An open type tissue culture method for wrinkled leaves

Abstract

This application provides an open tissue culture method for *Gnaphalium affine*, comprising the following steps: S1: sterilizing *Gnaphalium affine* explants to obtain pretreated explants; S2: inserting the pretreated explants into a first modified MS medium for a first-stage culture to obtain first-stage explants; S3: transferring the first-stage explants to a second modified MS medium for a second-stage culture to obtain second-stage explants; S4: transferring the second-stage explants to a third modified MS medium for a third-stage culture to obtain primary seedlings. By sequentially adding cell-free fermentation filtrate and PPM to the three-stage modified MS medium, efficient culture of *Gnaphalium affine* explants in an open environment is achieved, reducing contamination rate and browning degree, while promoting explant healing, adventitious bud differentiation, and root primordia formation, thus improving the seedling survival rate of open tissue culture of *Gnaphalium affine*.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, specifically to an open tissue culture method for *Gnaphalium affine*. Background Technology

[0002] Wrinkled-leaf vegetables (such as broccoli, cabbage, and lettuce) are important leafy vegetables belonging to the Brassicaceae or Asteraceae families, possessing high edible value and market demand. With the expansion of cultivation, traditional seed propagation methods are gradually showing shortcomings in maintaining varietal purity, seedling uniformity, and disease resistance. Tissue culture, as a highly efficient asexual reproduction method, holds significant promise for the rapid propagation and seedling renewal of superior wrinkled-leaf vegetable varieties.

[0003] In existing technologies, tissue culture of *Vigna angularis* (wrinkled leaf vegetable) mostly employs a closed, aseptic tissue culture method conducted under ultra-clean bench conditions. This typically requires high-pressure sterilization, strict aseptic inoculation procedures, and large amounts of chemical disinfectants to control contamination. However, *Vigna angularis* itself has a relatively loose tissue structure, a large number of endophytic bacteria, and a long culture period, making it highly susceptible to bacterial or fungal contamination during the culture process. This can cause browning and necrosis of explants, severely impacting seedling survival rates.

[0004] Furthermore, under closed, aseptic tissue culture conditions, the preparation and operation of the culture medium are cumbersome, highly dependent on equipment, and have high production costs, making it difficult to meet the needs of large-scale promotion. Especially in wrinkled leaf vegetables, due to the tendency of explants to brown and low callus induction efficiency, even under strict aseptic conditions, there are still problems such as high contamination rate, unstable seedling rate, and poor seedling quality.

[0005] While ensuring seedling efficiency, reducing the risk of contamination and decreasing reliance on strict aseptic conditions are urgent problems that need to be solved in the current tissue culture technology of wrinkled leaf vegetables. Summary of the Invention

[0006] This application provides an open tissue culture method for *Vigna angularis*, aiming to solve the problems of high contamination rate, severe browning, low seedling efficiency, and high dependence on strict aseptic conditions in the culture of *Vigna angularis* explants in the prior art. This method enables efficient tissue culture seedling cultivation of *Vigna angularis* in a non-strictly aseptic environment, improves seedling rate and seedling quality, reduces production costs, and facilitates industrialization.

[0007] In a first aspect, this application provides an open tissue culture method for *Vigna angularis*, comprising the following steps:

[0008] S1: Disinfect the explants of *Vigna angularis* to obtain pretreated explants;

[0009] S2: The pretreated explants are inserted into the first modified MS medium for the first stage of culture to allow the explant incisions to heal and obtain the first stage explants;

[0010] S3: The first-stage explants were transferred to the second modified MS medium for the second-stage culture, which allowed the explants to differentiate into adventitious shoots, thus obtaining the second-stage explants;

[0011] S4: The second-stage explants were transferred to the third-stage modified MS medium for third-stage culture, which induced the explants to form root primordia and obtain primary seedlings.

[0012] The first modified MS medium, the second modified MS medium, and the third modified MS medium each independently include MS medium, plant hormones, cell-free fermentation filtrate, and PPM.

[0013] The contents of cell-free fermentation filtrate and PPM in the first, second, and third modified MS media decreased sequentially.

[0014] The cell-free fermentation filtrate includes cell-free Bacillus fermentation filtrate and cell-free Trichoderma fermentation filtrate.

[0015] According to this application, by sequentially adding cell-free fermentation filtrate and PPM to a three-stage modified MS medium, efficient culture of *Corydalis rubra* explants in an open environment was achieved, reducing the contamination rate and browning degree, while promoting explant healing, adventitious bud differentiation and root primordia formation, thus improving the seedling rate of *Corydalis rubra* in open tissue culture.

[0016] In the open tissue culture of *Caragana korshinskii*, explants undergo a continuous physiological process from material collection to seedling formation, involving wound healing, adventitious bud induction, and root primordium differentiation. Because the physiological state and sensitivity to the external environment of explants differ significantly at different stages, using the same culture medium formulation throughout the entire culture cycle often leads to high contamination rates, severe browning, or low differentiation efficiency. Therefore, this application employs a staged culture approach, using modified MS medium prepared for each of the three stages, with a gradual reduction in the content of cell-free fermentation filtrate and PPM to adapt to the physiological needs of explants at different stages.

