Culture medium and culture method for efficiently inducing and stably proliferating black rose calluses

Abstract

The invention provides a culture medium for efficiently inducing and stably proliferating black rose calluses and a culture method, and belongs to the technical field of plant tissue culture. According to the scheme provided by the invention, the key technical problems in the black rose in-vitro culture can be effectively solved, a specific induction culture medium formula is adopted, the callus induction rate as high as 80% can be realized, and an excellent culture system is successfully established. The specific proliferation culture medium formula provided by the invention can realize efficient amplification of calluses, the proliferation rate can reach 1.98 under the optimal proportion, and an excellent growth promotion effect is shown. Therefore, stable and large-scale black rose callus culture becomes possible, and a brand-new solution independent of traditional culture is provided for meeting the requirement of the market for the special variety. The invention lays a solid material foundation for the subsequent development of products with high added value, and greatly expands the commercial application potential and industrial value of black roses.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a highly efficient culture medium and method for inducing stable proliferation of black rose callus. Background Technology

[0002] Black Baccarat ® Meidebenne is a dark-colored cut rose introduced by Meidebenne International in France in 2003. With its signature deep purple-red to near-black, velvety petals, it has become one of the closest commercial varieties to the concept of a "black rose" on the market. This variety not only boasts elegant blooms and a long vase life, but also possesses significant breeding research value. Furthermore, its petals are rich in anthocyanin-like active ingredients, offering significant cosmetic benefits, making it a unique plant resource that combines ornamental value, commercial potential, and functional characteristics.

[0003] However, due to its demanding cultivation requirements and relatively limited yield, this variety has long maintained a degree of commercial scarcity in the cut flower market. Currently, Baccarat Black Roses lack a large-scale, standardized cultivation system in China, and existing raw materials mainly rely on imports, resulting in high costs and severely restricting its industrial application in high-value-added sectors such as cosmetics. Furthermore, as a cut flower variety, Baccarat Black Roses often involve the extensive use of pesticides during cultivation to meet the demands of the ornamental market, leading to significant pesticide residue risks. In post-harvest distribution, preservatives containing fungicides, ethylene inhibitors, and growth regulators are commonly used to extend shelf life and maintain flower color. The residues of these substances seriously affect the compliance of Black Roses as raw materials in cosmetics. Therefore, if the existing cut flower production system continues, the raw materials obtained will not meet the stringent safety and traceability requirements of the cosmetics industry.

[0004] Plant tissue culture is an important biotechnology for the rapid propagation and sustainable utilization of precious plant resources. In practical applications, different species, and even different varieties of the same species, often have significantly different requirements for culture conditions due to their unique genetic backgrounds, secondary metabolite compositions, and endogenous hormone levels, especially for ornamental varieties with high levels of special metabolites such as pigments and aromatic substances. Existing rose tissue culture techniques are mostly based on common varieties, but when directly applied to distinctive varieties such as black roses, problems such as difficulty in callus induction, slow growth, severe browning, or rapid decline in proliferative capacity after subculturing often occur. Therefore, a highly efficient and stable dedicated callus culture protocol for black roses still needs to be developed. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient culture medium and method for inducing stable proliferation of black rose callus, which solves the technical problems of low induction rate, unstable proliferation efficiency, and difficulty in establishing a sustainable in vitro culture system for black rose callus.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a black rose callus induction culture medium, comprising MS basal medium, sucrose, agar, and plant growth regulators. The plant growth regulators, in final concentrations, include 6-benzylaminopurine, naphthaleneacetic acid, and thidiazuron, wherein the concentration of 6-benzylaminopurine is 0.1–1.0 mg / L, the concentration of naphthaleneacetic acid is 0.1–1.0 mg / L, and the concentration of thidiazuron is 1.0–3.0 mg / L.

[0008] Preferably, the pH value of the black rose callus induction culture medium is 5.6~6.0.

[0009] Preferably, the concentration of the agar is 6.0~9.0 g / L and the concentration of the sucrose is 20~40 g / L, based on the final concentration.

