious buds; (3) Adventitious buds are inoculated into a proliferation medium for proliferation culture to obtain proliferated seedlings; (4) The propagated seedlings were inoculated into the rooting medium for rooting culture to obtain test tube seedlings of Coptis chinensis huanjiangensis.

[0005] Preferably, the adventitious bud culture medium comprises MS + 0.5~1.5 mg / L TDZ + 6.0~10 g / L agar + 28~32 g / L sucrose.

[0006] Preferably, the adventitious bud culture time is 26-30 days, and the adventitious bud culture temperature is 23-27℃.

[0007] Preferably, the proliferation medium comprises MS + 0.5~2.5 mg / L 6-KT + 0.2~0.6 mg / L NAA + 6.0~10 g / L agar + 28~32 g / L sucrose.

[0008] Preferably, the proliferation culture time is 25-35 days and the proliferation culture temperature is 23-27℃.

[0009] Preferably, the rooting medium comprises 1 / 2 MS + 0.5~1.5 mg / L IAA + 0.3~0.7 mg / L IBA + 6.0~10 g / L agar + 28~32 g / L sucrose.

[0010] Preferably, the rooting culture time is 20-30 days, and the rooting culture temperature is 23-27℃.

[0011] Preferably, the explants of *Coptis huanjiangensis* are seeds germinated from *Coptis huanjiangensis*.

[0012] This invention provides a method for hardening and transplanting seedlings of Coptis chinensis from Huanjiang. When the root length of the test-tube seedlings obtained according to the culture method reaches 3-4 cm, the seedlings are hardened for 3-5 days to obtain hardened test-tube seedlings, and the hardened test-tube seedlings are then transplanted. The substrate used for transplanting consists of nutrient soil, vermiculite, and perlite in a mass ratio of 1 to 5:1:1.

[0013] As a preferred option, disinfection treatment is also performed on the hardened-off test-tube seedlings before transplanting.

[0014] Compared with existing technologies, this invention has the following beneficial effects: By using specific adventitious bud culture medium, proliferation culture medium, and rooting culture medium to cultivate *Coptis chinensis* test-tube seedlings, and through the selection of hardening-off and transplanting substrates, the proliferation coefficient of *Coptis chinensis* test-tube seedlings can reach 3.0, the rooting rate can reach 96.3%, and the survival rate after hardening-off and transplanting can reach 91.5%. The culture method provided by this invention can be used for the preservation of *Coptis chinensis* germplasm resources, providing a fundamental guarantee for the subsequent germplasm improvement of the medicinal herb *Coptis chinensis*. Attached Figure Description

[0015] Figure 1 These are seeds germinated from Coptis chinensis in Huanjiang. Figure 2 These are sterile seedlings of Coptis chinensis from Huanjiang. Figure 3 The growth status of adventitious buds in Coptis chinensis cultured in Huanjiang after 7 days; Figure 4 The growth status of adventitious buds after 14 days of cultivation in Huanjiang Coptis chinensis; Figure 5 The growth status of adventitious buds after 21 days of cultivation in Huanjiang Coptis chinensis; Figure 6 The growth status of adventitious buds after 28 days of cultivation in Huanjiang Coptis chinensis; Figure 7 These are seedlings of Coptis chinensis propagated in Huanjiang. Figure 8 These are test-tube seedlings of Coptis chinensis from Huanjiang. Figure 9 This is a picture of the transplanting of Coptis chinensis seedlings in Huanjiang. Detailed Implementation

[0016] This invention provides a method for culturing test-tube seedlings of Coptis chinensis from Huanjiang, comprising the following steps: (1) After disinfecting the explants of Coptis chinensis from Huanjiang, they were inoculated into MS basal medium for culture to obtain sterile seedlings; (2) Sterile seedlings were inoculated into an adventitious bud culture medium to obtain adventitious buds; (3) Adventitious buds are inoculated into a proliferation medium for proliferation culture to obtain proliferated seedlings; (4) The propagated seedlings were inoculated into the rooting medium for rooting culture to obtain test tube seedlings of Coptis chinensis huanjiangensis.

