A method for rapid propagation of Tripterygium wilfordii tissue culture

Abstract

The present invention relates to the technical field of tissue culture, and in particular to a method for rapid propagation of trifoliate green leaf tissue culture. A method for rapid propagation of trifoliate green leaf tissue culture comprises the following steps: selection and sterilization of explants; preparation of antimicrobial agents; improved induction culture medium and induction culture; improved proliferation culture medium and proliferation culture; improved rooting culture medium and rooting culture; transplantation of trifoliate green leaf seedlings. The present invention performs preliminary sterilization of axillary bud stem segments by disodium ethylenediaminetetraacetic acid solution and ultraviolet sterilization, then disinfects by sodium hypochlorite solution, then disinfects by mercuric chloride solution, and finally performs deep disinfection by a composite disinfection method of shock disinfection in hydrogen peroxide, thereby improving the disinfection effect of axillary bud stem segments, reducing the contamination rate of axillary bud stem segments contaminating a culture medium, promoting the growth and development of trifoliate green leaf plants by improving the culture medium, and rooting, thereby improving the survival rate of transplantation of trifoliate green leaf plant seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and in particular to a method for rapid propagation of trifoliate green tissue culture. Background Art

[0002] Three-leaf green, also known as horned blackberry and stone mouse, is a plant of the genus Three-leaf Clerodendrum of the Vitaceae family. Three-leaf green has been gradually used in the treatment of liver cancer, lung cancer, leukemia and AIDS in clinical practice, and is known as a safe and non-toxic "plant penicillin". However, due to excessive human excavation of three-leaf green and the strict requirements of three-leaf green on the growth environment, the wild resources of three-leaf green have been sharply reduced and are now in an endangered state. At the same time, with the more in-depth research and development of the medicinal ingredients of three-leaf green, its wild three-leaf green is far from meeting the increasing market demand year by year. The rapid proliferation of bottle seedlings using tissue culture technology can provide a large number of seedlings in a short period of time.

[0003] The explant selection, sterilization method, culture medium formula and expected effects of the currently reported rapid propagation systems of Tripterygium wilfordii tissues are all different. The explants sterilized by the existing conventional sterilization methods will still contain some bacteria, and there is a problem of incomplete and insufficient sterilization, which will cause the contaminated explants to contaminate the culture medium during the culture process and affect the culture effect. In addition, the conventionally used culture medium has the problems of low proliferation coefficient and low rooting rate, and will also result in a low survival rate of the seedlings after culture.

[0004] In order to solve the above-mentioned technical difficulties, the present invention studies a method for rapid propagation of Tripterygium wilfordii tissue culture, which improves the sterilization effect of explants, reduces the contamination rate of culture medium and increases the proliferation coefficient and rooting rate of Tripterygium wilfordii clustered shoots. Summary of the invention

[0005] In order to solve the above-mentioned technical difficulties, the present invention studies a method for rapid propagation of Tripterygium wilfordii tissue culture, which improves the sterilization effect of explants, reduces the contamination rate of culture medium and increases the proliferation coefficient and rooting rate of Tripterygium wilfordii clustered shoots.

[0006] A method for rapid propagation of Tripterygium wilfordii tissue culture, specifically comprising the following steps:

[0007] S1: Selection and sterilization of explants

[0008] Pick harmless axillary bud stem segments of Tripterygium wilfordii, clean them, perform preliminary disinfection on them, and then perform deep disinfection and sterilization on them to obtain sterilized explant axillary bud stem segments, dry the sterilized explant axillary bud stem segments, and then cut them into segments, each segment is guaranteed to contain one axillary bud;

[0009] S2: Preparation of antimicrobial agents

[0010] After the nano zinc oxide is modified, the modified nano zinc oxide, chitosan and tea polyphenols are magnetically stirred to obtain an antibacterial agent;

[0011] S3: Improved induction medium and induction culture

[0012] Using MS medium as the basic medium, adding 6-BA, agar, sucrose, and an antimicrobial agent into the MS basic medium, mixing and sterilizing to obtain a modified induction medium, inoculating the explant axillary bud stem segments cut into small segments onto the medium to obtain induced new shoots;

[0013] S4: Improved proliferation medium and proliferation culture

[0014] Put fresh bananas and potatoes into a crusher, add coconut juice to crush, add activated carbon powder to the crushed material, mix and stir evenly under the assistance of ultrasound to obtain a mixture, use MS medium as a basic medium, add BA, NAA, PVP and the mixture to the MS basic medium, mix and sterilize evenly to obtain a modified proliferation medium, take out explants with buds under sterile conditions, separate the buds with a scalpel, and inoculate them on the proliferation medium to obtain the proliferated three-leaf green clustered buds;

[0015] S5: Improved rooting medium and rooting culture

[0016] Grind and sieve activated carbon particles to obtain activated carbon powder, add sucrose and nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture, use 1 / 2MS medium as the basic medium, add IBA, NAA and the mixture to the 1 / 2MS basic medium, mix and sterilize evenly to obtain a modified rooting medium, inoculate clustered buds on the rooting medium and culture to obtain rooted clover plants;

[0017] S6: Transplanting of three-leaf green seedlings

[0018] After placing the rooted Tripterygium wilfordii plant culture medium in a natural environment for a period of time, take out the Tripterygium wilfordii plant seedlings, clean the base of the Tripterygium wilfordii plant, transplant the Tripterygium wilfordii plant seedlings to a mixed matrix of perlite and peat soil, cultivate for a period of time, and then transplant them into the field.

