A cultivation method and a seedling hardening and transplanting method for tube seedlings of cyclocarya paliurus

Abstract

The application provides a culture method and a seedling hardening and transplanting method of Huangjiang Coptis test-tube seedlings, and belongs to the technical field of plant tissue culture. The application comprises the following steps: (1) after disinfecting Huangjiang Coptis explants, the explants are inoculated into MS basic culture medium for culture to obtain sterile seedlings; (2) the sterile seedlings are inoculated into adventitious bud culture medium for adventitious bud culture to obtain adventitious buds; (3) the adventitious buds are inoculated into proliferation culture medium for proliferation culture to obtain proliferation seedlings; and (4) the proliferation seedlings are inoculated into rooting culture medium for rooting culture to obtain Huangjiang Coptis test-tube seedlings. The adventitious bud culture medium, the proliferation culture medium and the rooting culture medium are used for culturing the Huangjiang Coptis test-tube seedlings, through selection of a seedling hardening and transplanting matrix, the proliferation coefficient of the test-tube seedlings reaches 3.0, the rooting rate reaches 96.3%, and the seedling hardening and transplanting survival rate reaches 91.5%. The culture method can be used for preservation of Huangjiang Coptis germplasm resources, and provides a basic guarantee for subsequent improvement of Huangjiang Coptis medicinal material germplasm.

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:

[0005] This invention provides a method for culturing test-tube seedlings of Coptis chinensis from Huanjiang, comprising the following steps:

[0006] (1) After disinfecting the explants of Coptis chinensis from Huanjiang, they were inoculated into MS medium for culture to obtain sterile seedlings;

[0007] (2) Sterile seedlings were inoculated into an adventitious bud culture medium to obtain adventitious buds;

[0008] (3) Adventitious buds are inoculated into a proliferation medium for proliferation culture to obtain proliferated seedlings;

[0009] (4) The propagated seedlings were inoculated into the rooting medium for rooting culture to obtain test tube seedlings of Coptis chinensis huanjiangensis.

[0010] 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.

[0011] Preferably, the adventitious bud culture time is 26-30 days, and the adventitious bud culture temperature is 23-27℃.

[0012] 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.

[0013] Preferably, the proliferation culture time is 25-35 days and the proliferation culture temperature is 23-27℃.

[0014] 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.

[0015] Preferably, the rooting culture time is 20-30 days, and the rooting culture temperature is 23-27℃.

[0016] Preferably, the explants of *Coptis huanjiangensis* are seeds germinated from *Coptis huanjiangensis*.

[0017] 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.

[0018] The substrate used for transplanting consists of nutrient soil, vermiculite, and perlite in a mass ratio of 1 to 5:1:1.

[0019] As a preferred option, disinfection treatment is also performed on the hardened-off test-tube seedlings before transplanting.

[0020] 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

[0021] Figure 1 These are seeds germinated from Coptis chinensis in Huanjiang.

[0022] Figure 2 is aseptic seedling of Huanglian;

[0023] Figure 3 is the growth state of adventitious bud of Huanglian cultured for 7 days;

[0024] Figure 4 is the growth state of adventitious bud of Huanglian cultured for 14 days;

[0025] Figure 5 is the growth state of adventitious bud of Huanglian cultured for 21 days;

[0026] Figure 6 is the growth state of adventitious bud of Huanglian cultured for 28 days;

[0027] Figure 7 is the proliferation seedling of Huanglian;

[0028] Figure 8 is the test tube seedling of Huanglian;

[0029] Figure 9 is the seedling training and transplanting diagram of Huanglian. DETAILED DESCRIPTION

[0030] The application provides a culture method of test tube seedling of Huanglian, which comprises the following steps:

[0031] (1) after disinfecting the explant of Huanglian, the explant is inoculated into MS basic culture medium for culture to obtain aseptic seedling;

[0032] (2) the aseptic seedling is inoculated into adventitious bud culture medium for adventitious bud culture to obtain adventitious bud;

[0033] (3) the adventitious bud is inoculated into proliferation culture medium for proliferation culture to obtain proliferation seedling;

[0034] (4) the proliferation seedling is inoculated into rooting culture medium for rooting culture to obtain the test tube seedling of Huanglian.