[0017] In this invention, the explants of Coptis chinensis from Huanjiang are disinfected and then cultured in MS basal medium to obtain sterile seedlings.

[0018] In this invention, the explant of *Coptis huanjiangensis* is the seed of *Coptis huanjiangensis*. The germination method of the seed of *Coptis huanjiangensis* is as follows: the seed of *Coptis huanjiangensis* is mixed with moist vermiculite and placed in a low-temperature storage cabinet at 2~6℃ until the seed germinates. The mass ratio of the seed of *Coptis huanjiangensis* to moist vermiculite is 1:4~6, preferably 1:4.5~5.5, more preferably 1:5. The moisture content of the moist vermiculite is 50%~70%, preferably 55%~65%, more preferably 60%. The temperature of the low-temperature storage cabinet is preferably 3~5℃, more preferably 4℃.

[0019] In this invention, the disinfectant solution used for the explants of *Coptis chinensis* comprises one or more of the following: 65%–85% ethanol, 1%–3% sodium hypochlorite solution, and sterile water; preferably, one or more of 70%–80% ethanol, 1.5%–2.5% sodium hypochlorite solution, and sterile water; more preferably, one or more of 75% ethanol, 2% sodium hypochlorite solution, and sterile water; the disinfection is performed by sequentially using ethanol and sodium hypochlorite solution; the disinfection time using ethanol is 20–40 seconds, preferably 25–35 seconds, more preferably 30 seconds; the disinfection time using sodium hypochlorite solution is 5–10 seconds. The disinfection time is 1 min, preferably 6-9 min, and more preferably 7 min; after disinfection, the sample is rinsed 2-4 times with sterile water; the basic culture time is 8-12 days, preferably 9-11 days, and more preferably 10 days; the light intensity of the basic culture is 2800-3200 lx, preferably 2900-3100 lx, and more preferably 3000 lx; ​​the light duration of the basic culture is 12-16 h, preferably 13-15 h, and more preferably 14 h; the humidity of the basic culture is 60%-80%, preferably 65%-75%, and more preferably 70%.

[0020] In this invention, aseptic seedlings are inoculated into an adventitious bud culture medium to obtain adventitious buds; the adventitious bud culture medium comprises MS + 0.5~1.5 mg / L TDZ + 6.0~10 g / L agar + 28~32 g / L sucrose, preferably MS + 0.7~1.2 mg / L TDZ + 7.0~9 g / L agar + 29~31 g / L sucrose, and more preferably MS + 1.0 mg / L TDZ + 8.0 g / L agar + 30 g / L sucrose. The adventitious bud culture time is 26-30 days, preferably 27-29 days, more preferably 28 days; the adventitious bud culture temperature is 23-27℃, preferably 24-26℃, more preferably 25℃; the adventitious bud culture light intensity is 2800-3200 lx, preferably 2900-3100 lx, more preferably 3000 lx; ​​the adventitious bud culture light duration is 12-16 hours, preferably 13-15 hours, more preferably 14 hours; and the adventitious bud culture humidity is 60%-80%, preferably 65%-75%, more preferably 70%.

[0021] In this invention, adventitious buds are inoculated into a proliferation medium for proliferation culture to obtain proliferating seedlings. The proliferation medium comprises MS + 0.5~2.5 mg / L 6-KT + 0.2~0.6 mg / L NAA + 6.0~10 g / L agar + 28~32 g / L sucrose, preferably MS + 1~2 mg / L 6-KT + 0.3~0.5 mg / L NAA + 7.0~9.0 g / L agar + 29~31 g / L sucrose, and more preferably MS + 1.5 mg / L 6-KT + 0.4 mg / L NAA. The culture medium consists of NAA (8.0 g / L agar) and sucrose (30 g / L). The culture time is 25-35 days, preferably 28-32 days, more preferably 30 days. The culture temperature is 23-27°C, preferably 24-26°C, more preferably 25°C. The light intensity is 2800-3200 lx, preferably 2900-3100 lx, more preferably 3000 lx. The light duration is 12-16 hours, preferably 13-15 hours, more preferably 14 hours. The humidity is 60%-80%, preferably 65%-75%, more preferably 70%.