[0019] Furthermore, step S1 of explant selection and sterilization treatment specifically includes the following steps:

[0020] S1.1: Pick 10-20 healthy stem segments of axillary buds of Tripterygium wilfordii that are free of pests and diseases and bacteria, and bring them back for later use;

[0021] S1.2: Clean the brought back axillary bud stem segments with detergent, rinse under running water for 20-30 minutes, wash with sterile water 2-3 times, place in 1-2mmol / L disodium ethylenediaminetetraacetic acid solution for 5-10 minutes, soak in sterile water for 10-20 minutes, dry, and sterilize with ultraviolet light for 20-30 minutes to obtain preliminarily sterilized axillary bud stem segments;

[0022] S1.3: Place the initially sterilized axillary bud stem segment in a 70-75% ethanol solution, soak for 10-15 seconds, rinse with sterile water, place the axillary bud stem segment in a 2-3% sodium hypochlorite solution for disinfection for 15-20 minutes, rinse with sterile water, place the axillary bud stem segment in a 0.1-0.2% mercuric chloride solution, add 2-3 drops of Tween, sterilize for 6-10 minutes, rinse with sterile water 2-3 times, finally place in 8-10% hydrogen peroxide for shaking disinfection for 50-60 seconds, and rinse with sterile water to obtain the sterilized explant axillary bud stem segment;

[0023] S1.4: Use a sterile scalpel to cut off both ends of the disinfected explant axillary bud stem segment, and use sterile filter paper to absorb the moisture. Then use a sterile scalpel and tweezers to cut it into small segments, ensuring that each segment contains one axillary bud, and set aside.

[0024] Furthermore, the preparation of the antibacterial agent in step S2 specifically comprises the following steps:

[0025] S2.1: Take 10-20 parts by weight of anhydrous ethanol, add 2-4 parts by weight of nano zinc oxide, and ultrasonically disperse for 10-20 minutes. Add 0.1-0.2 parts by weight of sorbic acid to the dispersion, heat in a water bath at 50-60°C for 10-20 minutes, centrifuge to obtain a precipitate, and vacuum dry the precipitate at 50-60°C for 10-12 hours to obtain modified nano zinc oxide;

[0026] S2.2: Take 2-4 parts by weight of modified nano zinc oxide, add 5-8 parts by weight of chitosan and 1-2 parts by weight of tea polyphenols, and stir magnetically for 10-20 minutes to obtain an antibacterial agent.

[0027] Furthermore, step S3 is to improve the induction medium and induce culture, which specifically includes the following steps:

[0028] S3.1: Using MS medium as the basic medium, add 0.5-1.0 mg / L 6-BA, 7-8 g / L agar, 25-30 g / L sucrose, 0.1-0.5 mg / L NAA and 1-2 g / L antimicrobial agent into the MS basic medium, mix well, and obtain the medium required for improved induction culture;

[0029] S3.2: sterilize the induced culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified induction medium;

[0030] S3.3: Inoculate the explant axillary bud stem segments cut into small sections onto the culture medium. The temperature in the culture room is 25-28°C, the light intensity is 35-40 μmol.m-2.s-1, and the culture is carried out for 15-20 days to obtain new shoots after induced culture.

[0031] Furthermore, step S4 improves the proliferation medium and the proliferation culture, specifically comprising the following steps:

[0032] S4.1: Put 10-20g of fresh banana and 5-10g of potato into a crusher, add 80-90ml / L of coconut juice and crush to obtain a mixed crushed product, grind the activated carbon particles to obtain activated carbon powder, add 1-2g / L of activated carbon powder to the mixed crushed product, mix and stir evenly under the assistance of ultrasound to obtain a mixture;

[0033] S4.2: Using MS medium as the basic medium, add 0.5-1.0 mg / L BA, 0.1-0.2 mg / L NAA, 0.5-1 g / L PVP, and 2-4 g / L mixture into the MS basic medium, mix well, and obtain the proliferation medium required for proliferation culture;

[0034] S4.3: sterilize the proliferation culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified proliferation medium;

[0035] S4.4: Remove the explants with buds under sterile conditions, separate the buds with a scalpel, and inoculate them into proliferation medium. The temperature of the culture room is 25-28℃. First, culture them in the dark for 5-10 days, and then culture them under a light intensity of 35-40μmol.m -2 .s -1 After culturing for 20-25 days, the proliferated clustered shoots of the trifoliate green were obtained.

[0036] Furthermore, step S5 improves the rooting medium and rooting culture, specifically comprising the following steps:

[0037] S5.1: Grind and crush 1-2 g of activated carbon particles and sieve to obtain activated carbon powder, add 15-20 g of sucrose and 200-230 mg of nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture;

[0038] S5.2: Using 1 / 2MS medium as the basic medium, add 1-2mg / L IBA, 0.2-0.5mg / L NAA and 15-20g / L mixture into the 1 / 2MS basic medium, mix well, and obtain the medium required for rooting culture;

[0039] S5.3: sterilize the rooting culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified rooting medium;

[0040] S5.4: Inoculate the clustered buds onto the rooting medium. The temperature in the culture room is 25-28°C and the light intensity is 35-40 μmol.m -2 .s -1 After 30-40 days of cultivation, rooted Tripterygium wilfordii plants were obtained.

[0041] Furthermore, the transplanting of the three-leaf seedlings in step S6 specifically includes the following steps:

[0042] S6.1: Place the culture medium of the rooted Tripterygium wilfordii plants in a natural environment for 5-6 days, sprinkle some water on the surface of the culture medium, and then place it for another 1-2 days;

[0043] S6.2: Take out the placed cloverleaf green plant seedlings, clean the base of the cloverleaf green plant, and transplant the cloverleaf green plant seedlings to a mixed substrate of perlite and peat soil, maintain the relative humidity of the environment for 28-30 days, and avoid direct sunlight for the first 12-14 days;

[0044] S6.3: After 28-30 days, transplant the clover seedlings into the field.

