Fritillaria tissue culture medium, preparation method, fritillaria tissue culture method and application

Abstract

The invention provides a fritillaria tissue culture medium, a preparation method, a fritillaria tissue culture method and application, and belongs to the technical field of plant tissue culture. It is found for the first time that low-concentration picloram can achieve efficient browning resistance of fritillary callus; compared with the prior art, the formula of the fritillaria tissue culture medium has the advantages that the browning resistance efficiency is remarkable, healthy proliferation of calluses is synchronously promoted, and the contradiction that'browning inhibition 'and'growth promotion' are difficult to consider at the same time in a traditional fritillaria tissue culture method is solved. The method is suitable for various fritillaria such as fritillaria unibiacteata, bulbus fritillariae cirrhosae and bulbus fritillariae thunbergii, and has universality and industrialization prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a Fritillaria tissue culture medium, its preparation method, and the Fritillaria tissue culture method and its uses. Background Technology

[0002] Fritillaria cirrhosa is a traditional Chinese medicine, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It has the effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, dispersing nodules and reducing swelling. It is commonly used for dry cough due to lung heat, dry cough with little phlegm, yin deficiency cough, hemoptysis, scrofula, mastitis, and lung abscess. Commercially available fritillaria cirrhosa is often divided into Songbei, Lubei, and Qingbei. Among them, Songbei is of the highest quality. Dark purple fritillaria cirrhosa is the main source of Songbei, but wild resources are scarce, making it a national second-class protected plant with restricted harvesting. Zhejiang fritillaria cirrhosa has also long been used medicinally as "Zhebei," with high annual market demand, and wild resources are also facing depletion. Under traditional breeding conditions, dark purple fritillaria cirrhosa takes 4-5 years to flower and bear fruit, while Zhejiang fritillaria cirrhosa can be harvested in 2-3 years, but it suffers from severe virus accumulation and rapid seed degeneration, both leading to low propagation efficiency and limited industry scale.

[0003] Plant tissue culture technology, based on the totipotency of plant cells, utilizes artificial culture media to promote the continuous proliferation of plant tissues. It plays an indispensable role in the protection of rare and endangered plants, crop breeding, rapid propagation, artificial seed production, and germplasm preservation. Rapid propagation can precisely compensate for the long growth and reproductive cycle of fritillaria.

[0004] Browning is a challenging problem in plant tissue culture, limiting rapid plant propagation and even causing large-scale tissue death. Current research indicates that browning is primarily caused by the oxidation of phenolic substances by polyphenol oxidase, producing quinones, which lead to tissue browning and death, severely impacting the survival rate and growth of tissue-cultured seedlings. Antioxidants or activated charcoal are commonly used to address this issue, but these measures have drawbacks such as affecting plant growth and adsorbing nutrients.

[0005] Severe browning is a common problem in Fritillaria tissue culture. Traditional methods mainly reduce browning by adding activated carbon, antioxidants, and frequently changing the culture medium. However, the addition of exogenous substances inevitably alters the composition of the culture medium to some extent, thus affecting tissue growth. It also increases workload and wastes culture medium. Summary of the Invention

[0006] To address the problem of easy browning and subsequent death during Fritillaria tissue culture, this invention controls browning by comprehensively adjusting culture conditions such as hormones, sucrose, and pH, thereby achieving rapid tissue growth and effectively solving the problem of low efficiency in Fritillaria tissue culture.

[0007] The present invention provides a fritillaria tissue culture medium, which comprises the following components: 4.74 g / L MS, 1 mg / L cyproconazole, 25 g / L sucrose, and 8 g / L agar; the pH of the fritillaria tissue culture medium is 6.5.

[0008] MS in the above-mentioned Fritillaria tissue culture medium is a solid powder.

[0009] Furthermore, the MS is composed of the following components in parts by weight: 4500-4550 parts of macro-inorganic salts, 60-70 parts of iron salts, 30-50 parts of trace inorganic salts, 100-110 parts of organic vitamins, and 0.5-5 parts of amino acids.

[0010] Further, the MS is composed of the following components in parts by weight: 1650 parts NH4NO3, 1900 parts KNO3, 440 parts CaCl2·2H2O, 370 parts MgSO4·7H2O, 170 parts KH2PO4, 27.8 parts FeSO4·7H2O, 37.3 parts Na2-EDTA, 22.3 parts MnSO4·4H2O, 8.6 parts ZnSO4·7H2O, 0.025 parts CoCl2·6H2O, 0.025 parts CuSO4·5H2O, 0.25 parts Na2MoO4·2H2O, 0.83 parts KI, 6.2 parts H3BO3, 0.5 parts nicotinic acid, 0.5 parts thiamine hydrochloride (VB1), 0.1 parts pyridoxine hydrochloride (VB6), 100 parts inositol, and 2 parts glycine.