[0035] In the application, after disinfecting the explant of Huanglian, the explant is inoculated into MS basic culture medium for culture to obtain aseptic seedling.

[0036] In the application, the explant of Huanglian is the germinated seed of Huanglian, and the germination method of the germinated seed of Huanglian is that the seed of Huanglian is mixed with wet vermiculite and placed in a low-temperature storage cabinet at 2-6 ℃, and the seed is stored until germination; the mass ratio of the seed of Huanglian to wet vermiculite is 1:4-6, preferably 1:4.5-5.5, and more preferably 1:5; the humidity of the wet vermiculite is 50%-70%, preferably 55%-65%, and more preferably 60%; and the temperature of the low-temperature storage cabinet is preferably 3-5 ℃, and more preferably 4 ℃.

[0037] In the present application, the liquid medicine used for disinfecting the Coptis teeta external explants comprises one or more of 65% to 85% ethanol, 1 to 3% sodium hypochlorite solution and sterile water, preferably one or more of 70% to 80% ethanol, 1.5 to 2.5% sodium hypochlorite solution and sterile water, and further preferably one or more of 75% ethanol, 2% sodium hypochlorite solution and sterile water; the disinfection is performed by using ethanol and sodium hypochlorite solution in sequence; the disinfection time using ethanol is 20 to 40s, preferably 25 to 35s, and further preferably 30s; the disinfection time using sodium hypochlorite solution is 5 to 11min, preferably 6 to 9min, and further preferably 7min; the disinfection is followed by rinsing with sterile water for 2 to 4 times; the basic culture time is 8 to 12d, preferably 9 to 11d, and further preferably 10d; the basic culture light intensity is 2800 to 3200lx, preferably 2900 to 3100lx, and further preferably 3000lx; the basic culture light time is 12 to 16h, preferably 13 to 15h, and further preferably 14h; and the basic culture humidity is 60% to 80%, preferably 65% to 75%, and further preferably 70%.

[0038] In the present application, the sterile seedlings are inoculated into adventitious bud medium to perform adventitious bud culture, so as to obtain adventitious buds; the adventitious bud medium comprises MS+0.5 to 1.5mg / L TDZ+6.0 to 10g / L agar+28 to 32g / L sucrose, preferably MS+0.7 to 1.2mg / L TDZ+7.0 to 9.g / L agar+29 to 31g / L sucrose, and further preferably MS+1.0mg / L TDZ+8.0g / L agar+30g / L sucrose. The adventitious bud culture time is 26 to 30d, preferably 27 to 29d, and further preferably 28d; the adventitious bud culture temperature is 23 to 27℃, preferably 24 to 26℃, and further preferably 25℃; the adventitious bud culture light intensity is 2800 to 3200lx, preferably 2900 to 3100lx, and further preferably 3000lx; the adventitious bud culture light time is 12 to 16h, preferably 13 to 15h, and further preferably 14h; and the adventitious bud culture humidity is 60% to 80%, preferably 65% to 75%, and further preferably 70%.

[0039] In the present application, the adventitious buds are inoculated into the proliferation culture medium for proliferation culture to obtain the proliferation seedlings. The proliferation culture 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+8.0 g / L agar+30 g / L sucrose. The proliferation culture time is 25-35 d, preferably 28-32 d, and more preferably 30 d. The proliferation culture temperature is 23-27℃, preferably 24-26℃, and more preferably 25℃. The proliferation culture light intensity is 2800-3200 lx, preferably 2900-3100 lx, and more preferably 3000 lx. The proliferation culture light time is 12-16 h, preferably 13-15 h, and more preferably 14 h. The proliferation culture humidity is 60%-80%, preferably 65%-75%, and more preferably 70%.