[0045] Furthermore, the variety of Sanyeqing in step S1.1 is Zhongze No. 1.

[0046] Furthermore, in step S6.2, the relative humidity of the environment is 70-80%.

[0047] Furthermore, in step S6.2, the mixing ratio of perlite to peat soil is 1:1-2.

[0048] The beneficial effects are as follows: 1. The present invention performs preliminary sterilization on the axillary bud stem segments by using a disodium ethylenediaminetetraacetic acid solution and ultraviolet sterilization, then disinfects with a sodium hypochlorite solution, then disinfects with a mercuric chloride solution, and finally performs deep disinfection by a composite disinfection method of shock disinfection in hydrogen peroxide, thereby improving the disinfection effect of the axillary bud stem segments and preventing the axillary bud stem segments from contaminating the culture medium.

[0049] 2. The present invention obtains an improved induction medium by adding an antibacterial agent into the induction medium. The improved induction medium has a certain antibacterial effect. By culturing axillary bud stem segments with the improved induction medium, the probability of axillary bud stem segments contaminating the medium can be reduced, and the contamination rate can be reduced. At the same time, chitosan and tea polyphenols can promote the growth and development of axillary bud stem segments while having antibacterial effects.

[0050] 3. The present invention can promote the growth and development of the clover plants by improving the proliferation culture medium. The activated carbon particles can absorb harmful substances secreted by the plants. Coconut juice can effectively avoid the vitrification rate under high cytokinin conditions during the proliferation process. Bananas and potatoes provide the clover plants with the required nutrients to promote their growth and increase the survival rate of transplanted clover seedlings. At the same time, providing a dark environment for proliferation is beneficial to the proliferation and growth of the clover plants.

[0051] 4. The present invention can promote the rooting of the cloverleaf green plants by adding activated carbon particles and nano-silicon dioxide. Nano-silicon dioxide can promote tissue growth and organogenesis, and improve the morphology of the plants, thereby increasing the rooting rate of the cloverleaf green plants. The activated carbon particles can absorb harmful substances secreted by the plants and induce the rooting of the cloverleaf green plants. The addition of activated carbon particles and nano-silicon dioxide produces a synergistic effect, so that the rooting rate of the cloverleaf green plants is greatly improved, and the survival rate of transplanted cloverleaf green plant seedlings can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The present invention is a flowchart of a method for rapid propagation of Tripterygium wilfordii tissue culture adopted in an embodiment of the present invention.

[0053] Figure 2 It is a comparative table chart of the contamination rate and disinfection mortality rate of Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention.

[0054] Figure 3 It is a table chart comparing the proliferation coefficient and survival rate of Comparative Example 4 of the present invention.

[0055] Figure 4 It is a comparative table diagram of rooting rate and survival rate of comparative example 5 of the present invention. DETAILED DESCRIPTION

[0056] The following will be combined with the drawings in 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.

[0057] Example 1

[0058] A method for rapid propagation of Tripterygium wilfordii tissue culture, such as Figure 1 As shown, the specific steps include:

[0059] S1: Selection and sterilization of explants

[0060] S1.1: Pick 10 healthy and healthy stem segments of the axillary buds of "Zhongze No. 1" Sanyeqing that are free of pests and diseases and bacteria, and bring them back for use;

[0061] S1.2: Clean the brought back axillary bud stem segments with detergent, rinse under running water for 20 minutes, wash twice with sterile water, place in 1mmol / L disodium ethylenediaminetetraacetic acid solution for 5 minutes, soak in sterile water for 10 minutes, dry, and sterilize with ultraviolet light for 20 minutes to obtain preliminarily sterilized axillary bud stem segments;

[0062] S1.3: Place the initially sterilized axillary bud stem segment in a 70% ethanol solution, soak for 10 seconds, rinse with sterile water, place the axillary bud stem segment in a 2% sodium hypochlorite solution for 15 minutes, rinse with sterile water, place the axillary bud stem segment in a 0.1% mercuric chloride solution, add 2 drops of Tween, sterilize for 6 minutes, rinse with sterile water twice, finally place in 8% hydrogen peroxide for 50 seconds of shaking disinfection, and rinse with sterile water to obtain the sterilized explant axillary bud stem segment;

[0063] S1.4: Use a sterile scalpel to cut off both ends of the disinfected explant axillary bud stem segment, and use sterile filter paper to absorb the moisture. Then use a sterile scalpel and tweezers to cut it into small segments, ensuring that each segment contains one axillary bud, and set aside.

[0064] S2: Preparation of antimicrobial agents

[0065] S2.1: Take 10 parts by weight of anhydrous ethanol, add 2 parts by weight of nano zinc oxide, and disperse by ultrasonic for 10 minutes. Add 0.1 parts by weight of sorbic acid to the dispersion, heat in a water bath at 50°C for 10 minutes, centrifuge to obtain a precipitate, and vacuum dry the precipitate at 50°C for 10 hours to obtain modified nano zinc oxide;

[0066] S2.2: Take 2 parts by weight of modified nano zinc oxide, add 5 parts by weight of chitosan and 1 part by weight of tea polyphenols, and stir magnetically for 10 minutes to obtain an antibacterial agent.

