Culture medium for open tissue culture of camellia oleifera and culture method thereof

Abstract

The invention discloses a culture medium for open tissue culture of camellia oleifera and a culture method thereof, and belongs to the technical field of plant tissue culture. The culture medium for open type tissue culture of camellia oleifera comprises a basic culture medium and a compound bacteriostatic agent added into the basic culture medium, wherein the compound bacteriostatic agent is prepared from the following components in concentration in the culture medium: 0.1 to 0.5 g / L of tea polyphenol derivative, 30 to 50 mg / L of polyhexamethylene biguanide hydrochloride, 80 to 130 mg / L of plant essential oil compound, 0.05 to 0.2 g / L of chitosan and 0.05 to 0.15 g / L of dipotassium glycyrrhizinate. Through the combined action of various raw materials with bacteriostatic action, an excellent anti-pollution effect is synergistically achieved. The culture medium special for open tissue culture of camellia oleifera and the culture method of the culture medium are developed, and the culture medium and the culture method have good application value for breaking through the bottleneck of high-quality seedling breeding of camellia oleifera and promoting the camellia oleifera industry to develop towards the low-cost, high-efficiency and large-scale direction.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture medium and culture method for open tissue culture of Camellia oleifera. Background Technology

[0002] Camellia oleifera Abel., an important evergreen woody oil-bearing tree species in the genus Camellia of the family Theaceae, has a wide cultivation area. The camellia oil obtained by pressing its seeds is rich in unsaturated fatty acids, vitamin E, squalene, and other functional components, possessing extremely high nutritional and economic value. Besides edible uses, camellia oil also shows broad application prospects in medicine, chemicals, and cosmetics. Therefore, promoting the high-quality development of the camellia oil industry is of great significance for increasing income for camellia oil growers and developing a green economy.

[0003] The sustainable development of the camellia oleifera industry highly depends on the efficient and large-scale propagation of high-quality seedlings. Currently, camellia oleifera seedling cultivation mainly relies on traditional methods such as seedling planting, bud grafting, and cutting. While seedling planting is simple to operate and has a well-developed root system, it has inherent defects such as severe segregation of traits in offspring, varietal degeneration, and inconsistent ripening of tea and fruit, resulting in low yield and low efficiency in camellia oleifera stands. Bud grafting can maintain the superior traits of the scion variety and is currently the mainstream seedling cultivation method, but its operation is limited by the season, and grafting compatibility issues between the rootstock and scion may lead to inconsistent seedling growth, while also posing a risk of virus transmission from the parent plant. Cutting propagation can maintain the traits of the parent plant, but it generally suffers from low propagation coefficients, difficulty in rooting, and high consumption of parent tree resources.

[0004] Plant tissue culture, as one of the core methods of modern biotechnology, can theoretically overcome the bottlenecks of traditional seedling cultivation methods. Based on the totipotency of plant cells, it allows for rapid cloning and propagation of seedlings in the laboratory under conditions unrestricted by season, thus obtaining a large number of superior plants with consistent genetic backgrounds and free from specific pathogens in a short period. However, traditional Camellia oleifera tissue culture technology heavily relies on a strictly aseptic operating environment. All culture media, instruments, and operating spaces must be thoroughly sterilized using equipment such as autoclaves and laminar flow hoods. This process is not only costly in terms of equipment investment and energy consumption, but also involves cumbersome and complex procedures and requires extremely high levels of professional skills from operators. This "aseptic constraint" greatly limits the large-scale application and promotion of tissue culture technology in the industrialized seedling cultivation of Camellia oleifera, and further improvements are still needed.

[0005] To lower the technical barriers and production costs of tissue culture, open tissue culture technology has emerged. The core of this technology lies in inhibiting microbial contamination from the environment by adding appropriate antibacterial agents to the culture medium, allowing inoculation, culturing, and other operations to be performed in a non-completely sterile, ordinary environment.