[0040] In the present application, the proliferation seedlings are inoculated into the rooting culture medium for rooting culture to obtain the H. henryi test tube seedlings. Preferably, the proliferation seedlings are cut into single plants, and the single plants are respectively inoculated into the rooting culture medium for rooting culture. The rooting culture medium comprises 1 / 2MS+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, preferably 1 / 2MS+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 more preferably 1 / 2MS+1.0 mg / L IAA+0.5 mg / L IBA+8.0 g / L agar+30 g / L sucrose. The rooting culture time is 20-30 d, preferably 23-27 d, and more preferably 35 d. The rooting culture temperature is 23-27℃, preferably 24-26℃, and more preferably 25℃. The rooting culture light intensity is 2800-3200 lx, preferably 2900-3100 lx, and more preferably 3000 lx. The rooting culture light time is 12-16 h, preferably 13-15 h, and more preferably 14 h. The rooting culture humidity is 60%-80%, preferably 65%-75%, and more preferably 70%.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Example 1

[0046] A method for culturing test-tube seedlings of Coptis chinensis from Huanjiang, the steps of which are as follows:

[0047] (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 );

[0048] (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. Figure 3~Figure 6 );

[0049] (3) The adventitious buds are inoculated into a proliferation medium of MS+1.5 mg / L 6-KT+0.4 mg / L NAA+8.0 g / L agar+30 mg / L sucrose (pH 5.8), and only one new bud is inoculated into each medium, and the culture is carried out at a light intensity of 3000 lx, a temperature of 25±2°C, a humidity of 70%, a light culture time of 14 h, and a dark culture time of 10 h for 28 days to obtain the H. jiangjiangensis proliferation seedlings Figure 7

[0050] (4) The H. jiangjiangensis proliferation seedlings obtained are cut into single plants and transferred into a rooting medium of 1 / 2MS+IAA 1.0 mg / L+IBA 0.5 mg / L+8.0 g / L agar+30 g / L sucrose (pH 5.8), and the culture is carried out at a light intensity of 3000 lx, a temperature of 25±2°C, a humidity of 70%, a light culture time of 14 h, and a dark culture time of 10 h for 30 days, and the H. jiangjiangensis test tube seedlings are obtained when the roots of the seedlings grow to about 3 cm, the agar at the base of the seedlings is washed, the seedlings are soaked in a 1:800 fungicide solution for 30 min, and the seedlings are transplanted into a seedling raising pot containing a seedling raising substrate Figure 8 Figure 9 , the culture is carried out at a light intensity of 3500 lx, a temperature of 25±2°C, a humidity of 75%, a light culture time of 14 h, and a dark culture time of 10 h for 30 d to obtain the H. jiangjiangensis seedlings, and the seedling raising substrate is nutrient soil:vermiculite:perlite=3:1:1.

[0051] Test Example 1: Influence of sterilization time on the growth of H. jiangjiangensis explants

[0052] The seeds of H. jiangjiangensis are mixed with wet vermiculite at a ratio of 1:5, placed in a 4°C low-temperature storage cabinet, and stored for 5-8 months until the seeds germinate. The germinated seeds of H. jiangjiangensis are used as the tissue culture explants, inoculated into the MS basic medium after preliminary cleaning and sterilization, and sterile seedlings of H. jiangjiangensis are obtained by cultivation. The sterilization method is as follows: the seeds are sterilized with 75% alcohol for 30 s, washed with sterile water for 2 times, sterilized with a 2% sodium hypochlorite (NaClO) solution for 5-11 min, and washed with sterile water for 4 times; the surface water is absorbed on sterile filter paper, and the seeds are inoculated into the MS basic medium for 10 days. The sterile rate, contamination rate, and mortality rate are counted, and the results are shown in Table 1.

[0053] Table 1: Influence of different sterilization times of 2% sodium hypochlorite on the growth of H. jiangjiangensis explants

[0054]

[0055] ​​From Table 1, the disinfection time can directly affect the growth of the H. jiangxiensis explants. With the extension of the disinfection time, the number of contamination will decrease, but the number of death will also increase, indicating that the longer the disinfection time is, the better it is not. When the disinfection time is 7 min, the mortality rate is the lowest, and the aseptic rate is the highest. Therefore, when the explants are disinfected with 2% sodium hypochlorite solution, the disinfection time of 7 min is selected.

[0056] Test Example 2

[0057] The H. jiangxiensis test tube seedlings were cultured by the method in Example 1, with the difference being only that the hormone concentrations and ratios in the proliferation medium were different. The proliferation coefficients of the hormone concentrations and ratios in the proliferation medium were calculated, and the specific concentrations and ratios and the results are shown in Table 2.