[0067] S3: Improved induction medium and induction culture

[0068] S3.1: Take MS medium as the basic medium, add 0.5 mg / L 6-BA, 7-8 g / L agar, 25 g / L sucrose, 0.1 mg / L NAA and 1 g / L antimicrobial agent into the MS basic medium, mix well, and obtain the medium required for improved induction culture;

[0069] S3.2: sterilize the induced culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified induction medium;

[0070] S3.3: Cut the explant axillary bud stem segments into small pieces and inoculate them into the culture medium. The temperature of the culture room is 25℃ and the light intensity is 35μmol.m -2 .s -1 , cultured for 15 days, and obtained new shoots after induced culture;

[0071] S4: Improved proliferation medium and proliferation culture

[0072] S4.1: Put 10g of fresh banana and 5g of potato into a crusher, add 80mL of coconut juice and crush to obtain a mixed crushed product, grind the activated carbon particles to obtain activated carbon powder, add 1g of activated carbon powder to the mixed crushed product, mix and stir evenly under the assistance of ultrasound to obtain a mixture;

[0073] S4.2: Using MS medium as the basic medium, add 0.5 mg / L BA, 0.1 mg / L NAA, 0.5 g / L PVP, and 2 g / L mixture into the MS basic medium, mix well, and obtain the proliferation medium required for proliferation culture;

[0074] S4.3: sterilize the proliferation culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified proliferation medium;

[0075] S4.4: The explants with buds were removed under sterile conditions, the buds were separated with a scalpel, and inoculated into proliferation medium. The temperature of the culture room was 25°C, and the cells were cultured in the dark for 5 days, and then cultured under a light intensity of 35 μmol.m -2 .s -1 After culturing for 20 days, the proliferated clumps of Tripterygium wilfordii were obtained;

[0076] S5: Improved rooting medium and rooting culture

[0077] S5.1: Grind and sieve 1 g of activated carbon particles to obtain activated carbon powder, add 15 g of sucrose and 200 mg of nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture;

[0078] S5.2: Using 1 / 2MS medium as the basic medium, add 1 mg / L IBA, 0.2 mg / L NAA and 15 g / L mixture into the 1 / 2MS basic medium, mix well, and obtain the medium required for rooting culture;

[0079] S5.3: sterilize the rooting culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified rooting medium;

[0080] S5.4: Inoculate the clustered buds onto the rooting medium in a culture room at 25°C and a light intensity of 35-40 μmol.m -2 .s -1After culturing for 30 days, rooted Tripterygium wilfordii plants were obtained;

[0081] S6: Transplanting of three-leaf green seedlings

[0082] S6.1: Place the culture medium of the rooted Tripterygium wilfordii plants in a natural environment for 5 days, sprinkle some water on the surface of the culture medium, and then place it for another day;

[0083] S6.2: Take out the placed cloverleaf green plant seedlings, clean the base of the cloverleaf green plant, and transplant the cloverleaf green plant seedlings to a mixed substrate of perlite and peat soil in a ratio of 1:1. Maintain the relative humidity of the environment at 70% for 28 days, and avoid direct sunlight for the first 12 days;

[0084] S6.3: After 28 days, transplant the clover seedlings into the field.

[0085] Example 2

[0086] A method for rapid propagation of Tripterygium wilfordii tissue culture, such as Figure 1 As shown, the specific steps include:

[0087] S1: Selection and sterilization of explants

[0088] S1.1: Pick 20 healthy and healthy stem segments of the axillary buds of Zhongze No. 1 Sanyeqing, which are free of pests and diseases and bacteria, and bring them back for use;

[0089] S1.2: Clean the brought back axillary bud stem segments with detergent, rinse under running water for 20 minutes, wash twice with sterile water, place in 2mmol / L disodium ethylenediaminetetraacetic acid solution for 5 minutes, soak in sterile water for 10 minutes, dry, and sterilize with ultraviolet light for 20 minutes to obtain preliminarily sterilized axillary bud stem segments;

[0090] S1.3: Place the initially sterilized axillary bud stem segment in a 75% ethanol solution, soak for 10 seconds, rinse with sterile water, place the axillary bud stem segment in a 3% sodium hypochlorite solution for 15 minutes, rinse with sterile water, place the axillary bud stem segment in a 0.2% mercuric chloride solution, add 3 drops of Tween, sterilize for 6 minutes, rinse with sterile water twice, finally place in 10% hydrogen peroxide for 50 seconds of shaking disinfection, and rinse with sterile water to obtain the sterilized explant axillary bud stem segment;

[0091] S1.4: Use a sterile scalpel to cut off both ends of the disinfected explant axillary bud stem segment, and use sterile filter paper to absorb the moisture. Then use a sterile scalpel and tweezers to cut it into small segments, ensuring that each segment contains one axillary bud, and set aside.

[0092] S2: Preparation of antimicrobial agents

[0093] S2.1: Take 20 parts by weight of anhydrous ethanol, add 4 parts by weight of nano zinc oxide, and disperse by ultrasonic for 10 minutes. Add 0.2 parts by weight of sorbic acid to the dispersion, heat in a water bath at 50°C for 10 minutes, centrifuge to obtain a precipitate, and vacuum dry the precipitate at 50°C for 10 hours to obtain modified nano zinc oxide;

[0094] S2.2: Take 4 parts by weight of modified nano zinc oxide, add 8 parts by weight of chitosan and 2 parts by weight of tea polyphenols, and stir magnetically for 10 minutes to obtain an antibacterial agent.