[0010] The present invention also provides a black rose callus proliferation culture medium, comprising MS basal medium, sucrose, agar, and plant growth regulators, wherein the plant growth regulators include 6-benzylaminopurine, thiazuron, and naphthaleneacetic acid, and the final concentrations are as follows: 6-benzylaminopurine has a concentration of 0.1-1.0 mg / L, thiazuron has a concentration of 0.5-1.5 mg / L, and naphthaleneacetic acid has a concentration of 0.1-1.0 mg / L.

[0011] Preferably, the concentration ratio of 6-benzylaminopurine, thiazolinone, and naphthaleneacetic acid is selected from one of the following three combinations: (1) The concentration of 6-benzylaminopurine was 1.0 mg / L, the concentration of thiazolinone was 0.5 mg / L, and the concentration of naphthaleneacetic acid was 0.5 mg / L; (2) The concentration of 6-benzylaminopurine was 0.5 mg / L, the concentration of thiazolinone was 0.5 mg / L, and the concentration of naphthaleneacetic acid was 1.0 mg / L; (3) The concentration of 6-benzylaminopurine was 0.5 mg / L, the concentration of thiabendazole was 1.0 mg / L, and the concentration of naphthaleneacetic acid was 0.5 mg / L.

[0012] Preferably, the pH value of the black rose callus proliferation culture medium is 5.6~6.0.

[0013] Preferably, the concentration of the agar is 6.0~9.0 g / L and the concentration of the sucrose is 20~40 g / L, based on the final concentration.

[0014] The present invention also provides the application of the above-mentioned black rose callus induction medium and black rose callus proliferation medium in the culture of black rose callus.

[0015] This invention also provides a method for culturing black rose callus, comprising the following steps: Black rose explants were inoculated into the black rose callus induction medium provided by the present invention and cultured in the dark to obtain induced callus. The induced callus tissue was transferred to the above-mentioned black rose callus proliferation culture medium provided by the present invention for subculture to obtain proliferated black rose callus tissue.

[0016] Preferably, the black rose explant is a petal; The sterilization process includes soaking in an alcohol solution with a volume percentage of 60% to 80% for 30 to 60 seconds, and soaking in a sodium hypochlorite solution with a concentration of 8% to 12% for 1 to 3 minutes. The temperature for the dark culture is 22~24℃, and the dark culture period is 25~35 days; The subculture was carried out in the dark at a temperature of 22-24°C.

[0017] The technical effects and advantages of this invention are as follows: This invention provides a highly efficient callus induction and proliferation technology specifically for black rose, achieving significant beneficial effects. This solution effectively addresses key technical challenges in the in vitro culture of black rose. Using a specific induction medium formulation, a callus induction rate of up to 80% can be achieved, successfully establishing an excellent culture system. Furthermore, through an optimized proliferation medium formulation, highly efficient callus expansion can be achieved, with a proliferation rate reaching 1.98 at the optimal ratio, demonstrating excellent growth-promoting effects. This makes stable, large-scale black rose callus culture possible, providing a novel solution independent of traditional cultivation to meet market demand for this specialty variety. More importantly, the callus obtained by this invention can serve as a stable and reliable raw material source for subsequent extraction of its unique active ingredients, laying a solid material foundation for developing high-value-added products and greatly expanding the commercial application potential and industrial value of black rose. Attached Figure Description

[0018] Figure 1 This is a photograph of a black rose plant obtained in an embodiment of the present invention. Detailed Implementation

[0019] This invention provides a black rose callus induction medium. The black rose callus induction medium comprises MS basal medium, sucrose, agar, and plant growth regulators. MS basal medium is a widely used basic medium in plant tissue culture, and its formulation is well known to those skilled in the art, providing comprehensive inorganic salts and vitamins for plant cell growth and division. Sucrose is the most commonly used carbon and energy source in plant tissue culture, providing the necessary energy and carbon skeleton for cellular metabolic activities. Agar, a polysaccharide extracted from red algae, mainly acts as a coagulant in the medium, forming a gel at room temperature, providing physical support for explants and callus, and helping to retain the slow release of water and nutrients.