[0017] In the first stage of culture, the explants have just undergone disinfection and cutting, and the exposed wounds result in significant tissue damage, making them highly susceptible to bacterial invasion. The primary goal at this stage is to promote wound healing and maintain explant viability. Therefore, a relatively high proportion of cell-free fermentation filtrate and PPM is added to the first modified MS medium to establish a strong antibacterial environment and effectively reduce the risk of exogenous contamination. Simultaneously, plant hormones in the medium provide cell division signals, aiding in the repair of damaged tissue. After this stage, the explant wounds heal, and some tissues form preliminary callus structures.

[0018] In the second stage of culture, the explants enter the adventitious bud induction period, which requires strong cell division and differentiation signals. If high levels of antibacterial agents are maintained, it will inhibit cell metabolism and bud differentiation, leading to a decrease in the adventitious bud induction rate. Therefore, the content of cell-free fermentation filtrate and PPM is appropriately reduced in the second modified MS medium, so that the explants are more likely to differentiate into buds under lower antibacterial pressure. The effect of this stage is the appearance of a large number of adventitious buds, and the explants gradually transform from callus tissue state to bud structure.

[0019] In the third stage of culture, the explants need to be further induced from buds to develop root primordia and gradually form primary seedlings. If antibacterial agents are used in this stage, not only will root development be inhibited, but root malformation may also occur. Therefore, in the third modified MS medium, the PPM is further reduced or even removed, and only a small amount of cell-free fermentation filtrate is retained to maintain a basic antibacterial background, thereby providing a more suitable environment for the formation of root primordia. After this stage of culture, the explants complete the transformation from buds to primary seedlings and form seedlings with transplanting potential.

[0020] It should also be noted that PPM (Plant Preservative Mixture) is a broad-spectrum antibacterial agent that reduces the risk of contamination by inhibiting DNA replication and protein synthesis in bacteria and fungi. However, long-term or high-concentration use of PPM often inhibits the cellular metabolism of explants, leading to decreased callus viability, reduced adventitious shoot induction efficiency, and even abnormal root development. Therefore, this application combines cell-free fermentation filtrate with PPM to form a composite antibacterial system: PPM rapidly inhibits a large number of potentially contaminating microorganisms in the initial stage, while the cell-free fermentation filtrate, containing Bacillus subtilis and Trichoderma subtilis, is rich in various secondary metabolites secreted by microorganisms, including lipopeptides, enzymes, and antifungal active substances. These metabolites can still inhibit bacteria and fungi even without live bacteria, thus providing long-term and multi-target antibacterial activity. The two produce complementary and synergistic effects; this not only enhances the overall antibacterial strength but also effectively reduces dependence on high concentrations of PPM, thereby mitigating its adverse effects on explants.

[0021] In summary, the method provided in this application provides strong antibacterial protection in the early stage when the risk of contamination is highest; gradually reduces antibacterial pressure during the differentiation stage to release cell differentiation potential; and further reduces the amount of external antibacterial agents during the root primordia formation stage to ensure normal root development. Through this dynamic regulation, a balance between antibacterial and differentiation is achieved, thereby obtaining a high seedling rate even under open conditions.

[0022] It should also be noted that MS medium has a meaning known in the art. In the embodiments of this application, MS medium is purchased MS medium dry powder (without sucrose and agar), and is prepared by adding water to a final volume of 30 g / L sucrose and 7 g / L agar.

[0023] In some embodiments, the cell-free Bacillus fermentation filtrate is prepared by the following method:

[0024] With 10 6 ~10 7 Bacillus was inoculated into LB liquid medium at an inoculum of CFU / mL and fermented aerobicly at 25-35°C for 36-60 h. The bacterial cells were removed by filtration to obtain cell-free Bacillus fermentation filtrate.

[0025] The cell-free Trichoderma fermentation filtrate was prepared by the following method:

[0026] With 10 5 ~10 6 Trichoderma spores were inoculated into PDB medium at an inoculum rate of 1 spore / mL and fermented aerobicly at 25-35℃ for 60-84h. The mycelium and mycelium were removed by filtration to obtain cell-free Trichoderma fermentation filtrate.

[0027] In some of the above embodiments, fermenting Bacillus in LB liquid medium promotes its rapid entry into the logarithmic growth phase and the secretion of large amounts of lipopeptide antibacterial substances and extracellular enzymes, thereby giving the resulting cell-free filtrate strong antibacterial activity. Fermenting Trichoderma in PDB medium facilitates the secretion of chitinase, β-1,3-glucanase, and various secondary metabolites, which can degrade the fungal cell wall and inhibit the growth of other microorganisms. By preparing the filtrate through separate fermentation and then filtering it for sterilization, the introduction of live bacteria into the culture system can be avoided, ensuring the antibacterial function of the filtrate while reducing the risk of secondary contamination in an open tissue culture environment.

[0028] Compared to using Bacillus fermentation filtrate or Trichoderma fermentation filtrate alone, the combined application of the two creates a complementary effect. Bacillus filtrate primarily targets bacterial contamination, while Trichoderma filtrate has a stronger inhibitory effect on fungal contamination. The combination provides more comprehensive antibacterial protection in an open environment, and under the primary antibacterial effect of PPM, they work together to achieve a significant inhibitory effect. Therefore, the combined use of Bacillus fermentation filtrate and Trichoderma fermentation filtrate in the PPM antibacterial system has yielded unexpectedly outstanding results, effectively reducing the contamination rate of *Gnaphalium affine* explants at all stages of culture, thereby further improving the seedling survival rate of *Gnaphalium affine* in open tissue culture.