[0006] Although open tissue culture technology has shown great application potential, research on Camellia oleifera, an important economic tree species, still needs further development. Existing research on Camellia oleifera tissue culture mainly focuses on using basic culture media combined with different growth regulators (such as 6-BA, NAA, IBA, etc.) for callus induction, adventitious bud differentiation, and rooting. However, these studies were primarily conducted under strictly aseptic traditional tissue culture systems. Furthermore, minimizing the impact of antibacterial agents on explant survival rates still requires continuous effort from researchers. Therefore, introducing the concept of open tissue culture into Camellia oleifera propagation, screening for highly effective, low-toxicity antibacterial agent formulations that do not affect the normal growth and differentiation of Camellia oleifera explants, and establishing a complete, stable, and low-cost open tissue culture rapid propagation technology system for Camellia oleifera have become urgent technical challenges to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a culture medium and method for open tissue culture of Camellia oleifera, thereby solving the aforementioned problems in the background art. This invention achieves excellent anti-contamination effects through the synergistic effect of multiple raw materials with antibacterial properties. This invention develops a culture medium and method specifically for open tissue culture of Camellia oleifera, which has significant application value in overcoming the bottleneck in the propagation of high-quality Camellia oleifera seedlings and promoting the development of the Camellia oleifera industry towards low cost, high efficiency, and large-scale production.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] One of the technical solutions of this invention is to provide a culture medium for open tissue culture of Camellia oleifera, characterized in that it includes a basal culture medium and a compound antibacterial agent added thereto, wherein the composition of the compound antibacterial agent and its concentration in the culture medium are as follows:

[0010] Tea polyphenol derivatives 0.1-0.5 g / L, polyhexamethylene biguanide hydrochloride 30-50 mg / L, plant essential oil complex 80-130 mg / L, chitosan 0.05-0.2 g / L, and dipotassium glycyrrhizate 0.05-0.15 g / L.

[0011] Preferably, the tea polyphenol derivative is epigallocatechin gallate.

[0012] Preferably, the plant essential oil complex contains pine needle oil, forsythia essential oil, and basil oil.

[0013] Preferably, the mass ratio of pine needle oil, forsythia essential oil and basil oil is 1.5-2.5:1-1.5:1.

[0014] Preferably, the chitosan is water-soluble carboxymethyl chitosan.

[0015] Preferably, the basal culture medium is MS medium or 1 / 2 MS medium.

[0016] Preferably, the basal culture medium further contains growth hormone, which is one or more of indole-3-acetic acid (IAA), 6-benzylaminopurine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellin (GA3), indolebutyric acid (IBA), and naphthaleneacetic acid (NAA).

[0017] Preferably, the basal culture medium also contains sucrose, agar, and crosporopyrrolidone.

[0018] The second technical solution of the present invention provides a method for preparing the culture medium for open tissue culture of Camellia oleifera as described above, comprising the following steps:

[0019] The basal culture medium was mixed with the compound antibacterial agent to obtain the culture medium for open tissue culture of Camellia oleifera.

[0020] The third technical solution of the present invention provides an application of the above-mentioned open tissue culture medium for Camellia oleifera in the field of open tissue culture of Camellia oleifera.

[0021] Fourth technical solution of the present invention: A method for open tissue culture of Camellia oleifera, comprising the following steps:

[0022] The sterilized Camellia oleifera explants were inoculated onto the above-mentioned open tissue culture medium for Camellia oleifera in an open environment and cultured.

[0023] The technical principle of this invention is as follows:

[0024] EGCG, a natural component extracted from Camellia oleifera plants, exhibits natural biocompatibility with the plant. Furthermore, EGCG can disrupt the cell membrane structure of microorganisms, demonstrating inhibitory effects on various fungi and bacteria. Simultaneously, this component possesses strong antioxidant capabilities, effectively scavenging free radicals and significantly reducing phenolic oxidative browning of explants.

[0025] However, the antibacterial effect achieved when using tea polyphenol derivatives alone as the antibacterial component is relatively weak. This is mainly because the primary antibacterial mechanism of EGCG lies in the binding of its phenolic hydroxyl groups to the phospholipid bilayer and membrane proteins of the microbial cell membrane, thereby altering membrane permeability. However, this effect is insufficient to penetrate and damage microorganisms with dense cell walls, making it difficult to cause fatal damage when used alone.

