Cultivation method of bletilla striata

Abstract

The invention provides a cultivation method of bletilla chinensis, and belongs to the technical field of plant tissue culture. According to the bletilla chinensis cultivation method, the pseudobulbs and roots of bletilla chinensis are adopted as explants, the callus is obtained through a continuous induction method, then the callus is used for regenerating a complete plant, the rapid propagation method of the endangered plant bletilla chinensis is established, a feasible scheme with an excellent effect can be provided for protecting the germplasm resources of bletilla chinensis, and the method is suitable for large-scale popularization and application. The problem that in the prior art, a bletilla striata tissue culture scheme depends on mature capsules or seeds, but limitation of slow growth, long seed maturation period, difficult seed germination and the like of plants cannot be avoided is solved, complete plants can be rapidly obtained in batches under the condition that no bletilla striata or mature capsules or seeds exist, and the survival rate of the bletilla striata is increased. The rapid, stable and continuous supply of the bletilla chinensis plants is realized, and a new method is provided for the germplasm resource protection of the bletilla chinensis.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for cultivating Bletilla striata. Background Art

[0002] Whitehead Bletilla sinensis (Rolfe) Schltr.) is a perennial herbaceous plant of the genus Bletilla in the Orchidaceae family. It was first discovered by British plant collector Augustine Henry in Mengzi, Yunnan in 1898 and published in the international botanical journal "Linnaeus's Botanical Journal" in 1903. Bletilla striata mostly grows on rock walls or cracks in rocks. Its natural fruiting rate is very low and the seed germination conditions are harsh. Coupled with factors such as climate change and human picking, Bletilla striata is close to extinction. Bletilla striata has been listed as an endangered species in the Red List of the World Conservation Union (IUCN) and is listed as an Appendix II species in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

[0003] Chinese patent CN115589948A discloses a non-symbiotic germination culture medium and propagation method of Glechoma longituba, wherein the culture medium is a modified KC culture medium, wherein the modified KC culture medium is modified by adding coconut powder and activated carbon powder to the KC basic culture medium, and the propagation method comprises the following steps: disinfection of Glechoma longituba fruit pods and seed treatment; non-symbiotic germination and cultivation of Glechoma longituba: sowing Glechoma longituba seeds on the modified KC culture medium for germination; subculture of Glechoma longituba seedlings: subculture of Glechoma longituba seedlings that have not turned brown after germination and cultivation when they grow to a height of 3-4 cm; transplantation and planting of Glechoma longituba seedlings: when the Glechoma longituba seedlings grow to the top of the culture bottle cap, they can be hardened out of the bottle for transplantation.

[0004] This scheme is a method of obtaining a complete plant using S. chinensis seeds as explants, which is similar to the tissue culture schemes of other Bletilla species in the prior art, and is a scheme of rapid propagation using mature capsules or seeds as explants. However, since the number of S. chinensis in nature is extremely small and there are almost no mature fruit pods, the reported methods have obvious defects and are not suitable for the rescue of S. chinensis germplasm resources, and have low practical significance. Summary of the invention

[0005] The purpose of the present invention is to provide a method for cultivating Scutellaria baicalensis, which provides an effective solution for protecting Scutellaria baicalensis germplasm resources.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for inducing callus tissue of Chinese white wormwood, comprising the following steps: Inoculating the explants of the Chinese white wormwood into an induction medium for induction culture to obtain Chinese white wormwood callus tissue; The induction medium is based on MS medium and contains sucrose and agar.

[0007] Preferably, the source of the S. cerana explant is the root of S. cerana and / or the pseudobulb of S. cerana.

[0008] Preferably, the final concentration of sucrose in the induction medium is 20-40 g / L; The final concentration of agar in the induction medium is 5-10 g / L; And / or, the induction culture comprises 5 to 15 subcultures, each subculture using the same culture medium; The subculture frequency of the subculture is 20 to 30 days / time; The white scutellaria and explants are sterilized and then inoculated; The reagents used for disinfection and sterilization include ethanol, sterile water or sodium hypochlorite solution; The culture conditions of the induction culture are: 20-25° C., dark culture.

[0009] The present invention also provides the Huabai and callus tissue obtained by the above-mentioned Huabai and callus tissue induction method.

