A culture medium for tissue culture of scutellaria baicalensis

Abstract

The invention belongs to the technical field of plant tissue culture, and specifically relates to a culture medium for tissue culture of spatholobi; the culture medium for tissue culture of spatholobi comprises: A, an induction culture medium: 1 / 2 improved MS culture medium + Huabao No. 1 (0.1-0.3) g / L + sucrose 25 g / L + agar (6.8-7.5 g) g / L + IBA (0.5-0.7) mg / L + NAA (0.5-0.7) mg / L; B, a proliferation culture medium : Improved MS medium + 6-BA (2.0-3.0) mg / L + NAA (0.5-1) mg / L + sucrose 30 g / L + agar powder (6.0-7.0) g / L + coconut juice (60-75) g / L; C. Rooting medium: Improved MS medium + 6-BA (0.5-1.5) mg / L + sucrose 20 g / L + agar powder (5.5-7.0) g / L + activated carbon (3-5) g / L.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a culture medium for spatholobi tissue culture. Background Art

[0002] Panlong ginseng, also known as Panlong ginseng, is a perennial terrestrial orchidaceae herbaceous plant of the genus Panlong ginseng. It has both medicinal value and high ornamental value. The dried whole herb of Panlong ginseng is a precious medicinal material, and its main effects are to nourish qi and yin, clear away heat and detoxify. It is used in folk medicine to treat diabetes, respiratory diseases (such as pneumonia and sore throat), as well as anti-virus (such as hepatitis B and herpes zoster), anti-tumor, etc.

[0003] The environment requirements of the sedge grass are very strict. At present, no large-scale sedge grass communities have been found. Due to the geographical environment and seasonal restrictions, it is difficult to achieve the purpose of rapid and efficient reproduction. Moreover, although there are many sedge grass seeds under natural conditions, it is difficult to germinate because it does not have a fully developed embryo. The technical means and methods of tissue culture are currently an effective measure to solve the problem of sedge grass reproduction, but most of the culture media used for tissue culture are added with chemically synthesized plant hormones to promote rapid plant reproduction and growth. Tissue culture seedlings often have poor stress resistance and high transplant mortality.

[0004] Therefore, we proposed a culture medium for tissue culture of Psoralea corylifolia. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a culture medium for tissue culture of Herba Lycopodii.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A culture medium for tissue culture of Cynanchum truncatum, the culture medium for tissue culture of Cynanchum truncatum comprising the following culture mediums used in three culture stages, and the composition of the culture medium in each stage is respectively:

[0008] A. Induction medium: 1 / 2 modified MS medium + Huabao No. 1 (0.1-0.5) g / L + sucrose 25 g / L + agar (6.8-7.5 g) g / L + IBA (0.5-1) mg / L + NAA (0.5-1) mg / L;

[0009] B. Proliferation medium: modified MS medium + 6-BA (2.0-3.0) mg / L + NAA (0.5-1) mg / L + sucrose 30 g / L + agar powder (6.0-7.0) g / L + coconut juice (60-75) g / L;

[0010] C. Rooting medium: modified MS medium + 6-BA (0.5-1.5) mg / L + sucrose 20 g / L + agar powder (5.5-7.0) g / L + activated carbon (3-5) g / L;

[0011] The formula of the improved MS culture medium is: ammonium nitrate 1645-1655 mg / L, potassium nitrate 1895-1905 mg / L, calcium chloride 435-445 mg / L, potassium dihydrogen phosphate 165-175 mg / L, potassium iodide 0.80-0.85 mg / L, boric acid 6.0-6.5 mg / L, disodium ethylenediaminetetraacetate 37.0-37.5 mg / L, manganese sulfate 22.0-22.5 mg / L. g / L, zinc sulfate 8.4-8.8mg / L, sodium molybdate 0.22-0.27mg / L, ferrous sulfate 27.5-28.0mg / L, niacin 0.5mg / L, VB60.5mg / L, glycine 2.0mg / L, inositol 95-105mg / L, pyridoxine hydrochloride 1.0mg / L, cobalt chloride 0.15mg / L; sugar concentration 50-60mg / L; pH 5.8-6.0.

[0012] Preferably, the pH of the culture medium in the three culture stages is 5.8-6.2.

[0013] Preferably, the tissue of the Herba Lycopodiellae used for culturing is the fleshy root of the Herba Lycopodiellae.

[0014] Beneficial effects of the present invention:

[0015] 1. The present invention uses the improved MS medium as the basic medium for induction, proliferation or rooting, improves the concentrations of nitrogen, potassium, calcium, copper, inositol, VB6 and sugar, reduces the yellowing of leaves and the short and narrow leaves during the tissue culture of Panlong ginseng, ultimately reduces the seedling loss rate and reduces the production cost; at the same time, when the improved MS medium is used, the compactness between clustered buds is reduced, which is more conducive to actual production operations and also reduces the production operation costs to a certain extent.