[0022] In this invention, propagated seedlings are inoculated into a rooting medium for rooting culture to obtain test-tube seedlings of *Coptis chinensis*. Preferably, the propagated seedlings are cut into individual plants, and each individual plant is inoculated into a rooting medium for rooting culture. The rooting medium comprises 1 / 2 MS + 0.5~1.5 mg / L IAA + 0.3~0.7 mg / L IBA + 6.0~10 g / L agar + 28~32 g / L sucrose; more preferably, 1 / 2 MS + 0.7~1.2 mg / L IAA + 0.4~0.6 mg / L IBA + 7.0~9.0 g / L agar + 29~31 g / L sucrose; and even more preferably, 1 / 2 MS + 1.0 mg / L IAA + 0.5 mg / L IBA. The rooting culture consists of IBA + 8.0 g / L agar + 30 g / L sucrose; the rooting culture time is 20-30 days, preferably 23-27 days, more preferably 35 days; the rooting culture temperature is 23-27℃, preferably 24-26℃, more preferably 25℃; the light intensity of the rooting culture is 2800-3200 lx, preferably 2900-3100 lx, more preferably 3000 lx; ​​the light duration of the rooting culture is 12-16 hours, preferably 13-15 hours, more preferably 14 hours; and the humidity of the rooting culture is 60%-80%, preferably 65%-75%, more preferably 70%.

[0023] This invention provides a method for hardening and transplanting seedlings of Coptis chinensis from Huanjiang. When the root length of the test-tube seedlings obtained according to the culture method reaches 3-4 cm, the seedlings are hardened for 3-5 days to obtain hardened test-tube seedlings, and the hardened test-tube seedlings are then transplanted. The substrate used for transplanting is composed of nutrient soil, vermiculite, and perlite in a mass ratio of 1 to 5:1:1. The preferred mass ratio of nutrient soil, vermiculite, and perlite in the substrate is 2 to 4:1:1, and more preferably 3:1:1.

[0024] In this invention, the disinfection treatment of the hardened test-tube seedlings is also included before transplanting. The disinfection method is to soak the test-tube seedlings in a 1:600~1000 chlorothalonil solution, preferably 1:700~900, more preferably 1:800, for a soaking time of 20~40 min, preferably 25~35 min, more preferably 30 min.

[0025] 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.