[0058] The proliferation coefficient = the number of strains after proliferation / the number of inoculated strains x 100%.

[0059] Table 2 Influence of different hormone concentrations and ratios on the proliferation culture of H. jiangxiensis

[0060]

[0061] From Table 2, the hormone concentrations 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 largest, which is 3.0.

[0062] Test Example 3

[0063] The H. jiangxiensis test tube seedlings were cultured by the method in Example 1, with the difference being only that the hormone concentrations and ratios in the rooting medium were different. The rooting rates of the hormone concentrations and ratios in the rooting medium were calculated, and the specific concentrations and ratios and the results are shown in Table 3.

[0064] The rooting rate = the number of rooted strains / the number of inoculated strains x 100%.

[0065] Table 3 Influence of different hormone concentrations and ratios on the rooting culture of H. jiangxiensis

[0066]

[0067] From Table 3, the type of the rooting medium can directly affect the rooting rate. When the hormone concentrations in the rooting medium are IAA 1.0 mg / L + IBA 0.5 mg / L, the rooting rate is the highest, which is 96.3%.

[0068] Test Example 4

[0069] The H. jiangxiensis test tube seedlings were cultured by the method in Example 1, with the difference being only that the seedling substrates and ratios were different. The survival rates of the H. jiangxiensis test tube seedlings transplanted in different substrate ratios were calculated, and the seedling substrates and ratios and the results are shown in Table 4.

[0070] Survival rate = number of surviving plants / number of seedling plants x 100%.

[0071] Table 4 Influence of different substrate ratios on the survival rate of H. jiangxiense seedling transplanting

[0072]

[0073] From Table 4, it can be seen that the components and ratios of the seedling substrate can directly affect the survival rate of H. jiangxiense seedling transplanting. When the nutrient soil: vermiculite: perlite in the seedling substrate is 3:1:1, the survival rate is the highest, which is 91.5%.

[0074] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A cultivation method of Coptis teeta tube seedlings, characterized in that, It comprises the following steps: (1) disinfecting the Coptis teeta outer explants and then inoculating them into MS basic medium for culture to obtain aseptic seedlings; (2) inoculating the aseptic seedlings into adventitious bud culture medium for adventitious bud culture to obtain adventitious buds; (3) inoculating the adventitious buds into proliferation culture medium for proliferation culture to obtain proliferation seedlings; (4) inoculating the proliferation seedlings into rooting culture medium for rooting culture to obtain Coptis teeta test-tube seedlings; the adventitious bud culture medium is composed of MS+0.5~1.5mg / L TDZ+6.0~10g / L agar+28~32g / L sucrose; the proliferation culture medium is composed of MS+0.5~2.5mg / L 6-KT+0.2~0.4mg / L NAA+6.0~10g / L agar+28~32g / L sucrose; the rooting culture medium is composed of 1 / 2MS+0.5~1.5mg / L IAA+0.3~0.7mg / L IBA+6.0~10g / L agar+28~32g / L sucrose; the Coptis teeta outer explants are Coptis teeta germinated seeds.

2. The culture method according to claim 1, characterized by, the adventitious bud culture time is 26~30d, and the adventitious bud culture temperature is 23~27℃.

3. The culturing method according to claim 1, wherein the proliferation culture time is 25~35d, and the proliferation culture temperature is 23~27℃.

4. The culturing method according to claim 1, wherein the rooting culture time is 20~30d, and the rooting culture temperature is 23~27℃.

5. The culturing method according to claim 1, wherein It also comprises a seedling hardening and transplanting step, wherein the seedling hardening method is as follows: when the root length of the Coptis teeta test-tube seedlings reaches 3~4cm, the seedlings are hardened for 3~5 days to obtain hardened test-tube seedlings, and then the hardened test-tube seedlings are transplanted; the transplanting substrate is composed of nutrient soil, vermiculite and perlite at a mass ratio of 1~5:1:

1.

6. The culturing method according to claim 5, wherein It also comprises a disinfection treatment of the hardened test-tube seedlings before transplanting.

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