A culture method for improving browning of peony callus

Abstract

This invention discloses a culture method for improving browning of peony callus, belonging to the field of plant tissue culture. This method involves adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the culture medium in both the callus subculture and adventitious shoot differentiation stages. Through the synergistic effect of these two substances, a large quantity of peony callus can be obtained. After subculture, the callus quality is significantly improved, the browning rate is significantly reduced compared to the control, the number of embryogenic calluses is increased, and the browning rate of peony callus is reduced to below 20%, laying a good foundation for the subsequent induction of clustered shoots. Simultaneously, experiments verified that the synergistic effect of the two substances is better than adding them alone.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically relating to a culture method for improving browning of peony callus. Background Technology

[0002] Peony, a perennial herbaceous plant belonging to the genus Paeonia in the family Ranunculaceae, is a famous ornamental and medicinal plant in my country. Its roots are used medicinally, and there are two types: "red peony" and "white peony." The former is good at clearing heat and cooling blood, dispersing blood stasis and relieving pain, and clearing liver fire; the latter is good at nourishing blood and regulating menstruation, and soothing the liver and relieving pain. Traditional methods of peony propagation include division and sowing. However, these methods have drawbacks such as long cycles, low efficiency, and low propagation coefficients, and cannot effectively maintain the superior traits of the parent plant, seriously affecting the large-scale production of peonies.

[0003] Plant tissue culture is one of the most effective methods for rapid plant propagation. Compared with traditional propagation methods, tissue culture can maintain the original variety and superior traits, increase its propagation coefficient, shorten the breeding cycle, and operate year-round without being affected by the seasons, greatly improving propagation efficiency and facilitating large-scale production. However, in the current process of peony tissue culture, explants turn blackish-brown under the influence of phenolic compounds secreted by the plant itself, a phenomenon known as browning, which can lead to plant death in severe cases. Using anti-browning agents can effectively reduce the occurrence of browning; commonly used anti-browning agents include vitamin C, polyvinylpyrrolidone, and AgNO3. In peony tissue culture, adding anti-browning agents can reduce browning to varying degrees, but the effect is limited. The addition of some anti-browning agents can also cause material distortion, hindering the growth of experimental materials and severely restricting the development of peony tissue culture. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the browning of peony callus. This method involves adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the culture medium during both the callus subculture and adventitious shoot differentiation stages. Through the synergistic effect of these two substances, a large quantity of peony callus can be obtained. Furthermore, after subculture, the quality of the callus is significantly improved, the browning rate is substantially reduced compared to the control, the number of embryogenic calluses is increased, and the browning rate of peony callus is reduced to below 20%, laying a good foundation for the subsequent induction of clustered shoots. Simultaneously, experiments have verified that the synergistic effect of the two substances is better than adding them alone.

[0005] This invention is achieved through the following technical solution:

[0006] A method for improving browning of peony callus includes adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the callus subculture medium during the callus subculture step.

[0007] Melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol were added to the callus adventitious shoot differentiation medium during the callus adventitious shoot differentiation culture step.

[0008] Preferably, the melatonin concentration is 0.1-1.0 mg / L;

[0009] The mass concentration of the 3-[(9H-purine-6-ylamino)methyl]phenol is 0.2-0.8 mg / L.

[0010] Preferably, the cultivation method includes the following steps:

[0011] S1. Explant selection and disinfection:

[0012] Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20-30 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 5-6 minutes, rinse with sterile water 4-5 times, cut into petiole segments or leaves of 2-3 square centimeters, and inoculate into callus induction medium. Inoculate about 4 explants per bottle. Callus can be induced after 40 days of culture.

[0013] S2, Callus subculture:

[0014] The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture.

[0015] S3, Adventitious shoot differentiation culture of callus tissue:

[0016] Subcultured callus tissues were inoculated into callus differentiation medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. Embryoid callus tissues were obtained after 50 days of culture.