[0026] Polyhexamethylene biguanide hydrochloride (PHMB), as a cationic polymer bactericide, mainly works by the positive charge carried by its long polymer chains binding to the negatively charged microbial cell membrane through electrostatic interaction, thereby disrupting the microbial membrane structure, causing leakage of cell contents, and causing aggregation of the leaked proteins, ultimately leading to the death of the microorganisms.

[0027] Furthermore, the EGCG added in this invention exhibits a significant synergistic effect with PHMB, which helps to further enhance the antibacterial effect. This is mainly because EGCG can initially disturb the microbial cell membrane, creating favorable conditions for the insertion and binding of high molecular weight PHMB polymer chains.

[0028] PHMB has a highly effective antibacterial effect, but it also poses a risk of damaging explant cells. When its concentration is too high, it can kill more surface cells of Camellia oleifera by disrupting the plant cell membrane structure and exacerbating browning. Therefore, its addition amount must be strictly controlled.

[0029] The main component of pine needle oil is pinene, which has strong inhibitory activity against a variety of fungi and can destroy mycelial growth, serving as a basic line of defense against fungi.

[0030] Forsythia essential oil is rich in phenylethyl glycosides such as forsythoside, which not only inhibit various bacteria and fungi but also have rapid penetration properties, quickly acting on microbial cell membranes. Furthermore, its effects are gentle, effectively reducing the potential irritation of complex essential oil systems to plant cells.

[0031] The main components of basil oil are linalool and methyl chamomile. These components can interfere with the membrane function and energy metabolism of microorganisms, exhibiting good antibacterial effects.

[0032] The plant essential oil complex of this invention contains pine needle oil, forsythia essential oil, and basil oil, which can enhance the anti-contamination effect through different disinfection and sterilization mechanisms. Furthermore, this component has a certain degree of volatility, which can be used to prevent contamination of plant parts not inoculated into the culture medium.

[0033] The present invention, through testing, found that as the concentration of the plant essential oil complex increased, the browning rate generally showed an upward trend, while the survival rate initially increased and then decreased. The increase in survival rate at low concentrations was achieved by enhancing the anti-pollution effect; however, with further increases in concentration, the impact on the physiological activity of the Camellia oleifera explants became more pronounced, leading to a decrease in survival rate.

[0034] The chitosan added in this invention is a natural cationic polysaccharide. The amino groups on its molecular chain can adsorb onto the anionic sites of microbial cell walls, altering cell membrane permeability and causing leakage of cell contents, with a particularly significant effect against fungi. Simultaneously, chitosan can form a physical barrier membrane in the culture medium. Furthermore, chitosan can act as an elicitor to induce a self-defense response and callus formation in Camellia oleifera explants, achieving a dual effect of antibacterial and growth-promoting action.

[0035] The addition of dipotassium glycyrrhizate helps to further stabilize plant cell membranes, alleviate oxidative stress, and provide intrinsic protection for the survival of Camellia oleifera explants.

[0036] The beneficial technical effects of the present invention are as follows:

[0037] This invention achieves excellent pollution prevention through the synergistic effect of multiple raw materials with antibacterial properties. This invention also develops a culture medium and method specifically for open tissue culture of Camellia oleifera, which has significant application value in overcoming the bottleneck in the propagation of high-quality Camellia oleifera seedlings and promoting the development of the Camellia oleifera industry towards low cost, high efficiency, and large-scale production.

[0038] The open tissue culture of Camellia oleifera obtained by using the culture medium designed in this invention has good growth indicators and can control the browning rate and contamination rate at a reasonable level. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The images show actual specimens of Camellia oleifera after open tissue culture according to the culture media of Examples 1-3. Among them, (a) is Example 1, (b) is Example 2, and (c) is Example 3.

[0041] Figure 2 This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 1.

[0042] Figure 3 This is a photograph of the Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 2.

[0043] Figure 4 This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 3.

[0044] Figure 5This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 6. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0046] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. It should be noted that any aspects of this invention not described in detail are conventional practices in the art and are not the focus of this invention.

[0048] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0049] Compared to traditional tissue culture, open tissue culture does not rely on autoclaving and clean benches, which simplifies the operation process, reduces equipment investment and power consumption, and improves the fault tolerance and feasibility of operation, opening up a new path for the industrialized and intensive production of tissue culture seedlings.