[0010] The present invention also provides a method for inducing a Scutellaria baicalensis plant, comprising the following steps: Inoculating the callus tissue of the Chinese white wormwood into a regeneration medium for regeneration induction culture to obtain Chinese white wormwood plants; The regeneration medium is based on MS medium and contains sucrose, agar, activated carbon, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid.

[0011] Preferably, the final concentration of sucrose in the regeneration medium is 20-40 g / L; The final concentration of agar in the regeneration medium is 5-10 g / L; The final concentration of activated carbon in the regeneration medium is 0.5-2 g / L; The final concentration of 6-benzylaminopurine in the regeneration medium is 0.5-2 mg / L; The final concentration of 2,4-dichlorophenoxyacetic acid in the regeneration medium is 1-3 g / L; Preferably, the Huabai and callus tissue are the above-mentioned Huabai and callus tissue obtained by the present invention.

[0012] Preferably, the regeneration induction culture process includes replacing the same culture medium 2 to 5 times; The frequency of replacing the same culture medium is 25 to 35 days / time; The culture conditions of the regeneration induction culture are: 20-25° C., relative humidity 60-80%, and light intensity 1000-2000 lx.

[0013] The present invention also provides a Scutellaria baicalensis plant obtained by the Scutellaria baicalensis plant induction method.

[0014] The present invention also provides a method for cultivating Chinese white radish seedlings in soil, comprising the following steps: The above-mentioned Chinese white wormwood plant is removed from the culture medium and transplanted into a nutrient medium, and cultured for more than 5 days to obtain Chinese white wormwood soil-cultured seedlings; The nutrient matrix contains nutrient soil and vermiculite.

[0015] Preferably, the weight ratio of nutrient soil to vermiculite in the nutrient matrix is ​​1.5-2.5:1; The culture conditions of the culture are: 20-25° C., and light intensity of 1000-2000 lx.

[0016] Beneficial effects of the present invention: The method for cultivating S. chinensis provided by the present invention adopts the pseudobulbs and roots of S. chinensis as explants, obtains callus tissue by a continuous induction method, and then regenerates complete plants from the callus tissue, thereby establishing a rapid propagation method for the endangered plant S. chinensis, which can provide a feasible plan with excellent effects for protecting S. chinensis germplasm resources, and can achieve rapid and batch acquisition of complete plants under the condition that there are no mature capsules or seeds of S. chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a technical roadmap for callus tissue and callus regeneration plants of S. Figure 2 The statistical chart of the success rate of induction of S. alba and different explants by different types of culture medium; Figure 3 This is a statistical chart of the effects of different culture media on the seedling time and number of regenerated plants of S. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1 Induction of callus tissue of Scutellaria baicalensis Rinse the Huabai and plants under running water for 30 min, soak them in 70% alcohol in a clean bench for 30 s, then wash them with sterile ultrapure water for 5 times, then soak them in 2% sodium hypochlorite (NaClO) solution (with 0.1% Tween 20) for 5 min, and then wash them with sterile ultrapure water for 5 times. Use sterile filter paper to absorb the surface moisture, and use a sterile scalpel to place the roots, leaves, and pseudobulbs on different induction culture media (see the table below). Treat 150 explants with each culture medium, and subculture them to new induction culture media every 25 days. Remove or cut off the browned explants with a scalpel each time they are subcultured. After 10 subcultures, the number of explants producing callus was counted. Callus induction was carried out in the dark at 23±2℃.

[0020] The callus induction culture: 1) MS medium: 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331). Adjust the pH value of the solution to 5.8 and sterilize at high temperature (121°C, 20 min) for later use.

[0021] 2) 1 / 2 MS medium: 2.3 g / L 1 / 2 MS medium powder (Coolaber, PM1061), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331). Adjust the pH value of the solution to 5.8 and sterilize at high temperature (121°C, 20 min) for later use.

[0022] 3) Kyoto medium: 24.5 g / L Kyoto medium powder (CHINOOK, CN260730), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331). Adjust the pH value of the solution to 5.8 and sterilize at high temperature (115°C, 20 min) for later use.

[0023] 4) RM medium: 32.74 g / L RM medium powder (CHINOOK, CN260638), containing sucrose and agar, no need to add, the solution pH is adjusted to 5.8, and sterilized at high temperature (115°C, 15min) for use.

[0024] 5) KC medium: 29 g / L KC medium powder (REBIO, T214), containing sucrose and agar, no need to add, adjust the pH of the solution to 5.1±0.2, sterilize at high temperature (115°C, 30min) and set aside.