[0095] S3: Improved induction medium and induction culture

[0096] S3.1: Take MS medium as the basic medium, add 1.0 mg / L 6-BA, 7-8 g / L agar, 30 g / L sucrose, 0.5 mg / L NAA and 2 g / L antimicrobial agent into the MS basic medium, mix well, and obtain the medium required for improved induction culture;

[0097] S3.2: sterilize the induced culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified induction medium;

[0098] S3.3: Cut the explant axillary bud stem segments into small pieces and inoculate them into the culture medium. The temperature of the culture room is 25℃ and the light intensity is 35μmol.m -2 .s -1 , cultured for 15 days, and obtained new shoots after induced culture;

[0099] S4: Improved proliferation medium and proliferation culture

[0100] S4.1: 20 g of fresh banana and 10 g of potato were put into a crusher, and 90 mL of coconut juice was added to crush the mixture to obtain a mixed crushed product. Activated carbon particles were ground to obtain activated carbon powder. 2 g of activated carbon powder was added to the mixed crushed product, and the mixture was mixed and stirred evenly under the assistance of ultrasound to obtain a mixture.

[0101] S4.2: Using MS medium as the basic medium, add 1.0 mg / L BA, 0.2 mg / L NAA, 1 g / LPVP, and 4 g / L mixture into the MS basic medium, mix well, and obtain the proliferation medium required for proliferation culture;

[0102] S4.3: sterilize the proliferation culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified proliferation medium;

[0103] S4.4: The explants with buds were removed under sterile conditions, the buds were separated with a scalpel, and inoculated into proliferation medium. The temperature of the culture room was 25°C, and the cells were cultured in the dark for 5 days, and then cultured under a light intensity of 35 μmol.m -2 .s -1After culturing for 20 days, the proliferated clumps of Tripterygium wilfordii were obtained;

[0104] S5: Improved rooting medium and rooting culture

[0105] S5.1: Grind and sieve 2 g of activated carbon particles to obtain activated carbon powder, add 20 g of sucrose and 230 mg of nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture;

[0106] S5.2: Using 1 / 2MS medium as the basic medium, add 2mg / L IBA, 0.5mg / L NAA and 20g / L mixture into the 1 / 2MS basic medium, mix well, and obtain the medium required for rooting culture;

[0107] S5.3: sterilize the rooting culture medium by high pressure sterilization at 120°C for 15 min to obtain a modified rooting medium;

[0108] S5.4: Inoculate the clustered buds onto the rooting medium. The temperature in the culture room is 25°C and the light intensity is 35 μmol.m -2 .s -1 After culturing for 30 days, rooted Tripterygium wilfordii plants were obtained;

[0109] S6: Transplanting of three-leaf green seedlings

[0110] S6.1: Place the culture medium of the rooted Tripterygium wilfordii plants in a natural environment for 5 days, sprinkle some water on the surface of the culture medium, and then place it for another day;

[0111] S6.2: Take out the placed cloverleaf green plant seedlings, clean the base of the cloverleaf green plant, and transplant the cloverleaf green plant seedlings to a mixed substrate of perlite and peat soil with a mixing ratio of 1:2. Maintain the relative humidity of the environment at 80% for 28 days, and avoid direct sunlight for the first 12 days;

[0112] S6.3: After 28 days, transplant the clover seedlings into the field.

[0113] Example 3

[0114] A method for rapid propagation of Tripterygium wilfordii tissue culture, such as Figure 1 As shown, the specific steps include:

[0115] S1: Selection and sterilization of explants

[0116] S1.1: Pick 10 healthy and healthy stem segments of the axillary buds of "Zhongze No. 1" Sanyeqing that are free of pests and diseases and bacteria, and bring them back for use;

[0117] S1.2: Clean the brought back axillary bud stem segments with detergent, rinse under running water for 30 minutes, wash with sterile water for 3 times, place in 1mmol / L disodium ethylenediaminetetraacetic acid solution for 10 minutes, soak in sterile water for 20 minutes, dry, and sterilize with ultraviolet light for 30 minutes to obtain preliminarily sterilized axillary bud stem segments;

[0118] S1.3: Place the initially sterilized axillary bud stem segment in a 70% ethanol solution, soak for 15 seconds, rinse with sterile water, place the axillary bud stem segment in a 2% sodium hypochlorite solution for 20 minutes, rinse with sterile water, place the axillary bud stem segment in a 0.1% mercuric chloride solution, add 2 drops of Tween, sterilize for 10 minutes, rinse with sterile water 3 times, finally place in 8% hydrogen peroxide for 60 seconds for shock disinfection, and rinse with sterile water to obtain the sterilized explant axillary bud stem segment;

[0119] S1.4: Use a sterile scalpel to cut off both ends of the disinfected explant axillary bud stem segment, and use sterile filter paper to absorb the moisture. Then use a sterile scalpel and tweezers to cut it into small segments, ensuring that each segment contains one axillary bud, and set aside.

[0120] S2: Preparation of antimicrobial agents

[0121] S2.1: Take 10 parts by weight of anhydrous ethanol, add 2 parts by weight of nano zinc oxide, and disperse by ultrasonic for 20 minutes. Add 0.1 parts by weight of sorbic acid to the dispersion, heat in a water bath at 60°C for 20 minutes, centrifuge to obtain a precipitate, and vacuum dry the precipitate at 60°C for 12 hours to obtain modified nano zinc oxide;

[0122] S2.2: Take 2 parts by weight of modified nano zinc oxide, add 5 parts by weight of chitosan and 1 part by weight of tea polyphenols, and stir magnetically for 20 minutes to obtain an antibacterial agent.