[0050] Currently, open tissue culture technology has successfully established rapid propagation systems for various plants, including *Magnolia denudata*, banana, potato, blueberry, and sugarcane. Existing research indicates that the key to this technology lies in the construction and optimization of the antibacterial agent system, including explant surface disinfection methods and the types and concentrations of antibacterial agents added to the culture medium. Based on current research findings, different plants, and even different culture stages of the same plant (primary, subculture, and rooting), respond differently to the types and concentrations of antibacterial agents, requiring targeted screening and formulation.

[0051] The culture medium for tissue culture not only provides sufficient nutrients for plant tissue culture, but also provides suitable conditions for the growth of bacteria, microorganisms, etc., which can easily cause contamination of plant tissues and reduce the efficiency of experiments.

[0052] As a plant of the Theaceae family, Camellia oleifera contains a higher content of polyphenols (such as tea polyphenols) than conventional plant explants. These substances exist as secondary metabolites in intact cells. When the explant is cut, the cells are damaged and rupture, releasing the phenolic substances from the vacuoles. These substances then come into contact with polyphenol oxidase (PPO) in the cytoplasm and oxygen in the air, undergoing an enzymatic oxidation reaction to produce brownish-black quinones. These quinones further polymerize, not only causing browning of the explant but also having a toxic effect on the cells, inhibiting enzyme activity, and ultimately leading to the death of the explant.

[0053] Browning of Camellia oleifera explants refers to the phenomenon where, during tissue culture, the explant cut site and even the entire tissue turn brown or black, eventually leading to death. This is mainly caused by the oxidation of phenolic substances, a metabolic disorder. Although browning manifests on the upper part of leaves or stem segments, the source and center of its biochemical reaction are usually located at the cut site where the explant contacts the culture medium. The EGCG in the culture medium of this invention helps to inhibit browning by competitively inhibiting polyphenol oxidase (PPO) and scavenging initial free radicals, directly nipping the initiation of the oxidation chain reaction at its source.

[0054] The Camellia oleifera bud stem segments used in the following embodiments and comparative examples of the present invention are of the Changlin 53 variety, which were sampled from the Camellia oleifera base in Yongxiu County, Jiujiang City, Jiangxi Province, and refrigerated at 4°C after sampling for later use.

[0055] All raw materials used in the following embodiments and comparative examples of the present invention are commercially available products.

[0056] Example 1

[0057] Culture medium for open tissue culture of Camellia oleifera:

[0058] It consists of a basal culture medium and a compound antibacterial agent added thereto, with the concentrations of each ingredient in the culture medium as follows:

[0059] The components of the compound antibacterial agent and their concentrations in the culture medium are as follows: (epigallocatechin gallate 0.4 g / L, polyhexamethylene biguanide hydrochloride 42 mg / L, plant essential oil complex 115 mg / L, chitosan 0.10 g / L, dipotassium glycyrrhizate 0.12 g / L; wherein, the plant essential oil complex is composed of pine needle oil, forsythia essential oil and basil oil in a mass ratio of 2:1.4:1), sucrose 15 g / L, agar 10 g / L, PVPP (crosslinked polypropylene pyrrolidone) 2.5 g / L, IAA (indole-3-acetic acid) 1.0 mg / L.

[0060] The specific preparation steps are as follows:

[0061] Take MS medium, add sucrose, agar and PVPP to the medium, then boil the medium, adjust the pH to 5.5-6.0, and wait for the raw material system to cool naturally to 50℃. Add compound antibacterial agent and IAA, mix well, quantitatively dispense into tissue culture bottles and seal, and allow to cool and solidify naturally to obtain the culture medium for open tissue culture of Camellia oleifera.

[0062] Example 2

[0063] A culture medium for open tissue culture of Camellia oleifera, comprising a basal culture medium and a compound antibacterial agent added thereto, wherein the concentrations of each raw material in the culture medium are as follows:

[0064] The compound antibacterial agent consists of: epigallocatechin gallate 0.3 g / L, polyhexamethylene biguanide hydrochloride 40 mg / L, plant essential oil complex 110 mg / L, chitosan 0.1 g / L, and dipotassium glycyrrhizate 0.12 g / L; wherein the plant essential oil complex is composed of pine needle oil, forsythia essential oil, and basil oil in a mass ratio of 2:1.5:1), sucrose 15 g / L, agar 10 g / L, PVPP (crosslinked polypropylene pyrrolidone) 2.5 g / L, and IAA (indole-3-acetic acid) 1.0 mg / L.