[0020] The plant growth regulators include 6-benzylaminopurine, naphthaleneacetic acid (NAA), and thidiazuron. 6-Benzylaminopurine (6-BA) is a synthetic cytokinin commonly used to promote cell division. Naphthaleneacetic acid (NAA) is a synthetic auxin commonly used to induce cell dedifferentiation. Thidiazuron (TDZ) is a phenylurea cytokinin with high activity and multiple regulatory functions in plant tissue culture. In the culture medium of this invention, the concentrations of each component are final concentrations. Specifically, the concentration range of 6-benzylaminopurine is from 0.1 mg / L to 1.0 mg / L, for example, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1.0 mg / L. A further preferred concentration range is 0.3 mg / L to 0.7 mg / L. A more preferred concentration is 0.5 mg / L. The concentration range of naphthaleneacetic acid is 0.1 mg / L to 1.0 mg / L, for example, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1.0 mg / L. A further preferred concentration range is 0.3 mg / L to 0.7 mg / L. A more preferred concentration is 0.5 mg / L. The concentration range of thiabendazole is from 1.0 mg / L to 3.0 mg / L, for example, 1.0 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 2.0 mg / L, 2.2 mg / L, 2.5 mg / L, 2.8 mg / L, or 3.0 mg / L. A further preferred concentration range is 1.5 mg / L to 2.5 mg / L. A more preferred concentration is 2.0 mg / L.

[0021] Further, the pH range of the black rose callus induction medium is 5.6 to 6.0, for example, 5.6, 5.65, 5.7, 5.75, 5.8, 5.85, 5.9, 5.95, or 6.0. Preferably, the pH range is 5.7 to 5.9. More preferably, the pH is 5.8. The pH of the medium can be adjusted using an acid (such as hydrochloric acid) or a base (such as sodium hydroxide) solution.

[0022] Further, the concentration of the agar in the black rose callus induction medium, based on a final concentration, ranges from 6.0 g / L to 9.0 g / L, for example, 6.0 g / L, 6.5 g / L, 7.0 g / L, 7.5 g / L, 8.0 g / L, 8.5 g / L, or 9.0 g / L. Preferably, the concentration ranges from 7.0 g / L to 8.0 g / L. More preferably, the concentration is 7.5 g / L. The concentration of the sucrose in the black rose callus induction medium ranges from 20 g / L to 40 g / L, for example, 20 g / L, 22 g / L, 25 g / L, 28 g / L, 30 g / L, 32 g / L, 35 g / L, 38 g / L, or 40 g / L. Preferably, the concentration ranges from 25 g / L to 35 g / L. More preferably, the concentration is 30 g / L.

[0023] This invention also provides a black rose callus proliferation medium. The black rose callus proliferation medium comprises MS basal medium, sucrose, agar, and a plant growth regulator. In the proliferation medium of this invention, the concentration of 6-benzylaminopurine ranges from 0.1 mg / L to 1.0 mg / L, for example, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1.0 mg / L. A further preferred concentration range is 0.3 mg / L to 0.7 mg / L. The concentration range of thiabendazole is from 0.5 mg / L to 1.5 mg / L, for example, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, or 1.5 mg / L. A further preferred concentration range is 0.8 mg / L to 1.2 mg / L. The concentration range of naphthaleneacetic acid is from 0.1 mg / L to 1.0 mg / L, for example, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, or 1.0 mg / L. A further preferred concentration range is 0.3 mg / L to 0.7 mg / L.

[0024] In a preferred embodiment, the concentration ratio of 6-benzylaminopurine, thiazuron, and naphthaleneacetic acid (NAA) is selected from one of the following three specific combinations: The first combination is 1.0 mg / L 6-benzylaminopurine, 0.5 mg / L thiazuron, and 0.5 mg / L NAA; the second combination is 0.5 mg / L 6-benzylaminopurine, 0.5 mg / L thiazuron, and 1.0 mg / L NAA; and the third combination is 0.5 mg / L 6-benzylaminopurine, 1.0 mg / L thiazuron, and 0.5 mg / L NAA. These three specific combinations of plant growth regulators achieve optimal results.