[0029] It should be noted that LB liquid medium and PDB medium have the meanings known in the art. As an example, in the embodiments of this application, LB liquid medium is a liquid medium with 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0±0.2; PDB medium is obtained by boiling 200 g of potatoes in water for 30 min, filtering the potato pieces, adding 20 g of glucose to dissolve, making up to 1000 mL, and then sterilizing with high pressure steam at 121 °C for 20 min at 100 kPa.

[0030] In some embodiments, the Bacillus includes Bacillus subtilis and Bacillus licheniformis, and the inoculation ratio of Bacillus subtilis and Bacillus licheniformis is 1:0.5~1.5; the Trichoderma includes Trichoderma harzianum.

[0031] In some of the above embodiments, Bacillus subtilis has the ability to secrete lipopeptide antibacterial substances, which have a significant inhibitory effect on bacterial contamination; Bacillus licheniformis can produce a variety of enzymes and volatile substances, which enhance the antagonistic effect against fungi and miscellaneous bacteria; by combining the two in a certain proportion, richer and more complementary metabolites can be produced during the fermentation process, so that the resulting cell-free fermentation filtrate has a broader antibacterial spectrum and more stable antibacterial activity.

[0032] The selected Trichoderma is *Trichoderma harzianum*, which not only secretes cell wall degrading enzymes such as chitinase and β-1,3-glucanase, but also produces a variety of secondary metabolites, exhibiting a significant inhibitory effect on fungal contamination, especially filamentous fungi. When used in combination with Bacillus filtrate, it achieves a dual mechanism of action of "bacterial inhibition + fungal inhibition," forming a broad-spectrum and stable antibacterial system, further reducing the contamination rate of *Gnaphalium affine* explants under open tissue culture conditions, thereby improving the seedling survival rate.

[0033] As an example, the Bacillus subtilis used in this application is... Bacillus subtilis ATCC 6051, Bacillus licheniformis is Bacillus licheniformis ATCC 14580, Trichoderma harzianum is Trichoderma harzianum ACCC 30371.

[0034] In some embodiments, the volume ratio of cellless Bacillus fermentation filtrate to cellless Trichoderma fermentation filtrate in the cellless fermentation filtrate is (1.5~2.5):1.

[0035] In some of the above embodiments, the Bacillus fermentation filtrate is mainly rich in lipopeptide antibacterial substances, extracellular enzymes, and various antibacterial metabolites, which have a significant inhibitory effect on bacterial contamination. The Trichoderma fermentation filtrate contains chitinase, β-1,3-glucanase, and fungal antagonistic secondary metabolites, which can effectively degrade and inhibit filamentous fungi. The two are complementary in function, and when used in combination, they can simultaneously cover both bacteria and fungi, the two main sources of contamination, thus forming a broad-spectrum antibacterial environment. At the same time, when the volume ratio of cellless Bacillus fermentation filtrate to cellless Trichoderma fermentation filtrate is controlled within the range of (1.5~2.5):1, it can ensure that bacterial contamination is fully inhibited while still maintaining effective control of fungal contamination. The metabolites of the two maintain a good balance. This ratio range can achieve the best match for dual inhibition of bacteria and fungi, further improving the overall anti-contamination ability and seedling rate of the *Gnaphalium affine* explants in the open tissue culture process.

[0036] In some embodiments, the first modified MS medium is obtained by the following method:

[0037] The first modified MS medium was obtained by adding 1~2 mg / L 6-benzylaminopurine, 0.4~0.6 mg / L naphthaleneacetic acid, 0.1~0.3 mg / L thiazoline, 40~60 mL / L cellless Bacillus fermentation filtrate, and 0.04%~0.06% PPM to MS medium.

[0038] The second modified MS medium was obtained by the following method:

[0039] The second modified MS medium was obtained by adding 1~2 mg / L 6-benzylaminopurine, 0.4~0.6 mg / L naphthaleneacetic acid, 0.1~0.3 mg / L thiazoline, 15~25 mL / L cellless Bacillus fermentation filtrate, and 0.01%~0.03% PPM to MS medium.

[0040] The third modified MS medium was obtained by the following method:

[0041] The third modified MS medium was obtained by adding 1-2 mg / L 6-benzylaminopurine, 0.4-0.6 mg / L naphthaleneacetic acid, 0.1-0.3 mg / L thiazoline, 5-14 mL / L cellless Bacillus fermentation filtrate, and 0-0.002% PPM to MS medium.