[0017] S4. The embryonic callus tissue obtained above is cultured in vitro to complete the process.

[0018] Preferably, in S1, the callus induction culture medium formula is as follows:

[0019] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0020] Preferably, in S2, the callus subculture medium formulation is as follows:

[0021] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + NAA 0.1 mg / L + thidiazuron 0.2-0.8 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0022] Preferably, in S3, the formula for the callus adventitious shoot differentiation culture medium is as follows:

[0023] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + GA3 0.6-1.5 mg / L + NAA 0.1-0.3 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0024] Preferably, in step S4, the culture temperature is 20-25°C.

[0025] Preferably, in step S4, the cultivation is carried out under a light intensity of 1200-2200 lux.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] This invention provides a method for improving the browning of peony callus. This method involves adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the culture medium during both the callus subculture and adventitious shoot differentiation stages. Through the synergistic effect of these two substances, a large quantity of peony callus can be obtained. After subculture, the callus quality is significantly improved, the browning rate is substantially reduced compared to the control, the number of embryogenic calluses is increased, and the browning rate of peony callus is reduced to below 20%, laying a good foundation for the subsequent induction of clustered shoots. Furthermore, experiments have verified that the synergistic effect of the two substances is better than adding them alone. Attached Figure Description

[0028] Figure 1 This is a flowchart of the culture method for improving browning of peony callus in Example 1. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] One specific embodiment of the present invention is as follows:

[0031] A method for improving browning of peony callus includes adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the culture medium in the callus subculture step and the callus adventitious shoot differentiation step, respectively.

[0032] Preferably, the melatonin concentration is 0.1-1.0 mg / L;

[0033] The mass concentration of the 3-[(9H-purine-6-ylamino)methyl]phenol is 0.2-0.8 mg / L.

[0034] Preferably, the cultivation method includes the following steps:

[0035] S1. Explant selection and disinfection:

[0036] Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20-30 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 5-6 minutes, rinse with sterile water 4-5 times, cut into petiole segments or leaves of 2-3 square centimeters, and inoculate into callus induction medium. Inoculate about 4 explants per bottle. Callus can be induced after 40 days of culture.

[0037] S2, Callus subculture:

[0038] The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture.

[0039] S3, Adventitious shoot differentiation culture of callus tissue:

[0040] Subcultured callus tissues were inoculated into callus differentiation medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. Embryoid callus tissues were obtained after 50 days of culture.

[0041] S4. The embryonic callus tissue obtained above is cultured in vitro to complete the process.

[0042] Preferably, in S1, the callus induction culture medium formula is as follows:

[0043] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0044] Preferably, in S2, the callus subculture medium formulation is as follows:

[0045] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + NAA 0.1 mg / L + thidiazuron 0.2-0.8 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0046] Preferably, in S3, the formula for the callus adventitious shoot differentiation culture medium is as follows:

[0047] 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + GA3 0.6-1.5 mg / L + NAA 0.1-0.3 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

[0048] Preferably, in step S4, the culture temperature is 20-25°C.

[0049] Preferably, in step S4, the cultivation is carried out under a light intensity of 1200-2200 lux.

[0050] Example 1:

[0051] A culture method for improving browning of peony callus includes the following steps: (Specific process follows) Figure 1 As shown:

[0052] S1. Explant selection and disinfection:

[0053] Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 6 minutes, rinse with sterile water 4 times, cut into petiole segments or leaves of 2 square centimeters, and inoculate into callus induction medium. Inoculate about 4 explants per bottle. Callus can be induced after 40 days of culture.

[0054] S2, Callus subculture:

[0055] The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture.

[0056] S3, Adventitious shoot differentiation culture of callus tissue:

[0057] Subcultured callus tissues were inoculated into callus differentiation medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. Embryoid callus tissues were obtained after 50 days of culture.

[0058] S4. The embryonic callus tissue obtained above is cultured in vitro to complete the process.