[0025] Further, the pH range of the black rose callus proliferation medium is 5.6 to 6.0, for example, 5.6, 5.65, 5.7, 5.75, 5.8, 5.85, 5.9, 5.95, or 6.0. Preferably, the pH range is 5.7 to 5.9. More preferably, the pH is 5.8.

[0026] Further, the concentration of the agar in the black rose callus proliferation medium, based on a final concentration, ranges from 6.0 g / L to 9.0 g / L, for example, 6.0 g / L, 6.5 g / L, 7.0 g / L, 7.5 g / L, 8.0 g / L, 8.5 g / L, or 9.0 g / L. Preferably, the concentration ranges from 7.0 g / L to 8.0 g / L. More preferably, the concentration is 7.5 g / L. The concentration of the sucrose in the black rose callus proliferation medium ranges from 20 g / L to 40 g / L, for example, 20 g / L, 22 g / L, 25 g / L, 28 g / L, 30 g / L, 32 g / L, 35 g / L, 38 g / L, or 40 g / L. Preferably, the concentration ranges from 25 g / L to 35 g / L. More preferably, the concentration is 30 g / L.

[0027] This invention also provides the application of the aforementioned black rose callus induction medium and black rose callus proliferation medium in the cultivation of black rose callus. The term "application" refers to the use of the aforementioned medium in the field of plant tissue culture technology for the induction, proliferation, subculturing, or maintenance culture of black rose callus. The black rose callus can be used for various purposes, such as, but not limited to, in vitro preservation of plant germplasm resources, recipient material for genetic transformation, establishment of cell lines for secondary metabolite production, experimental material for physiological and biochemical research, or intermediate material for rapid propagation.

[0028] This invention also provides a method for culturing black rose callus. The method includes the following steps: First, black rose explant material is provided, and after appropriate surface sterilization treatment, it is inoculated into the black rose callus induction medium as described above. Then, under specific culture conditions, it is cultured in the dark. After a certain period of time, callus tissue can be induced at the site where the explant contacts the culture medium, thus obtaining induced callus tissue. Subsequently, the induced callus tissue is separated or cut and transferred to the black rose callus proliferation medium as described above. Finally, under suitable culture conditions, subculture is performed to allow the callus tissue to continuously grow and proliferate, thereby obtaining proliferated black rose callus tissue.

[0029] Furthermore, the black rose explant can be derived from various organs or tissues of the black rose plant, such as petals, leaves, stem segments, petioles, or receptacles. In one specific embodiment, the explant is a petal.

[0030] Furthermore, the sterilization process is a necessary step in plant tissue culture to obtain sterile explants. It typically involves treating the explant surface with a disinfectant. A preferred sterilization procedure includes sequentially using two disinfectants: first, immersion in a 60% to 80% (v / v) aqueous solution of alcohol for 30 to 60 seconds, e.g., 30, 40, 50, or 60 seconds. Alcohol rapidly penetrates and kills some surface microorganisms. Subsequently, immersion in an 8% to 12% (NaClO) aqueous solution for 1 to 3 minutes, e.g., 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, or 3 minutes. Sodium hypochlorite is a potent and commonly used surface sterilizer for plant materials. To improve sterilization effectiveness, a small amount of surfactant, such as Tween 20, can be added to the sodium hypochlorite solution. After each disinfectant treatment, the explants should be thoroughly washed multiple times with sterile water to remove any residual disinfectant.

[0031] Further, the dark culture in the induction step refers to culture in a light-free or near-light-free environment. The temperature range for dark culture is 22°C to 24°C, for example, 22°C, 22.5°C, 23°C, 23.5°C, or 24°C. Preferably, the temperature is 23±1°C. The period of dark culture, i.e., the time from explant inoculation to successful callus induction and transferability, ranges from 25 days to 35 days, for example, 25 days, 26 days, 28 days, 30 days, 32 days, or 35 days. Preferably, the period is approximately 30 days.