[0042] In some of the above embodiments, the three types of modified MS culture media all contain a certain proportion of 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and thiazolinone (TDZ). The combination of the three can simultaneously provide cell division signals, promote callus activation, and induce bud formation, thereby meeting the hormone requirements of *Gnaphalium affine* explants at each stage of wound healing, adventitious bud differentiation, and root primordia formation. At the same time, the cell-free Bacillus fermentation filtrate added to the culture medium forms a complex antibacterial system with PPM. Its content gradually decreases as the culture stage progresses. In the first stage, it is maintained at a high level to provide strong antibacterial protection to ensure smooth wound healing. In the second stage, it is moderately reduced to avoid inhibiting cell differentiation and to facilitate the induction of a large number of adventitious buds. In the third stage, PPM is further reduced or even basically removed, with only a small amount of fermentation filtrate retained to maintain a basic level of antibacterial activity, thereby creating more suitable conditions for the normal occurrence of root primordia.

[0043] The above-mentioned three-stage modified MS medium formula not only meets the needs of the gradual transformation of wrinkled leaf vegetable explants from callus to bud and then to root at the hormone level, but also achieves a balance between pollution control and differentiation induction in the dynamic regulation of antibacterial agents, thereby significantly improving the seedling rate of wrinkled leaf vegetable under open tissue culture conditions.

[0044] In some embodiments, step S1 includes:

[0045] Rinse the explants of *Vigna angularis* with running water 2-4 times, then soak them in a 60-80 wt% alcohol solution for 40-50 seconds, then disinfect them with a 0.05 wt%-0.15 wt% mercuric chloride solution for 6-10 minutes, then rinse them with sterile water 2-4 times, and cut them to a length of 0.4-0.6 cm to obtain pretreated explants.

[0046] In some of the above embodiments, rinsing with running water can remove dust, soil, and some attached microorganisms from the surface of the explants, reducing the initial contamination load; alcohol treatment can quickly destroy the cell membranes of microorganisms on the surface of the explants, improving the efficiency of subsequent disinfection; mercuric chloride solution is a powerful bactericide that can deeply kill residual bacteria and fungi, significantly reducing exogenous contamination; multiple rinses with sterile water can remove residual alcohol and mercuric chloride from the surface of the explants; cutting the disinfected explants to a length of 0.4~0.6cm is beneficial for exposing appropriate incisions, ensuring the contact surface for subsequent healing and differentiation, and avoiding an excessively large incision area that would increase the risk of contamination; through the above steps, pretreated explants with essentially sterile surfaces and good activity can be obtained, providing a reliable starting point for the subsequent three-stage culture.

[0047] In some implementations, step S2 includes:

[0048] S2: Insert the pretreated explants into the first modified MS medium and culture them for 6-8 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the first stage explants.

[0049] In some of the above embodiments, the main purpose of the first-stage culture is to promote the healing of explant incisions and maintain tissue viability. The temperature range of 20-30℃ is close to the suitable growth environment of *Gnaphalium affine*, which can maintain cell metabolic activity and reduce environmental stress. The light duration is controlled at 14-18 h / d to simulate long-day conditions, which helps the explants maintain photosynthetic activity while avoiding excessive light that could cause tissue dehydration or browning. The light intensity is set at 1800-2200 lx to provide adequate light for the explants and promote cell recovery and division. The culture time is controlled at 6-8 days to ensure sufficient healing of the incision tissue while avoiding the risk of contamination from prolonged stay of the explants in the culture medium. By removing contaminated explants in a timely manner during the culture process, the spread of contaminants can be reduced, and the survival rate of the entire culture population can be improved. Through the above culture, the explant incisions can heal well, and some preliminary callus tissue can be formed, thus obtaining first-stage explants suitable for entering the adventitious bud induction period.

[0050] In some implementations, step S3 includes:

[0051] S3: Insert the first-stage explants into the second modified MS medium and culture them for 12-20 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the second-stage explants.

[0052] In some of the above embodiments, the main purpose of the second-stage culture is to induce explant differentiation into adventitious shoots; the temperature range of 20-30℃ maintains the activity of cell division and differentiation; the light duration is maintained at 14-18 h / d to continuously provide light signals, stimulate cell differentiation, and promote shoot formation; the light intensity is controlled at 1800-2200 lx to provide suitable energy support for the formation of adventitious shoots; the culture time is set to 12-20 days to ensure that the explants undergo sufficient differentiation induction time in the second modified MS medium, allowing the callus tissue to gradually form multiple shoots; timely removal of contaminated explants can effectively reduce the spread of miscellaneous bacteria and ensure the overall seedling survival rate of the population culture; through the above culture, the explants can gradually transform from the callus state into a shoot structure, thereby obtaining a large number of adventitious shoots in the second-stage explants.

[0053] In some implementations, step S4 includes:

[0054] S4: Insert the second-stage explants into the third modified MS medium and culture them for 12-20 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the primary seedlings.

[0055] In some of the above embodiments, the main purpose of the third stage of cultivation is to induce adventitious buds to further differentiate into root primordia and develop into primary seedlings. The temperature range of 20-30℃ can maintain the metabolic activity required for root primordia formation. The light duration is controlled at 14-18 h / d to promote the establishment of photosynthetic physiology in seedlings, enhance energy supply, and facilitate the coordinated development of roots and aboveground parts. The light intensity is set at 1800-2200 lx to provide sufficient energy for root induction. The cultivation time is controlled at 12-20 days to provide sufficient time for root primordia development and primary seedling formation. By promptly removing contaminated explants during the cultivation process, the spread of miscellaneous bacteria can be prevented, ensuring the healthy growth of the overall population. After the above cultivation, the explants can complete the transformation from bud to seedling stage, forming primary seedlings with transplanting potential.