[0059] In S1, the formula for the callus induction culture medium is as follows:

[0060] 1 / 2 MS medium + calcium pantothenate 1.0 mg / L + adenine 1.0 mg / L + L-cysteine ​​0.5 mg / L + biotin 0.1 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 26 g / L + plant gel 2.2 g / L.

[0061] In S2, the formula for the callus subculture medium is as follows:

[0062] 1 / 2 MS medium + calcium pantothenate 0.5 mg / L + adenine 0.2 mg / L + L-cysteine ​​1.0 mg / L + biotin 0.1 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + NAA 0.1 mg / L + thidiazuron 0.4 mg / L + sucrose 28 g / L + plant gel 2.2 g / L.

[0063] In S3, the formula for the callus adventitious shoot differentiation culture medium is as follows:

[0064] 1 / 2 MS medium + calcium pantothenate 0.5 mg / L + adenine 0.2 mg / L + L-cysteine ​​1.0 mg / L + biotin 0.1 mg / L + melatonin 0.5 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.6 mg / L + GA3 0.8 mg / L + NAA 0.2 mg / L + sucrose 28 g / L + plant gel 2.2 g / L.

[0065] In S4, the culture temperature is 25°C.

[0066] In S4, the culture is carried out under a light intensity of 1200 lux.

[0067] Example 2:

[0068] A culture method for improving browning of peony callus includes the following steps:

[0069] S1. Explant selection and disinfection:

[0070] Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 6 minutes, rinse with sterile water 4 times, cut into petiole segments or leaves of 2 square centimeters, and inoculate into callus induction medium. Inoculate about 4 explants per bottle. Callus can be induced after 40 days of culture.

[0071] S2, Callus subculture:

[0072] The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture.

[0073] S3, Adventitious shoot differentiation culture of callus tissue:

[0074] Subcultured callus tissues were inoculated into callus differentiation medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. Embryoid callus tissues were obtained after 50 days of culture.

[0075] S4. The embryonic callus tissue obtained above is cultured in vitro to complete the process.

[0076] In S1, the formula for the callus induction culture medium is as follows:

[0077] 1 / 2 MS medium + calcium pantothenate 0.1 mg / L + adenine 0.1 mg / L + L-cysteine ​​0.1 mg / L + biotin 0.05 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 22 g / L + plant gel 1.5 g / L.

[0078] In S2, the formula for the callus subculture medium is as follows:

[0079] 1 / 2 MS medium + calcium pantothenate 0.1 mg / L + adenine 0.1 mg / L + L-cysteine ​​0.1 mg / L + biotin 0.05 mg / L + melatonin 0.1 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2 mg / L + NAA 0.1 mg / L + thidiazuron 0.2 mg / L + sucrose 22 g / L + plant gel 1.5 g / L.

[0080] In S3, the formula for the callus adventitious shoot differentiation culture medium is as follows:

[0081] 1 / 2 MS medium + calcium pantothenate 0.1 mg / L + adenine 0.1 mg / L + L-cysteine ​​0.1 mg / L + biotin 0.05 mg / L + melatonin 0.1 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2 mg / L + GA3 0.6 mg / L + NAA 0.1 mg / L + sucrose 22 g / L + plant gel 1.5 g / L.

[0082] In S4, the culture temperature is 20°C.

[0083] In S4, the cultivation is carried out under a light intensity of 2200 lux.

[0084] Example 3:

[0085] A culture method for improving browning of peony callus includes the following steps:

[0086] S1. Explant selection and disinfection:

[0087] Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 6 minutes, rinse with sterile water 4 times, cut into petiole segments or leaves of 2 square centimeters, and inoculate into callus induction medium. Inoculate about 4 explants per bottle. Callus can be induced after 40 days of culture.

[0088] S2, Callus subculture:

[0089] The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture.