[0032] Furthermore, the subculture step can also be performed under dark conditions. The temperature range for subculture is 22°C to 24°C, for example, 22°C, 22.5°C, 23°C, 23.5°C, or 24°C. Preferably, the temperature is 23±1°C. The subculture cycle can be adjusted according to the growth rate of the callus and experimental requirements, for example, it can be 18-25 days. By repeating the transfer and subculture steps, long-term stable preservation and large-scale expansion of black rose callus can be achieved.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Black rose petal callus induction Black rose petals were rinsed under running water for 30 minutes. In a clean bench, the petals were then immersed in 70% alcohol for 30 seconds, followed by rinsing five times with sterile ultrapure water. Next, the petals were immersed in a 10% sodium hypochlorite (NaClO) solution (with 0.1% Tween 20 added) for 2 minutes, followed by rinsing five times with sterile ultrapure water. The surface moisture was blotted dry with sterile filter paper. The petals were then cut into 1cm x 1cm cubes using a sterile scalpel and placed on induction medium, with 6 cubes per dish. This was repeated three times. The callus induction rate was calculated over a one-month period. Callus induction was performed using dark culture at 23±1℃. The callus induction rate was calculated as: (Number of explants producing callus / Number of inoculated explants) × 100%.

[0035] The callus induction medium contained 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). The solution was adjusted to pH 5.8 and sterilized at high temperature (121℃, 20 min) before use. When the temperature of the basic medium dropped to approximately 65℃, plant growth regulators 6-BA (6-Benzylaminopurine) 0.5 mg / L, NAA (Naphthylaceticacid) 0.5 mg / L, and TDZ (Thidiazuron) 2.0 mg / L were added. Following the above method, the callus induction rate is shown in Table 1 below. Table 1. Callus induction rate of black rose under the same cycle with different culture medium formulations.

[0036] Example 2 Black rose callus proliferation Five g of the callus induced in Example 1 was taken per bottle, and the callus proliferation coefficient was calculated over a 21-day period. This was used to confirm the culture medium for the proliferation of Black Rose callus.

[0037] The second induction medium contained 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). The solution was adjusted to pH 5.8 and sterilized at high temperature (121℃, 20 min) before use. When the temperature of the basic medium dropped to about 65℃, plant growth regulators 6-BA (6-Benzylaminopurine), TDZ (Thidiazuron), and NAA (Naphthylacetic acid) were added to maintain the stability of black rose callus for subculturing.

[0038] The callus tissue was cultured in the dark at 23±1℃ for 21 days, and the proliferation of black rose callus was measured. The callus proliferation rate was calculated as (fresh weight of callus 21 days after inoculation - fresh weight of callus before inoculation) / fresh weight of callus before inoculation × 100%. The results are shown in Table 2 below. Table 2. Proliferation coefficient of black rose callus under different hormone ratios

[0039] Comparative Example 1 Black rose callus proliferation Five g of the callus induced in Example 1 was taken per bottle, and the callus proliferation coefficient was calculated over a 21-day period. This was used to confirm the culture medium for the proliferation of Black Rose callus.

[0040] The second induction medium contained 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). The solution was adjusted to pH 5.8 and sterilized at high temperature (121℃, 20 min) before use. When the temperature of the basic medium dropped to about 65℃, plant growth regulators 6-BA (6-Benzylaminopurine), ZT (Zeatin), IAA (Indole-3-aceticacid), and 2,4-D (2,4-Dichlorophenoxyacetic acid) were added to maintain the stability of black rose callus for subculturing.

[0041] The callus tissue was cultured in the dark at 23±1℃ for 21 days, and the proliferation of black rose callus was measured. The callus proliferation rate was calculated as (fresh weight of callus 21 days after inoculation - fresh weight of callus before inoculation) / fresh weight of callus before inoculation × 100%. The results are shown in Table 3 below. Table 3. Proliferation coefficient of black rose callus under different hormone ratios

[0042] Example 3 Black Rose callus propagation plants Five g of the callus induced in Example 1 was taken per bottle, and the callus proliferation coefficient was calculated over a 21-day period. This was used to confirm the culture medium for the proliferation of Black Rose callus.

[0043] The second induction medium contained 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). The solution was adjusted to pH 5.8 and sterilized at high temperature (121℃, 20 min) before use. When the temperature of the basic medium dropped to about 65℃, plant growth regulators 6-BA (6-Benzylaminopurine) 0.5 mg / L, TDZ (Thidiazuron) 1.0 mg / L, and NAA (Naphthylacetic acid) 0.5 mg / L were added to promote the development and budding of black roses.