[0065] The specific preparation steps are as follows:

[0066] Take MS medium, add sucrose, agar and PVPP to the medium, then boil the medium, adjust the pH to 5.5-6.0, and wait for the raw material system to cool naturally to 50℃. Add compound antibacterial agent and IAA, mix well, quantitatively dispense into tissue culture bottles and seal, and allow to cool and solidify naturally to obtain the culture medium for open tissue culture of Camellia oleifera.

[0067] Example 3

[0068] A culture medium for open tissue culture of Camellia oleifera, comprising a basal culture medium and a compound antibacterial agent added thereto, wherein the concentrations of each raw material in the culture medium are as follows:

[0069] The compound antibacterial agent consists of: epigallocatechin gallate 0.2 g / L, polyhexamethylene biguanide hydrochloride 50 mg / L, plant essential oil complex 130 mg / L, chitosan 0.05 g / L, and dipotassium glycyrrhizate 0.15 g / L; wherein the plant essential oil complex is composed of pine needle oil, forsythia essential oil, and basil oil in a mass ratio of 2.5:1:1), sucrose 15 g / L, agar 10 g / L, PVPP (crosslinked polypropylene pyrrolidone) 2.5 g / L, and NAA (naphthaleneacetic acid) 1.5 mg / L.

[0070] The specific preparation steps are as follows:

[0071] Take MS medium, add sucrose, agar and PVPP to the medium, then boil the medium, adjust the pH to 5.5-6.0, and wait for the raw material system to cool naturally to 50℃. Add compound antibacterial agent and NAA, mix well, quantitatively dispense into tissue culture bottles and seal, and allow to cool and solidify naturally to obtain the culture medium for open tissue culture of Camellia oleifera.

[0072] Comparative Example 1

[0073] The only difference from Example 1 is that the addition of tea polyphenol derivatives is omitted and an equal mass of polyhexamethylene biguanide hydrochloride is added.

[0074] Comparative Example 2

[0075] The only difference from Example 1 is that the addition of polyhexamethylene biguanide hydrochloride is omitted and an equal mass of tea polyphenol derivative is added.

[0076] Comparative Example 3

[0077] The only difference from Example 1 is that the addition of tea polyphenol derivatives and polyhexamethylene biguanide hydrochloride is omitted.

[0078] Comparative Example 4

[0079] The only difference from Example 1 is that the addition of pine needle oil and forsythia essential oil is omitted, and an equal amount of basil oil is added.

[0080] Comparative Example 5

[0081] The only difference from Example 1 is that the amount of plant essential oil complex added is changed to 50 mg / L.

[0082] Comparative Example 6

[0083] The only difference from Example 1 is that the amount of plant essential oil complex added is changed to 200 mg / L.

[0084] Comparative Example 7

[0085] The only difference from Example 1 is that the addition of dipotassium glycyrrhizate is omitted.

[0086] Comparative Example 8

[0087] The only difference from Example 1 is that the addition of the compound antibacterial agent was omitted to serve as a positive control.

[0088] Application Example 1

[0089] A method for open tissue culture of Camellia oleifera, comprising the following steps:

[0090] Clean the tea bud stem segments thoroughly to remove surface mud and other contaminants. Then, soak them in a 2% sodium hypochlorite aqueous solution for 10 minutes. After removal, wash them three times with deionized water to obtain sterilized tea bud stem segments. Inoculate the sterilized tea bud stem segments, morphologically with the lower end facing down, into the culture media prepared in Examples 1-3. Transfer them to a culture room and treat them in the dark for 7 days, then switch to light cultivation. After 7 days of cultivation, the contamination rate, survival rate, and browning rate are statistically analyzed. The tests are repeated 20 times, and the average value is taken. The relevant calculation formulas are shown below:

[0091] Contamination rate (%) = (Contaminated stem segments with buds / Total number of inoculated stem segments with buds) × 100%

[0092] Browning rate (%) = (Number of browned stem segments / Total number of stem segments with buds) × 100%

[0093] Survival rate (%) = (Number of surviving stem segments with buds / Total number of inoculated stem segments with buds) × 100%

[0094] The comparison and verification were conducted under the same test conditions by simply replacing the culture medium in each example of Application Example 1 with the culture medium of Comparative Examples 1-8. The test results are shown in Table 1.