[0026] Example 1

[0027] A method for culturing test-tube seedlings of Coptis chinensis from Huanjiang, the steps of which are as follows: (1) The seeds of Coptis chinensis (natural population of Coptis chinensis in Huanjiang, Hechi City, Guangxi Zhuang Autonomous Region) were mixed with wet vermiculite at a ratio of 1:5 and stored in a 4-degree low-temperature storage cabinet until the seeds germinated (5-8 months). The germinated seeds of Coptis chinensis were used. Figure 1 As explants for tissue culture, after preliminary cleaning and disinfection, they were inoculated into MS basal medium to cultivate sterile Coptis chinensis seedlings. The disinfection method was as follows: disinfection with 75% alcohol for 30 seconds in a laminar flow hood, rinsing twice with sterile water, then disinfection with 2% sodium hypochlorite (NaClO) solution for 7 minutes, rinsing four times with sterile water; placing them on sterile filter paper to absorb surface moisture, inoculating into MS basal medium, and culturing for 10 days to obtain sterile Coptis chinensis seedlings from Huanjiang. Figure 2 ); (2) Aseptic seedlings of *Coptis chinensis* were inoculated onto MS medium containing 1.0 mg / L TDZ agar, 8.0 g / L agar, and 30 mg / L sucrose (pH 5.8). The culture was carried out at a light intensity of 3000 lx, a temperature of 25 ± 2℃, and a humidity of 70%. The light culture time was set to 14 h, followed by 10 h in the dark, for a total of 30 days to obtain adventitious buds. Figures 3-6 ); (3) Adventitious buds were inoculated into MS medium (pH 5.8) containing 1.5 mg / L 6-KT, 0.4 mg / L NAA, 8.0 g / L agar, and 30 mg / L sucrose. Only one new bud was inoculated into each bottle of medium. The light intensity was 3000 lx, the temperature was 25 ± 2℃, and the humidity was 70%. The light culture time was set to 14 h, the dark culture time to 10 h, and the culture time to 28 days to obtain Huanjiang Coptis chinensis propagation seedlings. Figure 7 ); (4) After cutting the obtained Huanjiang Huanglian propagation seedlings into individual plants, they were transferred to rooting medium (pH 5.8) consisting of 1 / 2 MS + IAA 1.0 mg / L + IBA 0.5 mg / L + 8.0 g / L agar + sucrose 30 g / L. The light intensity was 3000 lx, the temperature was between 25 ± 2℃, and the humidity was 70%. The light culture time was set to 14 h, the dark culture time to 10 h, and the culture time to 30 days. When the roots of the Huanjiang Huanglian test-tube seedlings reached about 3 cm in length, the cap of the tissue culture bottle was removed, and the seedlings were hardened off in a light incubator for 4 days to obtain Huanjiang Huanglian test-tube seedlings. Figure 8 Wash the agar off the base of the test tube seedlings, soak them in a 1:800 chlorothalonil solution for 30 minutes, and then transplant them into seedling pots containing seedling substrate. Figure 9 Seedlings of *Coptis chinensis* were obtained by setting the light intensity to 3500 lx, the temperature to 25±2℃, and the humidity to 75%, with a light culture time of 14 h and a dark culture time of 10 h, and a culture time of 30 days. The seedling substrate was nutrient soil: vermiculite: perlite = 3:1:1.

[0028] Experiment 1: Effect of disinfection time on the growth of explants of Coptis chinensis from Huanjiang County

[0029] The seeds of *Coptis huanjiangensis* were mixed with moist vermiculite at a ratio of 1:5 and stored in a 4°C low-temperature storage cabinet for 5-8 months until germination. Germinated seeds were used as explants for tissue culture. After preliminary cleaning and disinfection, they were inoculated into MS basal medium to cultivate sterile seedlings. The disinfection method was as follows: disinfection with 75% alcohol for 30 seconds in a clean bench, rinsing twice with sterile water, then disinfection with 2% sodium hypochlorite (NaClO) solution for 5-11 minutes, rinsing four times with sterile water; the seedlings were then placed on sterile filter paper to absorb surface moisture and inoculated into MS basal medium for 10 days. The sterility rate, contamination rate, and mortality rate were recorded, and the results are shown in Table 1.

[0030] Table 1. Effects of 2% sodium hypochlorite disinfection at different times on the growth of explants of Coptis chinensis from Huanjiang.

[0031] As shown in Table 1, the disinfection time directly affects the growth of explants of Coptis chinensis in Huanjiang. As the disinfection time increases, although the number of contaminants decreases, the number of deaths also increases, indicating that the longer the disinfection time, the better. When the disinfection time is 7 minutes, the mortality rate is the lowest and the sterility rate is the highest. Therefore, when using 2% sodium hypochlorite solution to disinfect explants, a disinfection time of 7 minutes should be selected.

[0032] Experimental Example 2

[0033] The method described in Example 1 was used to cultivate test-tube seedlings of Coptis chinensis from Huanjiang. The only difference was the hormone concentration and ratio in the proliferation medium. The proliferation coefficient was calculated when the hormone concentration and ratio in the proliferation medium were different. The specific concentrations, ratios and results are shown in Table 2.

[0034] Proliferation coefficient = (Number of plants after propagation / Number of inoculated plants) × 100%.

[0035] Table 2. Effects of different hormone concentrations and ratios on the proliferation culture of Coptis chinensis in Huanjiang.

[0036] As shown in Table 2, different concentrations of various hormones in the proliferation medium can directly affect the proliferation coefficient. When the concentration of 6-KT is 1.5 mg / L and the concentration of NAA is 0.4 mg / L, the proliferation coefficient is the highest at 3.0.