[0016] 2. The present invention adds a certain concentration of Huabao No. 1 to the induction culture medium, and improves the germination rate of fleshy roots during the induction differentiation process by compounding the improved MS culture medium and Huabao No. 1.

[0017] 3. The present invention adds a certain concentration of coconut juice to the proliferation culture medium. The modified MS culture medium and coconut juice are compounded to make the tender shoots stronger, so that the propagated seedlings can be directly used for rooting, omitting the seedling strengthening step and simplifying the production process. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Common MS medium formula:

[0020]

[0021]

[0022] Improved MS medium formula:

[0023]

[0024]

[0025] Example 1

[0026] Breeding and cultivation of sedge grass

[0027] S1. Take the fleshy roots of the herb and set aside;

[0028] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0029] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + Huabao No. 1 0.1g / L + sucrose 25g / L + agar 6.8g / L + IBA 0.5mg / L + NAA 0.5mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0030] S4, inoculating the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 2.0mg / L + NAA 0.5mg / L + sucrose 30g / L + agar powder 6.0g / L + coconut juice 60g / L; the proliferation culture conditions are: culture temperature 30°C, light exposure time 10h, light intensity 1500lx;

[0031] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: modified MS medium + 6-BA 0.5 mg / L + sucrose 20 g / L + agar powder 5.5 g / L + activated carbon 3 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0032] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0033] Example 2

[0034] Breeding and cultivation of sedge grass

[0035] S1. Take the fleshy roots of the herb and set aside;

[0036] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0037] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + Huabao No. 1 0.3g / L + sucrose 25g / L + agar 7.0g / L + IBA 0.7mg / L + NAA 0.7mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0038] S4. Inoculate the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 3.0 mg / L + NAA 1 mg / L + sucrose 30 g / L + agar powder 7.0 g / L + coconut juice 75 g / L; the proliferation culture conditions are: culture temperature 30°C, light exposure time 10 h, light intensity 1500 lx;

[0039] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: modified MS medium + 6-BA 1.5 mg / L + sucrose 20 g / L + agar powder 7.0 g / L + activated carbon 5 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0040] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0041] Comparative Example 1

[0042] Breeding and cultivation of sedge grass

[0043] S1. Take the fleshy roots of the herb and set aside;

[0044] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0045] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: MS medium + Huabao No. 1 0.3g / L + sucrose 25g / L + agar 7.0g / L + IBA 0.7mg / L + NAA 0.7mg / L; the conditions for induction culture are: culture temperature is 25℃, light time is 8h, and light intensity is 1100lx.

[0046] S4. Inoculate the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 3.0 mg / L + NAA 1 mg / L + sucrose 30 g / L + agar powder 7.0 g / L + coconut juice 75 g / L; the proliferation culture conditions are: culture temperature is 30°C, light time is 10 h, and light intensity is 1500 lx.

[0047] S5. Place the seedlings in a rooting medium to allow them to take root; the formula of the rooting medium is: modified MS medium + 6-BA 1.5 mg / L + sucrose 20 g / L + agar powder 7.0 g / L + activated carbon 5 g / L; the conditions for rooting culture are: culture temperature of 30°C, light exposure time of 12 h, and light intensity of 1700 lx.

[0048] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0049] Comparative Example 2

[0050] Breeding and cultivation of sedge grass

[0051] S1. Take the fleshy roots of the herb and set aside;

[0052] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0053] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + sucrose 25g / L + agar 6.8g / L + IBA 0.5mg / L + NAA 0.5mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0054] S4, inoculating the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 2.0mg / L + NAA 0.5mg / L + sucrose 30g / L + agar powder 6.0g / L + coconut juice 60g / L; the proliferation culture conditions are: culture temperature 30°C, light exposure time 10h, light intensity 1500lx;

[0055] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: modified MS medium + 6-BA 0.5 mg / L + sucrose 20 g / L + agar powder 5.5 g / L + activated carbon 3 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0056] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0057] Comparative Example 3

[0058] Breeding and cultivation of sedge grass

[0059] S1. Take the fleshy roots of the herb and set aside;

[0060] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0061] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + Huabao No. 1 0.1g / L + sucrose 25g / L + agar 6.8g / L + IBA 0.5mg / L + NAA 0.5mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0062] S4, inoculating the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: MS medium + 6-BA 2.0mg / L + NAA 0.5mg / L + sucrose 30g / L + agar powder 6.0g / L + coconut juice 60g / L; the proliferation culture conditions are: culture temperature 30°C, light exposure time 10h, light intensity 1500lx;

[0063] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: modified MS medium + 6-BA 0.5 mg / L + sucrose 20 g / L + agar powder 5.5 g / L + activated carbon 3 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0064] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0065] Comparative Example 4

[0066] Breeding and cultivation of sedge grass

[0067] S1. Take the fleshy roots of the herb and set aside;