[0095] Table 1

[0096] Group Pollution rate (%) Browning rate (%) Survival rate (%) Main observation phenomena Example 1 5 10 95 Tissue culture seedlings are growing vigorously, with normal leaf color and good control of pollution and browning. Comparative Example 1 0 60 35 Excessive PHMB addition led to browning at the base of many explants, reduced survival rate, and poor growth in most of the surviving explants. Comparative Example 2 30 15 65 The contamination rate increased, and colonies were found all over the surface of the culture medium. Comparative Example 3 55 25 40 The antibacterial effect is insufficient, and obvious contaminant colonies are visible on the surface. Comparative Example 4 30 10 75 The different disinfection and sterilization mechanisms of the various essential oil components have been lost, resulting in greater contamination. Comparative Example 5 25 5 80 Slightly poor pollution prevention effect Comparative Example 6 0 45 60 When explants show symptoms of pesticide damage, not only does the survival rate decrease, but growth also slows down. Comparative Example 7 5 30 75 Browning has increased significantly. Comparative Example 8 95 25 0 Almost all explants were contaminated, and all explants died; some explants died from rapid microbial contamination before they even had a chance to brown.

[0097] Figure 1 The images show actual specimens of Camellia oleifera after open tissue culture according to the culture media of Examples 1-3. Among them, (a) is Example 1, (b) is Example 2, and (c) is Example 3.

[0098] Figure 2 This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 1.

[0099] Figure 3 This is a photograph of the Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 2.

[0100] Figure 4 This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 3.

[0101] Figure 5 This is a photograph of Camellia oleifera after open tissue culture in accordance with the culture medium of Comparative Example 6.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for open tissue culture of Camellia oleifera, characterized in that, It includes a basal culture medium and a compound antibacterial agent added thereto, the composition of which and its concentration in the culture medium are as follows: Tea polyphenol derivatives 0.1-0.5 g / L, polyhexamethylene biguanide hydrochloride 30-50 mg / L, plant essential oil complex 80-130 mg / L, chitosan 0.05-0.2 g / L, and dipotassium glycyrrhizate 0.05-0.15 g / L.

2. The culture medium for open tissue culture of Camellia oleifera according to claim 1, characterized in that, The tea polyphenol derivative is epigallocatechin gallate.

3. The culture medium for open tissue culture of Camellia oleifera according to claim 1, characterized in that, The plant essential oil complex contains pine needle oil, forsythia essential oil, and basil oil.

4. The culture medium for open tissue culture of Camellia oleifera according to claim 3, characterized in that, The mass ratio of pine needle oil, forsythia essential oil and basil oil is 1.5-2.5:1-1.5:

1.

5. The culture medium for open tissue culture of Camellia oleifera according to claim 1, characterized in that, The chitosan is water-soluble carboxymethyl chitosan.

6. The culture medium for open tissue culture of Camellia oleifera according to claim 1, characterized in that, The basal culture medium is MS medium or 1 / 2 MS medium.

7. A method for preparing a culture medium for open tissue culture of Camellia oleifera according to any one of claims 1-6, characterized in that, Includes the following steps: The basal culture medium was mixed with the compound antibacterial agent to obtain the culture medium for open tissue culture of Camellia oleifera.

8. The application of the culture medium for open tissue culture of Camellia oleifera according to any one of claims 1-6 in the field of open tissue culture of Camellia oleifera.

9. A method for open tissue culture of Camellia oleifera, characterized in that, Includes the following steps: The sterilized Camellia oleifera explants were inoculated in an open environment onto the open tissue culture medium of Camellia oleifera as described in any one of claims 1-6, and cultured.

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