[0090] S3, Adventitious shoot differentiation culture of callus tissue:

[0091] Subcultured callus tissues were inoculated into callus differentiation medium, with 6 callus tissues per bottle, each about 2 square centimeters in size. Embryoid callus tissues were obtained after 50 days of culture.

[0092] S4. The embryonic callus tissue obtained above is cultured in vitro to complete the process.

[0093] In S1, the formula for the callus induction culture medium is as follows:

[0094] 1 / 2 MS medium + calcium pantothenate 1.5 mg / L + adenine 1.5 mg / L + L-cysteine ​​1.5 mg / L + biotin 0.2 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 30 g / L + plant gel 2.5 g / L.

[0095] In S2, the formula for the callus subculture medium is as follows:

[0096] 1 / 2 MS medium + calcium pantothenate 1.5 mg / L + adenine 1.5 mg / L + L-cysteine ​​1.5 mg / L + biotin 0.2 mg / L + melatonin 1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.8 mg / L + NAA 0.1 mg / L + thidiazuron 0.8 mg / L + sucrose 30 g / L + plant gel 2.5 g / L.

[0097] In S3, the formula for the callus adventitious shoot differentiation culture medium is as follows:

[0098] 1 / 2 MS medium + calcium pantothenate 1.5 mg / L + adenine 1.5 mg / L + L-cysteine ​​1.5 mg / L + biotin 0.2 mg / L + melatonin 1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.8 mg / L + GA3 1.5 mg / L + NAA 0.3 mg / L + sucrose 30 g / L + plant gel 2.5 g / L.

[0099] In S4, the culture temperature is 25°C.

[0100] In S4, the cultivation is carried out under a light intensity of 2200 lux.

[0101] Experimental example:

[0102] Experiment 1: Effects of different culture medium formulations on the subculturing of peony callus

[0103] Materials: Induced callus tissue from Paeonia lactiflora

[0104] Basic formula: 1 / 2 MS medium + calcium pantothenate 0.5 mg / L + adenine 0.2 mg / L + L-cysteine ​​1.0 mg / L + biotin 0.1 mg / L + NAA 0.1 mg / L + TDZ 0.4 mg / L + sucrose 28 g / L + plant gel 2.2 g / L.

[0105] Based on the basic formula, variables were added to create a peony culture medium.

[0106] The variables were: CK1: 0; treatment 1: melatonin 0.5 mg / L, treatment 2: metatopolin 0.6 mg / L, treatment 3: melatonin 0.5 mg / L + metatopolin 0.6 mg / L.

[0107] After culturing for 50 days at a temperature of 25℃ and a light intensity of 2000 lux, the results are as follows:

[0108] Results of subculture experiment of peony callus

[0109] deal with growth Browning rate Water staining rate state CK1 1 81％ 15％ + Process 1 1.2 60％ 30％ ++ Process 2 1.3 40％ 20％ ++ Process 3 2.0 10％ 0 ++++

[0110] (Growth: Relative values ​​of growth between different treatments and the control)

[0111] The "+" symbols above represent the state of the callus tissue. "+" indicates severe browning and water staining, with poor growth; "++" indicates relatively severe browning and water staining, with average growth; and "++++" indicates little browning, no water staining, and good growth.

[0112] Experiment 2: Effects of different culture medium formulations on peony callus differentiation

[0113] Materials: Peony callus tissue

[0114] Basic formula: 1 / 2 MS medium + calcium pantothenate 0.5 mg / L + adenine 0.2 mg / L + L-cysteine ​​1.0 mg / L + biotin 0.1 mg / L + melatonin 0.1-1.0 mg / L + metatopolin 0.2-0.8 mg / L + GA3 0.8 mg / L + NAA 0.2 mg / L + sucrose 286 g / L + plant gel 2.2 g / L.

[0115] Based on the basic formula, variables were added to create a peony culture medium.