[0056] In some embodiments, the wrinkled-leaf vegetable explants are selected from the petiole segments of broccoli, cabbage, or lettuce.

[0057] In some of the above embodiments, petiole segments, as explants, have the characteristics of relatively dense tissue structure and high mechanical support strength, which can maintain good integrity during cutting and transfer operations and reduce the risk of mechanical damage. At the same time, petiole tissue contains a certain number of potential meristematic cells, which have a high regeneration capacity and are more likely to induce the formation of callus, adventitious buds and root primordia under suitable culture conditions. Broccoli, cabbage and lettuce are all representative crops of wrinkled leaf vegetables, and their petiole segments show relatively low contamination rate, mild browning degree and high seedling efficiency in tissue culture. Compared with directly taking leaves or stem segments, petiole segment explants can maintain activity and stability better under open tissue culture conditions, thereby significantly improving the seedling survival rate of primary seedlings.

[0058] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0059] By sequentially adding acellular Bacillus fermentation filtrate and acellular Trichoderma fermentation filtrate to a three-stage modified MS medium, and using them in conjunction with PPM, effective anti-pollution protection of *Gnaphalium affine* explants was achieved in an open environment. While reducing the pollution rate and browning degree, the incision healing, adventitious bud differentiation, and root primordia formation of the explants were ensured, thus significantly improving the seedling rate of *Gnaphalium affine* under open tissue culture conditions and reducing the dependence on strict aseptic conditions. This method has strong potential for widespread application. Attached Figure Description

[0060] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0061] Figure 1 The broccoli 24060 grown in one embodiment of this application.

[0062] Figure 2 This is a petiole segment of broccoli 24060 used as an explant of wrinkled leaf vegetable in one embodiment of this application. Detailed Implementation

[0063] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0064] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0065] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0066] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0067] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0068] Bacillus subtilis ATCC 6051 (Wuhan Warner Biotechnology Co., Ltd.), and Bacillus licheniformis ATCC 14580 (Wuhan Warner Biotechnology Co., Ltd.) were used after cell rejuvenation.

[0069] Trichoderma harzianum ACCC 30371 (China Agricultural Microbial Culture Collection Center). Spores were collected after cell rejuvenation for later use.

[0070] LB liquid medium: a liquid medium containing 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 7.0±0.2, sterilized by autoclaving at 121℃ for 20 min at 100 kPa for later use;

[0071] PDB medium: Boil 200g of potatoes in water for 30 minutes, filter the potato pieces, add 20g of glucose to dissolve, bring the volume to 1000mL, and sterilize with high pressure steam at 121℃ for 20 minutes at 100kPa for later use.

[0072] MS medium: MS medium dry powder (without sucrose and agar) was prepared by adding water to a final volume of 30 g / L sucrose and 7 g / L agar.

[0073] Explants for wrinkled leaf vegetables: Broccoli 24060, sown from seed, with petioles harvested one month later as explants, such as... Figure 1 and Figure 2 As shown;

[0074] Preparation Example 1-1

[0075] Preparation of cell-free fermentation filtrate:

[0076] With 5×10 6 Bacillus subtilis was inoculated into LB liquid medium at an inoculum of CFU / mL and fermented at 30°C with aerobic shaking for 48 hours. After fermentation, the supernatant was collected by centrifugation at 8000g for 10 min at 4°C and then filtered through a 0.22μm sterile filter membrane to obtain cell-free Bacillus fermentation filtrate. The Bacillus subtilis and Bacillus licheniformis were used, and the inoculum ratio of Bacillus subtilis to Bacillus licheniformis was 1:1.

[0077] With 5×10 5Trichoderma harzianum spores were inoculated into PDB medium at an inoculum rate of 1 spore / mL and fermented at 28°C with aerobic shaking for 60-84 h. After fermentation, the blocky mycelia were removed with sterile gauze, and the supernatant was collected by centrifugation at 8000g, 10 min, and 4°C. The supernatant was then filtered through a 0.22μm sterile filter membrane to remove mycelia and mycelia, thus obtaining cell-free Trichoderma fermentation filtrate.

[0078] Cellless fermentation filtrate A was obtained by mixing cellless Bacillus fermentation filtrate and cellless Trichoderma fermentation filtrate at a volume ratio of 2:1.

[0079] Preparation Examples 1-2

[0080] Preparation of cell-free fermentation filtrate:

[0081] With 5×10 6 Bacillus was inoculated into LB liquid medium at an inoculum concentration of CFU / mL and fermented at 30°C with aerobic shaking for 48 hours. After fermentation, the supernatant was collected by centrifugation at 8000g for 10 min at 4°C and then filtered through a 0.22μm sterile filter membrane. The resulting filtrate was cell-free Bacillus fermentation filtrate. The Bacillus included Bacillus subtilis.

[0082] With 5×10 5 Trichoderma harzianum spores were inoculated into PDB medium at an inoculum rate of 1 spore / mL and fermented at 28°C with aerobic shaking for 60-84 h. After fermentation, the blocky mycelia were removed with sterile gauze, and the supernatant was collected by centrifugation at 8000g, 10 min, and 4°C. The supernatant was then filtered through a 0.22μm sterile filter membrane to remove mycelia and mycelia, thus obtaining cell-free Trichoderma fermentation filtrate.