[0123] S3: Improved induction medium and induction culture

[0124] S3.1: Take MS medium as the basic medium, add 0.5 mg / L 6-BA, 7-8 g / L agar, 25 g / L sucrose, 0.1 mg / L NAA and 1 g / L antimicrobial agent into the MS basic medium, mix well, and obtain the medium required for improved induction culture;

[0125] S3.2: sterilize the induced culture medium by high pressure sterilization at 125°C for 20 min to obtain a modified induction medium;

[0126] S3.3: Cut the explant axillary bud stem segments into small pieces and inoculate them into the culture medium. The temperature of the culture room is 28℃ and the light intensity is 40μmol.m -2 .s -1 , cultured for 20 days, and obtained new shoots after induced culture;

[0127] S4: Improved proliferation medium and proliferation culture

[0128] S4.1: Put 10g of fresh banana and 5g of potato into a crusher, add 80mL of coconut juice and crush to obtain a mixed crushed product, grind the activated carbon particles to obtain activated carbon powder, add 1g of activated carbon powder to the mixed crushed product, mix and stir evenly under the assistance of ultrasound to obtain a mixture;

[0129] S4.2: Using MS medium as the basic medium, add 0.5 mg / L BA, 0.1 mg / L NAA, 0.5 g / L PVP, and 2 g / L mixture into the MS basic medium, mix well, and obtain the proliferation medium required for proliferation culture;

[0130] S4.3: sterilize the proliferation culture medium by high pressure sterilization at 125°C for 20 min to obtain a modified proliferation medium;

[0131] S4.4: The explants with buds were removed under sterile conditions, the buds were separated with a scalpel, and inoculated into proliferation medium. The temperature of the culture room was 28°C, and the cells were cultured in the dark for 10 days, and then cultured under a light intensity of 40 μmol.m -2 .s -1 After culturing for 25 days, the proliferated clumps of Tripterygium wilfordii were obtained;

[0132] S5: Improved rooting medium and rooting culture

[0133] S5.1: Grind and sieve 1 g of activated carbon particles to obtain activated carbon powder, add 15 g of sucrose and 200 mg of nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture;

[0134] S5.2: Using 1 / 2MS medium as the basic medium, add 1 mg / L IBA, 0.2 mg / L NAA and 15 g / L mixture into the 1 / 2MS basic medium, mix well, and obtain the medium required for rooting culture;

[0135] S5.3: sterilize the rooting culture medium by high pressure sterilization at 125°C for 20 min to obtain a modified rooting medium;

[0136] S5.4: Inoculate the clustered buds onto the rooting medium. The temperature in the culture room is 28°C and the light intensity is 40 μmol.m -2 .s -1 After culturing for 40 days, rooted Tripterygium wilfordii plants were obtained;

[0137] S6: Transplanting of three-leaf green seedlings

[0138] S6.1: Place the culture medium of the rooted Tripterygium wilfordii plants in a natural environment for 6 days, sprinkle some water on the surface of the culture medium, and then place it for another 2 days;

[0139] S6.2: Take out the placed cloverleaf green plant seedlings, clean the base of the cloverleaf green plant, and transplant the cloverleaf green plant seedlings to a mixed substrate of perlite and peat soil with a mixing ratio of 1:2. Maintain the relative humidity of the environment at 80% for 30 days, and avoid direct sunlight for the first 14 days;

[0140] S6.3: After 30 days, transplant the clover seedlings into the field.

[0141] Comparative Example 1

[0142] Compared with Example 1, the difference of Comparative Example 1 is that, in Comparative Example 1, steps 1.2-1.3 are replaced by "immersing the explant in 70% alcohol for disinfection for 1 minute, then rinsing with sterile water 3 times, subsequently treating with 0.1% HgCl2 solution for 3 minutes, constantly stirring with a glass rod during the period, and finally rinsing with sterile water 6 times", and the other steps remain unchanged to cultivate the three-leaf green plants, which is recorded as Comparative Example 1.

[0143] Comparative Example 2

[0144] Compared with Example 1, the difference of Comparative Example 2 is that the antibacterial agent in step S2 and step S3.1 is removed in Comparative Example 2, and the other steps are unchanged to cultivate the clover plants, which is recorded as Comparative Example 2.

[0145] Comparative Example 3

[0146] Compared with Example 1, the difference of Comparative Example 3 is that, in Comparative Example 3, steps 1.2-1.3 are replaced by "immersing the explant in 70% alcohol for disinfection for 1 minute, then rinsing with sterile water 3 times, subsequently treating with 0.1% HgCl2 solution for 3 minutes, constantly stirring with a glass rod during the period, and finally rinsing with sterile water 6 times", and the antibacterial agent in step S2 and step S3.1 is removed, and the other steps remain unchanged to cultivate the three-leaf green plants, which is recorded as Comparative Example 3.

[0147] The contamination rate was obtained by the number of contaminated culture media / total number of culture media × 100%, and the disinfection mortality rate was obtained by the number of dead clover plants after disinfection / total number of clover plants × 100%. Two parallel experiments were performed to calculate the contamination rate and disinfection mortality rate of Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3, and the above data were tabulated for reference. Figure 2 It can be seen that the contamination rates of Example 1, Example 2 and Example 3 are much lower than those of Comparative Example 1, Comparative Example 2 and Comparative Example 3, which proves that the process flow in the embodiment can reduce the bacteria in the explants to the greatest extent, avoid the contamination of the culture medium by the axillary bud stem segments, and significantly improve the antibacterial property of the culture medium after adding the antibacterial agent to avoid contamination.