[0044] Black rose plants cultured at 23±1℃ for 21 days were observed to show obvious signs of new shoots emerging. Their growth status is as follows: Figure 1 As shown: As demonstrated by the above embodiments, this invention provides a culture system for efficiently inducing callus tissue from black rose petals, achieving its stable proliferation, and subsequently obtaining plantlets. A high callus induction rate can be obtained using a specific formulation of the induction medium. Furthermore, by optimizing the combination and ratio of plant growth regulators in the proliferation medium, the biomass accumulation of callus tissue can be significantly promoted, resulting in callus tissue material with good growth status and suitable for subculture, laying a solid foundation for subsequent research and applications.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A black rose callus induction culture medium, characterized in that, The medium contains MS basal medium, sucrose, agar, and plant growth regulators. The plant growth regulators include 6-benzylaminopurine, naphthaleneacetic acid, and thidiazuron, with the concentration of 6-benzylaminopurine being 0.1–1.0 mg / L, the concentration of naphthaleneacetic acid being 0.1–1.0 mg / L, and the concentration of thidiazuron being 1.0–3.0 mg / L.

2. The black rose callus induction culture medium according to claim 1, characterized in that, The pH value of the induction medium is 5.6~6.

0.

3. The black rose callus induction culture medium according to claim 1, characterized in that, The concentration of the agar is 6.0~9.0 g / L and the concentration of the sucrose is 20~40 g / L, based on the final concentration.

4. A black rose callus proliferation culture medium, characterized in that, The medium contains MS basal medium, sucrose, agar, and plant growth regulators, including 6-benzylaminopurine, thiazuron, and naphthaleneacetic acid, with the following final concentrations: 6-benzylaminopurine at 0.1–1.0 mg / L, thiazuron at 0.5–1.5 mg / L, and naphthaleneacetic acid at 0.1–1.0 mg / L.

5. The black rose callus proliferation culture medium according to claim 4, characterized in that, The concentration ratio of 6-benzylaminopurine, thiazolinone, and naphthaleneacetic acid is selected from one of the following three combinations: (1) The concentration of 6-benzylaminopurine was 1.0 mg / L, the concentration of thiazolinone was 0.5 mg / L, and the concentration of naphthaleneacetic acid was 0.5 mg / L; (2) The concentration of 6-benzylaminopurine was 0.5 mg / L, the concentration of thiazolinone was 0.5 mg / L, and the concentration of naphthaleneacetic acid was 1.0 mg / L; (3) The concentration of 6-benzylaminopurine was 0.5 mg / L, the concentration of thiazolinone was 1.0 mg / L, and the concentration of naphthaleneacetic acid was 0.5 mg / L.

6. The black rose callus proliferation culture medium according to claim 4 or 5, characterized in that, The pH value of the proliferation medium is 5.6~6.

0.

7. The black rose callus proliferation culture medium according to claim 4 or 5, characterized in that, The concentration of the agar is 6.0~9.0 g / L and the concentration of the sucrose is 20~40 g / L, based on the final concentration.

8. The use of the black rose callus induction medium according to any one of claims 1 to 3 and the black rose callus proliferation medium according to any one of claims 4 to 7 in the culture of black rose callus.

9. A method for culturing black rose callus, characterized in that, Includes the following steps: Black rose explants were inoculated into the black rose callus induction medium according to any one of claims 1 to 3 and cultured in the dark to obtain induced callus. The induced callus tissue was transferred to the black rose callus proliferation culture medium according to any one of claims 4 to 7 for subculture to obtain proliferated black rose callus tissue.

10. The cultivation method according to claim 9, characterized in that, The black rose explants were petals; The sterilization process includes soaking in an alcohol solution with a volume percentage of 60% to 80% for 30 to 60 seconds, and soaking in a sodium hypochlorite solution with a concentration of 8% to 12% for 1 to 3 minutes. The temperature for the dark culture is 22~24℃, and the dark culture period is 25~35 days; The subculture was carried out in the dark at a temperature of 22-24°C.

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