[0011] In the formulation of the specific embodiments of the present invention, "part" refers to parts by weight, and 1 part is 1 mg.

[0012] The present invention also provides a method for preparing the above-mentioned Fritillaria tissue culture medium, the method comprising the following steps: mixing MS, chlordane, sucrose, agar and water, adjusting the pH with a pH adjuster, and sterilizing to obtain the culture medium.

[0013] Furthermore, the sterilization temperature is 110~130℃, the pressure is 0.1~0.2Mpa, and the time is 10~30min.

[0014] Preferably, the sterilization temperature is 121°C, the pressure is 0.11 MPa, the time is 20 min, and no venting is performed.

[0015] Furthermore, the pH adjuster is selected from sodium hydroxide or hydrochloric acid.

[0016] The present invention also provides a method for fritillaria tissue culture, the method comprising the following steps: inoculating fritillaria tissue into the above-mentioned fritillaria tissue culture medium and culturing it in the dark at 15~30℃ for 5~50 days.

[0017] Furthermore, the method for culturing fritillaria tissue involves culturing it in the dark at 20-25°C for 7-45 days.

[0018] Furthermore, the fritillaria is selected from Fritillaria thunbergii, Fritillaria thunbergii, and Fritillaria cirrhosa; the tissue is callus tissue.

[0019] The present invention also provides the use of the above-mentioned Fritillaria tissue culture medium formulation or the above-mentioned Fritillaria tissue culture method in the browning resistance of easily browning plants.

[0020] The present invention also provides the use of the above-mentioned Fritillaria tissue culture medium formulation or the above-mentioned Fritillaria tissue culture method in the in vitro propagation, germplasm preservation or genetic transformation of Fritillaria.

[0021] Furthermore, the fritillaria is selected from Fritillaria thunbergii, Fritillaria thunbergii, and Fritillaria cirrhosa.

[0022] The core of this invention lies in providing a novel Fritillaria tissue culture medium formulation and its cultivation method. This formulation consists of two main parts: a core functional component and synergistic environmental factors. Through synergistic action, they jointly achieve highly efficient inhibition of browning. The core anti-browning component is the auxin analog Piclorm (PIC). The synergistic environmental factors are: a carbon source and osmotic pressure regulator: sucrose; and a pH regulator: sodium hydroxide (NaOH) or hydrochloric acid (HCl), used to adjust the pH value.

[0023] The technical solution of this invention is not a simple superposition of various technical features, but rather uses atrazine as the system's "brain," issuing core instructions to alter the browning sensitivity of cells; low sucrose as the system's "internal environmental basis," reducing external stress and ensuring that the "brain's" instructions can be executed efficiently; and high pH as the system's "external barrier," directly inhibiting the destructive effects of key enzymes from the external environment. With atrazine as the core and low sucrose and high pH as the two wings, these three elements work synergistically to act on the fritillaria explant, forming a three-dimensional, highly efficient browning inhibition network.

[0024] This invention provides an effective method for comprehensively inhibiting browning during the in vitro culture of Fritillaria thunbergii by finely controlling the culture medium environment (sucrose concentration, pH) and endogenous signals (hormone levels). It significantly reduces the browning rate and degree; improves the survival rate and regeneration capacity of explants; promotes the healthy growth of Fritillaria thunbergii tissue culture seedlings; and shortens the culture cycle. It provides reliable technical support for biotechnological applications such as rapid in vitro propagation, germplasm resource preservation, and genetic transformation of Fritillaria thunbergii. It is mainly applied to the tissue culture (primary explant culture, subculture proliferation), callus induction, and culture of Fritillaria thunbergii (such as *Fritillaria thunbergii* and *Fritillaria thunbergii*). This invention determines the optimal balance point of synergistic effects of atrazine, low sucrose, and high pH, ​​solving a key obstacle in Fritillaria thunbergii tissue culture. It has significant practical value and industrial significance for the large-scale, efficient propagation and germplasm innovation of Fritillaria thunbergii. Furthermore, it provides a new, universal, robust, and low-cost technical pathway for other easily browning plants, possessing both significant theoretical value and broad application prospects.

[0025] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. Strategic Innovation 1.1 Shifting from "post-adsorption" to "pre-prevention": Compared to the passive remedial measures of adding substances to adsorb or remove browning products after browning occurs in traditional methods, this invention adopts an active prevention strategy, which regulates the physiological state of cells and the culture environment before browning occurs, thereby fundamentally preventing browning from taking place.

[0026] 1.2 Optimize basic parameters: Browning resistance can be achieved by optimizing the concentration of picrotoxin (PIC), sucrose, and pH value, thus avoiding the influence of exogenous substances such as activated carbon (AC), ascorbic acid (VC), and polyvinylpyrrolidone (PVP) on plant growth and development.