[0148] Comparative Example 4

[0149] Compared with Example 1, the difference of Comparative Example 4 is that Comparative Example 4 removes steps S4.1-S4.3, replaces the improved proliferation medium in step S4.4 with "MS+6-BA4.0mg / L proliferation medium", and cultivates Tripterygium wilfordii plants in the remaining steps unchanged, which is recorded as Comparative Example 4.

[0150] The number of proliferated seedlings in Example 1, Example 2, Example 3 and Comparative Example 4 was recorded and the proliferation coefficient was calculated. The higher the proliferation coefficient, the better the effect of the proliferation medium. The survival rate was obtained by using the number of surviving plants / total number of plants × 100%. Two parallel experiments were performed to calculate the survival rates of Example 1, Example 2, Example 3 and Comparative Example 4. The above data were tabulated for reference. Figure 3 It can be seen that the proliferation coefficient and survival rate of Example 1, Example 2 and Example 3 are much higher than those of Comparative Example 4, proving that the improved proliferation medium is more conducive to the growth and development of explants and can improve the survival rate of transplanted clover seedlings.

[0151] Comparative Example 5

[0152] Compared with Example 1, the difference of Comparative Example 5 is that Comparative Example 5 removes steps S5.1-S5.3, replaces the modified rooting medium in step S5.4 with "1 / 2MS+IBA2.0mg / L rooting medium", and cultivates the Tripterygium wilfordii plants without changing the other steps, which is recorded as Comparative Example 5.

[0153] The rooting rate was obtained by the number of rooted plants / total number of plants × 100%, and the survival rate was obtained by the number of surviving plants / total number of plants × 100%. Two parallel experiments were performed to calculate the rooting rate and survival rate of Example 1, Example 2, Example 3 and Comparative Example 5, and the above data were tabulated for reference. Figure 4 It can be seen that the rooting rate and survival rate of Example 1, Example 2 and Example 3 are much higher than those of Comparative Example 5, proving that the improved rooting medium is more conducive to the rooting of the Tripterygium wilfordii plants and can improve the survival rate of transplanted Tripterygium wilfordii seedlings.

[0154] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for rapid propagation of Tripterygium wilfordii tissue culture, characterized in that: The specific steps include: S1: Selection and sterilization of explants Pick harmless axillary bud stem segments of Tripterygium wilfordii, clean them, perform preliminary disinfection on them, and then perform deep disinfection and sterilization on them to obtain sterilized explant axillary bud stem segments, dry the sterilized explant axillary bud stem segments, and then cut them into segments, each segment is guaranteed to contain one axillary bud; S2: Preparation of antimicrobial agents After the nano zinc oxide is modified, 2-4 weight parts of the modified nano zinc oxide are added with 5-8 weight parts of chitosan and 1-2 weight parts of tea polyphenols, and magnetic stirring is performed to obtain an antibacterial agent; S3: Improved induction medium and induction culture Using MS medium as the basic medium, adding 0.5-1.0 mg / L 6-BA, 7-8 g / L agar, 25-30 g / L sucrose, 0.1-0.5 mg / L NAA and 1-2 g / L antimicrobial agent into the MS basic medium, mixing and sterilizing to obtain a modified induction medium, inoculating the explant axillary bud stem segments cut into small sections onto the medium for cultivation to obtain new shoots after induction culture; S4: Improved proliferation medium and proliferation culture 10-20g of fresh bananas and 5-10g of potatoes are put into a crusher, and then 80-90ml / L of coconut juice is added to crush the mixture to obtain a mixed crushed product, 1-2g / L of activated carbon powder is added to the mixed crushed product, and the mixture is mixed and stirred evenly under the assistance of ultrasound to obtain a mixture, MS medium is used as a basic medium, 0.5-1.0mg / L BA, 0.1-0.2mg / L NAA, 0.5-1g / LPVP, and 2-4g / L of the mixture are added to the MS basic medium, and the mixture is mixed and sterilized to obtain a modified proliferation medium, and an explant with buds is taken out under sterile conditions, the buds are separated with a scalpel, and the buds are inoculated on the proliferation medium to obtain the proliferated three-leaf green clustered buds; S5: Improved rooting medium and rooting culture 1-2g of activated carbon particles are ground, crushed and sieved to obtain activated carbon powder, 15-20g of sucrose and 200-230mg of nano-silicon dioxide are added to the activated carbon powder, and the mixture is stirred evenly to obtain a mixture, 1 / 2MS medium is used as the basic medium, 1-2mg / LIBA, 0.2-0.5mg / LNAA and 15-20g / L of the mixture are added to the 1 / 2MS basic medium, the mixture is mixed evenly and sterilized to obtain a modified rooting medium, and the clustered buds are inoculated into the rooting medium to obtain rooted clover plants; S6: Transplanting of three-leaf green seedlings After placing the rooted Tripterygium wilfordii plant culture medium in a natural environment for a period of time, take out the Tripterygium wilfordii plant seedlings, clean the base of the Tripterygium wilfordii plant, transplant the Tripterygium wilfordii plant seedlings to a mixed matrix of perlite and peat soil, cultivate for a period of time, and then transplant them into the field.

2. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S1: selection and sterilization of explants, specifically including the following steps: S1.1: Pick 10-20 healthy stem segments of axillary buds of Tripterygium wilfordii that are free of pests and diseases and bacteria, and bring them back for later use; S1.2: Clean the brought back axillary bud stem segments with detergent, rinse under running water for 20-30 minutes, wash with sterile water 2-3 times, place in 1-2mmol / L disodium ethylenediaminetetraacetic acid solution for 5-10 minutes, soak in sterile water for 10-20 minutes, dry, and sterilize with ultraviolet light for 20-30 minutes to obtain preliminarily sterilized axillary bud stem segments; S1.3: Place the initially sterilized axillary bud stem segment in a 70-75% ethanol solution, soak for 10-15 seconds, rinse with sterile water, place the axillary bud stem segment in a 2-3% sodium hypochlorite solution for disinfection for 15-20 minutes, rinse with sterile water, place the axillary bud stem segment in a 0.1-0.2% mercuric chloride solution, add 2-3 drops of Tween, sterilize for 6-10 minutes, rinse with sterile water 2-3 times, finally place in 8-10% hydrogen peroxide for shaking disinfection for 50-60 seconds, and rinse with sterile water to obtain the sterilized explant axillary bud stem segment; S1.4: Use a sterile scalpel to cut off both ends of the disinfected explant axillary bud stem segment, and use sterile filter paper to absorb the moisture. Then use a sterile scalpel and tweezers to cut it into small segments, ensuring that each segment contains one axillary bud, and set aside.

3. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S2: Preparation of antibacterial agent, specifically comprising the following steps: S2.1: Take 10-20 parts by weight of anhydrous ethanol, add 2-4 parts by weight of nano zinc oxide, and ultrasonically disperse for 10-20 minutes. Add 0.1-0.2 parts by weight of sorbic acid to the dispersion, heat in a water bath at 50-60°C for 10-20 minutes, centrifuge to obtain a precipitate, and vacuum dry the precipitate at 50-60°C for 10-12 hours to obtain modified nano zinc oxide; S2.2: Take 2-4 parts by weight of modified nano zinc oxide, add 5-8 parts by weight of chitosan and 1-2 parts by weight of tea polyphenols, and stir magnetically for 10-20 minutes to obtain an antibacterial agent.

4. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S3 improves the induction medium and induces the culture, specifically comprising the following steps: S3.1: Using MS medium as the basic medium, add 0.5-1.0 mg / L 6-BA, 7-8 g / L agar, 25-30 g / L sucrose, 0.1-0.5 mg / L NAA and 1-2 g / L antimicrobial agent into the MS basic medium, mix well, and obtain the medium required for improved induction culture; S3.2: sterilize the induced culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified induction medium; S3.3: Inoculate the explant axillary bud stem segments cut into small sections onto the culture medium. The temperature in the culture room is 25-28°C, the light intensity is 35-40 μmol.m-2.s-1, and the culture is carried out for 15-20 days to obtain new shoots after induced culture.

5. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S4 improves the proliferation medium and the proliferation culture, specifically comprising the following steps: S4.1: Put 10-20g of fresh banana and 5-10g of potato into a crusher, add 80-90ml / L of coconut juice and crush to obtain a mixed crushed product, grind the activated carbon particles to obtain activated carbon powder, add 1-2g / L of activated carbon powder to the mixed crushed product, mix and stir evenly under the assistance of ultrasound to obtain a mixture; S4.2: Using MS medium as the basic medium, add 0.5-1.0 mg / L BA, 0.1-0.2 mg / L NAA, 0.5-1 g / L PVP, and 2-4 g / L mixture into the MS basic medium, mix well, and obtain the proliferation medium required for proliferation culture; S4.3: sterilize the proliferation culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified proliferation medium; S4.4: Take out the explants with buds under sterile conditions, separate the buds with a scalpel, and inoculate them into the proliferation culture medium. The temperature in the culture room is 25-28℃. First culture them in the dark for 5-10 days, and then culture them at a light intensity of 35-40μmol.m-2.s-1 for 20-25 days to obtain the proliferated clumps of Tripterygium wilfordii buds.

6. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S5 improves the rooting medium and performs rooting culture, specifically comprising the following steps: S5.1: Grind and crush 1-2 g of activated carbon particles and sieve to obtain activated carbon powder, add 15-20 g of sucrose and 200-230 mg of nano-silicon dioxide to the activated carbon powder, mix and stir evenly to obtain a mixture; S5.2: Using 1 / 2MS medium as the basic medium, add 1-2mg / L IBA, 0.2-0.5mg / L NAA and 15-20g / L mixture into the 1 / 2MS basic medium, mix well, and obtain the medium required for rooting culture; S5.3: sterilize the rooting culture medium by high pressure sterilization at 120-125°C for 15-20 min to obtain a modified rooting medium; S5.4: Inoculate the clustered buds onto the rooting medium. The temperature in the culture room is 25-28°C and the light intensity is 35-40 μmol.m -2 .s -1 After 30-40 days of cultivation, rooted Tripterygium wilfordii plants were obtained.

7. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 1, characterized in that: Step S6, transplanting of three-leaf seedlings, specifically comprises the following steps: S6.1: Place the culture medium of the rooted Tripterygium wilfordii plants in a natural environment for 5-6 days, sprinkle some water on the surface of the culture medium, and then place it for another 1-2 days; S6.2: Take out the placed cloverleaf green plant seedlings, clean the base of the cloverleaf green plant, and transplant the cloverleaf green plant seedlings to a mixed substrate of perlite and peat soil, maintain the relative humidity of the environment for 28-30 days, and avoid direct sunlight for the first 12-14 days; S6.3: After 28-30 days, transplant the clover seedlings into the field.

8. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 2, characterized in that: The variety of Sanyeqing in step S1.1 is Zhongze No.

1.

9. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 7, characterized in that: In step S6.2, the relative humidity of the environment is 70-80%.

10. The method for rapid propagation of Tripterygium wilfordii tissue culture according to claim 7, characterized in that: In step S6.2, the mixing ratio of perlite to peat soil is 1:1-2.

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