[0025] 6) VW medium: 1.63 g / L VW medium powder (CHINOOK, CN260637), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331). Adjust the pH value of the solution to 5.8 and sterilize at high temperature (121°C, 20 min) for later use.

[0026] The number of explants that produced callus tissue after 10 generations of continuous induction culture was counted. Figure 2 As shown: On the above-mentioned induction medium, callus could hardly be induced using S. calyx and leaves as explants; callus could be induced using S. calyx and roots as explants on the above-mentioned induction medium, and the callus initiation rates were as follows: MS medium 23.33% > KC and VW medium 20.67% > RM medium 18.67% > 1 / 2MS medium 16.67% > Kyoto medium 14.67%; callus could be induced using S. calyx and pseudobulbs as explants on the above-mentioned induction medium, and the callus initiation rates were slightly higher than those using leaves as explants, as follows: MS medium 26.67% > KC medium 22.67% > RM medium 22% > VW medium 18.67% > 1 / 2MS medium 18% > Kyoto medium 14%.

[0027] In summary, compared with the other five culture media, MS medium showed a better callus induction rate whether using roots or pseudobulbs as explants. Note: Callus induction rate = number of callus in explants / total number of explants × 100%.

[0028] Table 1 Callus induction

[0029] Example 2 Regeneration of Scutellaria baicalensis and intact plants from callus tissue 3 g of callus tissue induced in Example 1 was taken per bottle and spread into tissue culture bottles containing regeneration medium. 10 bottles were treated with each medium, and the corresponding medium was replaced every 30 days. Three generations were induced continuously, and the seedling emergence time (the time when the first seedling appeared) and the number of seedlings (90 days of culture) were counted to confirm the culture medium for inducing regeneration of complete plants of Huabai.

[0030] The regeneration culture medium: 1) MS medium: contains 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), 1.0 g / L activated carbon, 1.0 mg / L 6-benzylaminopurine (6-BA), 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D). The solution was adjusted to pH 5.8 and sterilized at high temperature (121°C, 20 min) before use.

[0031] 2) 1 / 2 MS medium: 2.3 g / L 1 / 2 MS medium powder (Coolaber, PM1061), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), 1.0 g / L activated carbon, 1.0 mg / L 6-benzylaminopurine (6-BA), 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D). Adjust the pH of the solution to 5.8 and sterilize at high temperature (121°C, 20 min) for later use.

[0032] 3) Kyoto medium: 24.5 g / L Kyoto medium powder (CHINOOK, CN260730), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), 1.0 g / L activated carbon, 1.0 mg / L 6-benzylaminopurine (6-BA), 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), adjust the pH of the solution to 5.8, sterilize at high temperature (115°C, 20 min) and set aside.

[0033] 4) RM medium: 32.74 g / L RM medium powder (CHINOOK, CN260638), containing sucrose and agar, no need to add, activated carbon 1.0 g / L, 6-benzylaminopurine (6-BA) 1.0 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, the solution was adjusted to pH 5.8, and sterilized at high temperature (115°C, 15 min) for use.

[0034] 5) KC medium: 29 g / L KC medium powder (REBIO, T214), containing sucrose and agar, no need to add, activated carbon 1.0 g / L, 6-benzylaminopurine (6-BA) 1.0 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2.0 mg / L, the solution pH is adjusted to 5.1±0.2, and sterilized at high temperature (115°C, 30min) before use.

[0035] 6) VW medium: 1.63 g / L VW medium powder (CHINOOK, CN260637), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), 1.0 g / L activated carbon, 1.0 mg / L 6-benzylaminopurine (6-BA), 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), adjust the pH of the solution to 5.8, sterilize at high temperature (121°C, 20 min) and set aside.

[0036] The callus tissue was placed in a temperature of 23±2℃, relative humidity of 60-80%, and light intensity of 1500lx for 45-90 days. The number of seedlings was counted after 90 days. After the statistics were completed, 3-5 regenerated seedlings were inoculated into the regeneration medium for seedling growth and rooting. After 30 days, soil planting could be carried out according to the growth and rooting conditions. The statistical results are as follows: Figure 3 As shown: Both in terms of seedling emergence time and seedling number, the MS medium has a significantly shorter time than other culture media (MS medium 50.1±3.21 days < VW medium 58.6±2.99 days < 1 / 2MS medium 60.4±2.46 days < KC medium 62.9±2.13 days < RM medium 64.5±3.21 days < Kyoto medium 68.1±2.13 days) and a larger number (MS medium 82±3.56 > VW medium 50.1±3.9 > 1 / 2MS medium 44±2.62 > RM medium 24.7±2.11 > KC medium 24.2±3.26 > Kyoto medium 10.2±1.55).