[0083] Cellless fermentation filtrate B was obtained by mixing cellless Bacillus fermentation filtrate and cellless Trichoderma fermentation filtrate at a volume ratio of 2:1.

[0084] Preparation Examples 1-3

[0085] Preparation of cell-free fermentation filtrate:

[0086] With 5×10 6 Bacillus was inoculated into LB liquid medium at an inoculum concentration of CFU / mL and fermented at 30°C with aerobic shaking for 48 hours. After fermentation, the supernatant was collected by centrifugation at 8000g for 10 min at 4°C and then filtered through a 0.22μm sterile filter membrane. The resulting filtrate was cell-free Bacillus fermentation filtrate. The Bacillus included Bacillus licheniformis.

[0087] With 5×10 5Trichoderma harzianum spores were inoculated into PDB medium at an inoculum rate of 1 spore / mL and fermented at 28°C with aerobic shaking for 60-84 h. After fermentation, the blocky mycelia were removed with sterile gauze, and the supernatant was collected by centrifugation at 8000g, 10 min, and 4°C. The supernatant was then filtered through a 0.22μm sterile filter membrane to remove mycelia and mycelia, thus obtaining cell-free Trichoderma fermentation filtrate.

[0088] Cellless fermentation filtrate C was obtained by mixing cellless Bacillus fermentation filtrate and cellless Trichoderma fermentation filtrate at a volume ratio of 2:1.

[0089] Preparation Examples 1-4

[0090] Preparation of cell-free fermentation filtrate:

[0091] The preparation method is largely the same as in Example 1-1, except that the cellless Bacillus fermentation filtrate and the cellless Trichoderma fermentation filtrate are mixed in a volume ratio of 1:1 to obtain cellless fermentation filtrate D.

[0092] Preparation Examples 1-5

[0093] Preparation of cell-free fermentation filtrate:

[0094] The preparation method is largely the same as in Example 1-1, except that only the cellless Bacillus fermentation filtrate is used as cellless fermentation filtrate E.

[0095] Preparation Examples 1-6

[0096] Preparation of cell-free fermentation filtrate:

[0097] The preparation method is largely the same as in Example 1-1, except that only the cellless Trichoderma fermentation filtrate is used as cellless fermentation filtrate F.

[0098] Preparation Example 2-1

[0099] Preparation of modified MS medium:

[0100] Add 1.5 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 0.2 mg / L thiazoline, 50 mL / L cellless Bacillus fermentation filtrate A, and 0.05% PPM to MS medium. After dissolving, pour directly onto plates without sterilization to obtain the first modified MS medium-A.

[0101] Add 1.5 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 0.2 mg / L thiazoline, 20 mL / L cellless Bacillus fermentation filtrate A, and 0.02% PPM to MS medium. After dissolution, pour directly onto plates without sterilization to obtain the second modified MS medium-A.

[0102] Add 1.5 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 0.2 mg / L thiazoline, and 10 mL / L cell-free Bacillus fermentation filtrate A to MS medium. After dissolution, pour directly onto plates without sterilization to obtain the third modified MS medium-A.

[0103] Preparation Example 2-2

[0104] Preparation of modified MS medium:

[0105] The preparation was largely the same as in Example 2-1, except that cellless Bacillus fermentation filtrate B was used instead of cellless Bacillus fermentation filtrate A to obtain the first modified MS medium-B, the second modified MS medium-B, and the third modified MS medium-B, respectively.

[0106] Preparation Examples 2-3

[0107] Preparation of modified MS medium:

[0108] The preparation was largely the same as in Example 2-1, except that cellless Bacillus fermentation filtrate C was used instead of cellless Bacillus fermentation filtrate A to obtain the first modified MS medium-C, the second modified MS medium-C, and the third modified MS medium-C, respectively.

[0109] Preparation Examples 2-4

[0110] Preparation of modified MS medium:

[0111] The preparation was largely the same as in Example 2-1, except that cellless Bacillus fermentation filtrate D was used instead of cellless Bacillus fermentation filtrate A to obtain the first modified MS medium-D, the second modified MS medium-D, and the third modified MS medium-D, respectively.

[0112] Preparation Examples 2-5

[0113] Preparation of modified MS medium:

[0114] The preparation method is largely the same as in Example 2-1, except that cellless Bacillus fermentation filtrate E is used instead of cellless Bacillus fermentation filtrate A to obtain the first modified MS medium-E, the second modified MS medium-E, and the third modified MS medium-E, respectively.

[0115] Preparation Examples 2-6

[0116] Preparation of modified MS medium:

[0117] The preparation was largely the same as in Example 2-1, except that cellless Bacillus fermentation filtrate F was used instead of cellless Bacillus fermentation filtrate A to obtain the first modified MS medium-F, the second modified MS medium-F, and the third modified MS medium-F, respectively.

[0118] Example 1

[0119] Open tissue culture method for cruciferous vegetables:

[0120] Experimental environment: Standard laboratory workbench surface; room floor mopped once a week; UV sterilization for 2 hours every three months; operators wear masks.