[0068] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0069] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + Huabao No. 1 0.1g / L + sucrose 25g / L + agar 6.8g / L + IBA 0.5mg / L + NAA 0.5mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0070] S4. Inoculate the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 2.0 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar powder 6.0 g / L; the proliferation culture conditions are: culture temperature 30°C, light duration 10 h, light intensity 1500 lx;

[0071] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: modified MS medium + 6-BA 0.5 mg / L + sucrose 20 g / L + agar powder 5.5 g / L + activated carbon 3 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0072] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0073] Comparative Example 5

[0074] Breeding and cultivation of sedge grass

[0075] S1. Take the fleshy roots of the herb and set aside;

[0076] S2. Soak the fleshy roots of the spatholobi in 65% alcohol for 30 seconds, then take them out and rinse them three times with sterile water; then disinfect them with 15% hydrogen peroxide for 15 minutes, and then rinse them three times with sterile water;

[0077] S3. Use sterile paper to absorb the water on the surface of the fleshy roots after sterilization, and then place them in an induction medium for induction culture for 15 days to differentiate the fleshy roots into differentiated buds; the formula of the induction medium is: 1 / 2 modified MS medium + Huabao No. 1 0.1g / L + sucrose 25g / L + agar 6.8g / L + IBA 0.5mg / L + NAA 0.5mg / L; the conditions of induction culture are: culture temperature 25℃, light exposure time 8h, light intensity 1100lx;

[0078] S4, inoculating the differentiated buds into a proliferation medium for proliferation culture to obtain seedlings; the proliferation medium formula is: modified MS medium + 6-BA 2.0mg / L + NAA 0.5mg / L + sucrose 30g / L + agar powder 6.0g / L + coconut juice 60g / L; the proliferation culture conditions are: culture temperature 30°C, light exposure time 10h, light intensity 1500lx;

[0079] S5. Place the seedlings in a rooting medium to allow the seedlings to take root; the formula of the rooting medium is: MS medium + 6-BA 0.5 mg / L + sucrose 20 g / L + agar powder 5.5 g / L + activated carbon 3 g / L; the conditions for rooting culture are: culture temperature 30°C, light exposure time 12 h, light intensity 1700 lx;

[0080] S6. Transplant the rooted seedlings to a well-permeable substrate and grow them for 1 week. Transplant them outdoors after the seedlings have developed well-developed root systems. The well-permeable substrate is a mixture of peat soil, perlite and sawdust, and the mass ratio of peat soil, perlite and sawdust is 4:1:1.

[0081] Table 1 is a statistical table of the germination rate, rooting rate and survival rate of the fleshy roots of the Herba Lycopodii in Examples 1-2 and Comparative Examples 1-5.

[0082]

[0083] Table 1

[0084] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0085] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A culture medium for tissue culture of Coleus chinensis, characterized in that: The culture medium for the tissue culture of the spatholobiaceae includes the following culture mediums used in the three culture stages, and the composition of the culture medium in each stage is respectively: A. Induction medium: 1 / 2 modified MS medium + Huabao No. 1 (0.1-0.3) g / L + sucrose 25 g / L + agar (6.8-7.5 g) g / L + IBA (0.5-0.7) mg / L + NAA (0.5-0.7) mg / L; B. Proliferation medium: modified MS medium + 6-BA (2.0-3.0) mg / L + NAA (0.5-1) mg / L + sucrose 30 g / L + agar powder (6.0-7.0) g / L + coconut juice (60-75) g / L; C. Rooting medium: modified MS medium + 6-BA (0.5-1.5) mg / L + sucrose 20 g / L + agar powder (5.5-7.0) g / L + activated carbon (3-5) g / L; The formula of the improved MS culture medium is: ammonium nitrate 1645-1655 mg / L, potassium nitrate 1895-1905 mg / L, calcium chloride 435-445 mg / L, potassium dihydrogen phosphate 165-175 mg / L, potassium iodide 0.80-0.85 mg / L, boric acid 6.0-6.5 mg / L, disodium ethylenediaminetetraacetic acid 37.0-37.5 mg / L, manganese sulfate 22.0-22.5 mg / L, zinc sulfate 8.4-8.8 mg / L, sodium molybdate 0.22-0.27 mg / L, iron sulfate 27.5-28.0 mg / L, nicotinic acid 0.5 mg / L, VB6 0.5 mg / L, glycine 2.0 mg / L, inositol 95-105 mg / L, pyridoxine hydrochloride 1.0 mg / L, cobalt chloride 0.15mg / L; sugar concentration 50-60mg / L; PH is 5.8-6.

0.

2. The medium for tissue culture of Coleus chinensis according to claim 1, characterized in that: The pH of the culture medium in the three culture stages was 5.8-6.

2.

3. The medium for tissue culture of Panicum truncatum according to claim 1, characterized in that: The tissue of the Herba Lycoris used for cultivation is the fleshy root of the Herba Lycoris radiata.

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