[0037] Example 3 Transplantation of Scutellaria baicalensis and regenerated plants The nutrient medium for transplanting Scutellaria baicalensis is a mixture of nutrient soil and 1-3mm vermiculite in a ratio of 2:1, and is irrigated with ultrapure water for later use. Take out the rooted Scutellaria baicalensis regeneration seedlings from the regeneration medium, rinse the medium between the roots with running water, transplant them into the nutrient medium, wrap the entire flower pot and Scutellaria baicalensis seedlings with plastic wrap, and culture them at a temperature of 23±2℃ and a light intensity of 1500lx for 10 days. After 10 days, remove the plastic wrap to obtain the soil-cultured Scutellaria baicalensis seedlings. For the specific process, see Figure 1 .

[0038] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inducing callus tissue of Scutellaria baicalensis, characterized in that: The following steps are involved: Inoculating the explants of the Chinese white wormwood into an induction medium for induction culture to obtain Chinese white wormwood callus tissue; The induction medium is based on MS medium and contains sucrose and agar.

2. The method for inducing callus of Chinese white wormwood according to claim 1, characterized in that: The sources of the S. cerana explants are the roots of S. cerana and / or the pseudobulbs of S. cerana.

3. The method for inducing callus of Chinese white spherical ... The final concentration of sucrose in the induction medium is 20-40 g / L; The final concentration of agar in the induction medium is 5-10 g / L; And / or, the induction culture comprises 5 to 15 subcultures, each subculture using the same culture medium; The subculture frequency of the subculture is 20 to 30 days / time; The white scutellaria and explants are sterilized and then inoculated; The reagents used for disinfection and sterilization include ethanol, sterile water or sodium hypochlorite solution; The culture conditions of the induction culture are: 20-25° C., dark culture.

4. The Huabai and callus tissue obtained by the Huabai and callus tissue induction method described in any one of claims 1 to 3.

5. A method for inducing Scutellaria baicalensis plants, characterized in that: The following steps are involved: Inoculating the callus tissue of the Chinese white wormwood into a regeneration medium for regeneration induction culture to obtain Chinese white wormwood plants; The regeneration medium is based on MS medium and contains sucrose, agar, activated carbon, 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid.

6. The method for inducing the Chinese white radish plant according to claim 5, characterized in that: The final concentration of sucrose in the regeneration medium is 20-40 g / L; The final concentration of agar in the regeneration medium is 5-10 g / L; The final concentration of activated carbon in the regeneration medium is 0.5-2 g / L; The final concentration of 6-benzylaminopurine in the regeneration medium is 0.5-2 mg / L; The final concentration of 2,4-dichlorophenoxyacetic acid in the regeneration medium is 1-3 g / L; Preferably, the Huabai and callus tissue are the Huabai and callus tissue described in claim 4.

7. The method for inducing the Chinese white radish plant according to claim 5, characterized in that: The regeneration induction culture process includes replacing the same culture medium 2 to 5 times; The frequency of replacing the same culture medium is 25 to 35 days / time; The culture conditions of the regeneration induction culture are: 20-25° C., relative humidity 60-80%, and light intensity 1000-2000 lx.

8. A Scutellaria baicalensis plant obtained by the Scutellaria baicalensis plant induction method according to any one of claims 5 to 7.

9. A method for cultivating Chinese white radish seedlings, characterized in that: The following steps are involved: Removing the culture medium from the Glechoma longituba plant of claim 8 and transplanting it into a nutrient medium, culturing it for more than 5 days to obtain Glechoma longituba soil-cultured seedlings; The nutrient matrix contains nutrient soil and vermiculite.

10. The method for cultivating Scutellaria baicalensis and soil-grown seedlings according to claim 9, characterized in that: The weight ratio of nutrient soil to vermiculite in the nutrient matrix is ​​1.5-2.5:1; The culture conditions of the culture are: 20-25° C., and light intensity of 1000-2000 lx.

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