[0121] The explants of *Vigna angularis* were rinsed three times with running water, soaked in 75wt% alcohol for 40-50 seconds, disinfected with 0.1% mercuric chloride solution for 8 minutes, rinsed three times with sterile water, and cut to a length of 0.5 cm to obtain pretreated explants.

[0122] Pretreated explants were inserted into the first modified MS medium-A, with 8 explants per dish, repeated 8 times, for a total of 64 explants. After inoculation, the explants were cultured for 7 days at a temperature of 25℃, a light intensity of 16h / d and a light intensity of 2000Lx. During the culture process, contaminated explants were removed.

[0123] Uncontaminated explants were transferred to modified MS medium-A and cultured for 14 days at 25°C, 16 h / d light, and 2000 Lx light intensity. Contaminated explants were removed during the culture process.

[0124] Uncontaminated explants were transferred to modified MS medium-A and cultured for 16 days at 25°C, 16 h / d light, and 2000 Lx light intensity. The number of primary seedlings that formed root primordia was counted and the emergence rate was calculated. The results are shown in Table 1.

[0125] Example 2

[0126] Open tissue culture method for cruciferous vegetables:

[0127] The method is largely the same as in Example 1, except that the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A are replaced by the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A, respectively.

[0128] Example 3

[0129] Open tissue culture method for cruciferous vegetables:

[0130] The method is largely the same as in Example 1, except that the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A are replaced by the first modified MS medium-C, the second modified MS medium-A, and the third modified MS medium-A, respectively.

[0131] Example 4

[0132] Open tissue culture method for cruciferous vegetables:

[0133] The method is largely the same as in Example 1, except that the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A are replaced by the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A, respectively.

[0134] Example 5

[0135] Open tissue culture method for cruciferous vegetables:

[0136] The method is largely the same as in Example 1, except that the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A are replaced by the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A, respectively.

[0137] Example 6

[0138] Open tissue culture method for cruciferous vegetables:

[0139] Similar to Example 1, except that: the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A are replaced by the first modified MS medium-A, the second modified MS medium-A, and the third modified MS medium-A, respectively.

[0140] Comparative Example 1

[0141] Open tissue culture method for cruciferous vegetables:

[0142] Experimental environment: Standard laboratory workbench surface; room floor mopped once a week; UV sterilization for 2 hours every three months; operators wear masks.

[0143] The explants of *Vigna angularis* were rinsed three times with running water, soaked in 75wt% alcohol for 40-50 seconds, disinfected with 0.1% mercuric chloride solution for 8 minutes, rinsed three times with sterile water, and cut to a length of 0.5 cm to obtain pretreated explants.

[0144] Pretreated explants were inserted into the first modified MS medium-A, with 8 explants per dish, repeated 8 times, for a total of 64 explants. After inoculation, the explants were cultured for 7 days at a temperature of 25℃, a light intensity of 16h / d and a light intensity of 2000Lx. During the culture process, contaminated explants were removed.

[0145] Uncontaminated explants were transferred to a new modified MS medium-A and cultured for 14 days at 25°C, 16 h / d light, and 2000 Lx light intensity. Contaminated explants were removed during the culture process.

[0146] Uncontaminated explants were transferred to a new modified MS medium-A and cultured for 16 days at 25°C, 16 h / d light, and 2000 Lx light intensity. The number of seedlings that formed root primordia was counted and the emergence rate was calculated. The results are shown in Table 1.

[0147] Comparative Example 2

[0148] Open tissue culture method for cruciferous vegetables:

[0149] It is largely the same as Comparative Example 1, except that the second modified MS medium-A is used instead of the first modified MS medium-A.

[0150] Comparative Example 3

[0151] Open tissue culture method for cruciferous vegetables:

[0152] It is largely the same as Comparative Example 1, except that the third modified MS medium-A is used instead of the first modified MS medium-A.

[0153] Table 1

[0154]

[0155] According to Table 1, each embodiment is better than Comparative Examples 1-3, indicating that the open tissue culture method for *Vigna angularis* provided in this application significantly improves the seedling rate and reduces the risk of contamination and browning under open conditions, thus possessing superior promotional value. The possible reasons are as follows: In Comparative Example 1, the first modified MS medium was consistently used. Although the antibacterial strength was high, it inhibited the occurrence of buds and root primordia in the later differentiation stage, resulting in a low seedling rate. In Comparative Example 2, the second modified MS medium was consistently used. Although the inhibitory effect on explants was lower than in Comparative Example 1, the level of antibacterial agent was not dynamically adjusted according to the physiological state of the explants, still resulting in insufficient differentiation efficiency and a seedling rate of only moderate level. In Comparative Example 3, the third modified MS medium was consistently used. Without PPM, the fermentation filtrate content was too low, failing to effectively inhibit exogenous contamination in the early stages, leading to a large number of explant deaths and the lowest seedling rate.