[0037] Experimental Example 3

[0038] The method described in Example 1 was used to cultivate test-tube seedlings of Coptis chinensis from Huanjiang. The only difference was the concentration and ratio of hormones in the rooting medium. The rooting rate was statistically analyzed when the hormone concentration and ratio in the rooting medium were different. The specific concentrations, ratios and results are shown in Table 3.

[0039] Rooting rate = Number of rooted plants / Number of inoculated plants × 100%.

[0040] Table 3. Effects of different hormone concentrations and ratios on rooting culture of Coptis chinensis in Huanjiang County.

[0041] As shown in Table 3, different types of rooting media can directly affect the rooting rate. The highest rooting rate of 96.3% was achieved when the hormone concentration in the rooting medium was IAA 1.0 mg / L + IBA 0.5 mg / L.

[0042] Test Example 4

[0043] The method described in Example 1 was used to cultivate test-tube seedlings of Coptis chinensis from Huanjiang. The only difference was the seedling substrate and its ratio. The survival rate of test-tube seedlings of Coptis chinensis from Huanjiang transplanted into different substrate ratios was statistically analyzed. The specific seedling substrates, ratios and results are shown in Table 4.

[0044] Survival rate = (Number of surviving plants / Number of hardened-off plants) × 100%.

[0045] Table 4. Effects of different substrate ratios on the survival rate of Coptis chinensis seedlings transplanted from Huanjiang.

[0046] As shown in Table 4, the composition and ratio of the seedling substrate can directly affect the survival rate of Huanjiang Coptis chinensis seedlings after transplanting. When the ratio of nutrient soil: vermiculite: perlite in the seedling substrate is 3:1:1, the survival rate is the highest at 91.5%.

[0047] 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 cultivation method of Coptis teeta tube seedlings, characterized in that, It comprises the following steps: (1) disinfecting the Coptis deltoidea C.Y.Wu et How ex Chu ex Huang explants and then inoculating them into MS basic medium to obtain sterile seedlings; (2) inoculating the sterile seedlings into adventitious bud medium to culture adventitious buds; (3) inoculating the adventitious buds into proliferation medium to culture and proliferate the adventitious buds and obtain proliferated seedlings; (4) inoculating the proliferated seedlings into rooting medium to culture and root the proliferated seedlings and obtain Coptis deltoidea C.Y.Wu et How ex Chu ex Huang test tube seedlings.

2. The culture method according to claim 1, characterized by, The adventitious bud medium comprises MS+0.5~1.5mg / L TDZ+6.0~10g / L agar+28~32g / L sucrose.

3. The culturing method according to claim 2, wherein, The adventitious bud culture is performed for 26~30d at a temperature of 23~27℃.

4. The culturing method according to claim 1, wherein The proliferation medium comprises MS+0.5~2.5mg / L 6-KT+0.2~0.6mg / L NAA+6.0~10g / L agar+28~32g / L sucrose.

5. The culture method according to claim 4, characterized by, The proliferation culture is performed for 25~35d at a temperature of 23~27℃.

6. The culturing method according to claim 1, wherein The rooting medium comprises 1 / 2MS+0.5~1.5mg / L IAA+0.3~0.7mg / L IBA+6.0~10g / L agar+28~32g / L sucrose.

7. The culture method according to claim 6, characterized by, The rooting culture is performed for 20~30d at a temperature of 23~27℃.

8. The culture method according to any one of claims 1 to 7, characterized by, The Coptis deltoidea C.Y.Wu et How ex Chu ex Huang explants are Coptis deltoidea C.Y.Wu et How ex Chu ex Huang germinated seeds.

9. A method for transplanting seedlings of Coptis teeta by hardening, characterized in that, The test tube seedlings obtained by the culture method of any one of claims 1~8 are acclimatized for 3~5 days when the root length reaches 3~4cm, and then the acclimatized test tube seedlings are transplanted. The substrate for the transplantation is composed of nutrient soil, vermiculite and perlite with a mass ratio of 1~5:1:

1.

10. The transplanting method according to claim 9, wherein The acclimatized test tube seedlings are further subjected to disinfection treatment before the transplantation.

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