[0116] The variables were: CK2: 0; treatment 4: melatonin 0.5 mg / L, treatment 5: metatopolin 0.6 mg / L, treatment 6: melatonin 0.5 mg / L + metatopolin 0.6 mg / L.

[0117] After culturing for 50 days at a temperature of 25℃ and a light intensity of 2000 lux, the results are as follows:

[0118] Results of peony callus differentiation culture experiment

[0119]

[0120] (Growth: Relative values ​​of growth between different treatments and the control)

[0121] The "+" symbols above represent the state of the callus tissue. "+" indicates severe browning and water staining, with poor growth; "++" indicates relatively severe browning and water staining, with average growth; and "++++" indicates little browning, no water staining, and good growth.

[0122] The data table above shows that the browning rate in CK1, the control treatment without this method, was between 81% and 95%. After using this method (treatment 1, treatment 2, and treatment 3), the browning rate can be reduced to 10% and 16%, respectively, which is below 20%.

[0123] Based on the extensive experiments conducted above, melatonin and metatopolin have a synergistic effect. The combined addition of 0.05-0.2 mg / L melatonin (0.1-1.0 mg / L) and 0.2-0.8 mg / L metatopolin is more effective than adding them separately.

[0124] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A culture method for improving browning of peony callus, characterized in that, This includes adding melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol to the callus subculture medium during the callus subculture step; And, melatonin and 3-[(9H-purine-6-ylamino)methyl]phenol were added to the callus adventitious shoot differentiation medium in the callus adventitious shoot differentiation culture step; The melatonin concentration is 0.1-1.0 mg / L; The mass concentration of the 3-[(9H-purin-6-ylamino)methyl]phenol is 0.2-0.8 mg / L; The cultivation method includes the following steps: S1. Explant selection and disinfection: Select disease-free peony buds, young leaves, and petioles from April to May as explants. Rinse with tap water for 2-5 hours, disinfect with 75% alcohol for 20-30 seconds in a clean bench, disinfect with 0.1% mercuric chloride for 5-6 minutes, rinse with sterile water 4-5 times, cut into petiole segments or leaves of 2-3 square centimeters, and inoculate into callus induction medium. Inoculate 4 explants per bottle. Callus can be induced after 40 days of culture. S2, Callus subculture: The induced callus tissue was inoculated into the subculture medium, with 6 callus tissues per bottle, each 2 square centimeters in size. A large amount of callus tissue could be obtained after 50 days of culture. S3, Adventitious shoot differentiation culture of callus tissue: Subcultured callus tissue was inoculated into callus differentiation medium, with 6 callus tissues per bottle, each 2 square centimeters in size. Embryoid callus tissue was obtained after 50 days of culture. S4. The embryoid callus tissue obtained above is cultured in vitro to complete the process. In S1, the formula for the callus induction culture medium is as follows: 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + 2,4-dichlorophenoxyacetic acid 0.2 mg / L + thidiazuron 0.4 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L; In S2, the formula for the callus subculture medium is as follows: 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + NAA 0.1 mg / L + thidiazuron 0.2-0.8 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L; In S3, the formula for the callus adventitious shoot differentiation culture medium is as follows: 1 / 2 MS medium + calcium pantothenate 0.1-1.5 mg / L + adenine 0.1-1.5 mg / L + L-cysteine ​​0.1-1.5 mg / L + biotin 0.05-0.2 mg / L + melatonin 0.1-1.0 mg / L + 3-[(9H-purin-6-ylamino)methyl]phenol 0.2-0.8 mg / L + GA3 0.6-1.5 mg / L + NAA 0.1-0.3 mg / L + sucrose 22-30 g / L + plant gel 1.5-2.5 g / L.

2. The culture method for improving browning of peony callus according to claim 1, characterized in that, In S4, the culture temperature is 20-25℃.

3. The culture method for improving browning of peony callus according to claim 1, characterized in that, In S4, the cultivation is carried out under a light intensity of 1200-2200 lux.

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