[0156] As shown in Examples 1-4, the composition and ratio of the microbial strains in the cell-free fermentation filtrate have a significant impact on the effect of open tissue culture of *Caragana korshinskii*. Compared with Examples 2 and 3, Example 1 additionally combined *Bacillus licheniformis* with *Bacillus*, which, together with the *Trichoderma* fermentation filtrate, resulted in a broader antibacterial spectrum and a better balance between contamination control and differentiation efficiency in explants. Therefore, the seedling rate of Example 1 was higher than that of Examples 2 and 3. Compared with Examples 4-6, changing the volume ratio from 2:1 (Example 1) to 1:1 (Example 4) weakens the early effect on… The inhibitory strength of bacterial contamination was reduced, but there was no benefit to the overall synergy, and the seedling rate decreased. When using acellular Bacillus fermentation filtrate alone (Example 5) or acellular Trichoderma fermentation filtrate (Example 6), the seedling rate was further reduced. This indicates that the type and ratio of acellular fermentation filtrate have a significant impact on the effect of open tissue culture. When Bacillus subtilis and Bacillus licheniformis are combined and mixed with Trichoderma fermentation filtrate at a volume ratio of 2:1, the three-stage reduction strategy is more effective and more suitable for open-condition tissue culture of wrinkled leaf vegetables.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. An open tissue culture method for *Vigna angularis*, characterized in that, Includes the following steps: S1: Disinfect the explants of *Vigna angularis* to obtain pretreated explants; S2: The pretreated explants are inserted into the first modified MS medium for the first stage of culture to allow the explant incisions to heal and obtain the first stage explants; S3: The first-stage explants were transferred to the second modified MS medium for the second-stage culture, which allowed the explants to differentiate into adventitious shoots, thus obtaining the second-stage explants; S4: The second-stage explants were transferred to the third-stage modified MS medium for third-stage culture, which induced the explants to form root primordia and obtain primary seedlings. The first modified MS medium was obtained by the following method: The first modified MS medium was obtained by adding 1~2 mg / L 6-benzylaminopurine, 0.4~0.6 mg / L naphthaleneacetic acid, 0.1~0.3 mg / L thiazoline, 40~60 mL / L cellless Bacillus fermentation filtrate, and 0.04%~0.06% PPM to MS medium. The second modified MS medium was obtained by the following method: The second modified MS medium was obtained by adding 1~2 mg / L 6-benzylaminopurine, 0.4~0.6 mg / L naphthaleneacetic acid, 0.1~0.3 mg / L thiazoline, 15~25 mL / L cellless Bacillus fermentation filtrate, and 0.01%~0.03% PPM to MS medium. The third modified MS medium was obtained by the following method: The third modified MS medium was prepared by adding 1-2 mg / L 6-benzylaminopurine, 0.4-0.6 mg / L naphthaleneacetic acid, 0.1-0.3 mg / L thiazoline, 5-14 mL / L cellless Bacillus fermentation filtrate, and 0-0.002% PPM to MS medium. The cell-free fermentation filtrate includes cell-free Bacillus fermentation filtrate and cell-free Trichoderma fermentation filtrate; the volume ratio of cell-free Bacillus fermentation filtrate to cell-free Trichoderma fermentation filtrate in the cell-free fermentation filtrate is (1.5~2.5):1; The cell-free Bacillus fermentation filtrate was prepared by the following method: With 10 6 ~10 7 Bacillus was inoculated into LB liquid medium at an inoculum concentration of CFU / mL and fermented aerobicly at 25-35°C for 36-60 hours. The bacterial cells were removed by filtration to obtain cell-free Bacillus fermentation filtrate. The Bacillus included Bacillus subtilis and Bacillus licheniformis, and the inoculum ratio of Bacillus subtilis to Bacillus licheniformis was 1:0.5-1.

5. The cell-free Trichoderma fermentation filtrate was prepared by the following method: With 10 5 ~10 6 Trichoderma spores were inoculated into PDB medium at an inoculum rate of 1 spore / mL and fermented aerobically at 25-35°C for 60-84 hours. The mycelium and mycelium were removed by filtration to obtain cell-free Trichoderma fermentation filtrate. The Trichoderma included Trichoderma harzianum.

2. The open tissue culture method according to claim 1, characterized in that, S1 includes: Rinse the explants of *Vigna angularis* with running water 2-4 times, then soak them in a 60-80 wt% alcohol solution for 40-50 seconds, then disinfect them with a 0.05 wt%-0.15 wt% mercuric chloride solution for 6-10 minutes, then rinse them with sterile water 2-4 times, and cut them to a length of 0.4-0.6 cm to obtain pretreated explants.

3. The open tissue culture method according to claim 1, characterized in that, S2 includes: S2: Insert the pretreated explants into the first modified MS medium and culture them for 6-8 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the first stage explants.

4. The open tissue culture method according to claim 1, characterized in that, S3 includes: S3: Insert the first-stage explants into the second modified MS medium and culture them for 12-20 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the second-stage explants.

5. The open tissue culture method according to claim 1, characterized in that, S4 includes: S4: Insert the second-stage explants into the third modified MS medium and culture them for 12-20 days at 20-30℃, 14-18h / d light intensity, and 1800-2200lx light intensity. Remove the contaminated explants during the culture process to obtain the primary seedlings.

6. The open tissue culture method according to claim 1, characterized in that, The explants of the wrinkled leaf vegetable were selected from the petiole segments of broccoli, cabbage, or lettuce.

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