[0027] 1.3 PIC Functional Repositioning: Low concentration (1 mg / L) of picaridin (PIC) mainly acts as an anti-browning signaling agent, downregulating key enzyme genes of phenol metabolism at the source and activating the endogenous antioxidant system, while also having a mild growth regulatory effect.

[0028] 1.4 Transparent and controllable composition: Eliminates the drift of culture medium efficacy caused by uncontrollable adsorption of activated carbon, ensuring high reproducibility and standardization of experiments.

[0029] 2. Overall benefits 2.1 Significant effect: Dual intervention of cell and enzyme, browning rate far exceeds that of single antioxidant treatment.

[0030] 2.1 Clear system: All components are clearly defined, there is no "black box" operation, which facilitates large-scale production and quality control.

[0031] 2.3 Growth-promoting compatibility: It avoids the loss of hormones and nutrients caused by the adsorption of substances such as activated carbon, ensures the integrity of the culture medium components, and provides the most suitable growth environment for explants while inhibiting browning, which significantly improves the survival rate and growth efficiency of Fritillaria tissue culture.

[0032] 2.4 Cost advantage: Using only conventional reagents, without the need for expensive special antioxidants or adsorbents, reduces production costs.

[0033] 2.5 Theoretical Innovation: This invention discovers that picrotoxin (PIC) has a novel and unconventional anti-browning function at low concentrations, providing a new approach and direction for solving the browning problem in plant tissue culture.

[0034] This invention is the first to discover that low concentrations (1 mg / L) of thiamethoxam can achieve highly efficient anti-browning of Fritillaria callus tissue. Furthermore, it establishes a ternary synergistic system of thiamethoxam-low sucrose-high pH. Compared with existing technologies, the Fritillaria tissue culture medium formulation of this invention not only shows significantly higher anti-browning efficiency but also simultaneously promotes healthy callus proliferation, resolving the contradiction in traditional methods of simultaneously inhibiting browning and promoting growth. This invention is applicable to various Fritillaria species, including *Fritillaria purpurea*, *Fritillaria cirrhosa*, and *Fritillaria thunbergii*, possessing universality and promising industrialization prospects.

[0035] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0036] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the experimental procedure.

[0038] Figure 2 The browning index of *Fritillaria purpurea* in different culture media is shown (ae in the figure is labeled using the Duncan method).

[0039] Figure 3 The percentage of browning at different levels in different culture media of *Fritillaria purpurea*.

[0040] Figure 4 The growth status of *Fritillaria purpurea* in different culture media.

[0041] Figure 5 The growth status of Fritillaria thunbergii in different culture media.

[0042] Figure 6 The browning index of Fritillaria thunbergii in different culture media (ae in the figure is labeled using the Duncan method). Detailed Implementation

[0043] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0044] MS medium solid powder was purchased from Haibo Biotechnology. The MS medium solid powder (excluding agar and sucrose) product number is HB8469-5, and the composition is shown in Table 1.

[0045] Table 1 Salt formulation of MS basal medium Example 1: Preparation of the Fritillaria tissue culture medium of the present invention The experimental procedure of this invention is as follows: Figure 1 As shown.

[0046] Preparation of Fritillaria cirrhosa tissue culture medium (1L system): Weigh 4.74g MS medium solid powder and 25g sucrose, dissolve in reverse osmosis (RO) water, add 1mg picolinate (PIC), adjust the pH of the solution system to 6.5 using NaOH or HCl, add 8g agar, sterilize at 121℃ and autoclave (0.11Mpa) for 20 minutes, place in a clean bench, and after the temperature drops to 55℃~65℃, dispense into approximately 50ml portions per dish, and allow to stand and cool to solidify for later use.

[0047] Example 2: Method for Tissue Culture of Fritillaria with Anti-Brown Properties Callus inoculation: Select healthy, normally growing callus tissue (Dark Purple Fritillaria, Curly Leaf Fritillaria, or Zhejiang Fritillaria), and cut it into pieces 0.5 cm in size with a scalpel. Small pieces, about 0.5 cm in size, are shaped in one cut to reduce mechanical damage caused by repeated cutting. The cut surfaces are then placed on the culture medium from Example 1 and incubated at 21°C in the dark for 45 days.

[0048] The following experimental examples demonstrate the beneficial effects of the present invention.

[0049] Experiment Example 1: Testing the anti-browning effect of different Fritillaria tissue culture media 1. Experimental Grouping Experimental groups were formed according to Table 2.

[0050] Table 2 Different experimental groups Among them, W1 is the culture medium formula described in paragraph 0044 of the instruction manual in CN117502239B; W2, based on section 0054 of the instruction manual in CN117502239B, removes the plant hormones NAA, 2,4-D, and TDZ from W1. W3 is the culture medium formulation described in section 0047 of the instruction manual in CN107494261B; W4 is the culture medium formulation described in section 0046 of the instruction manual in CN107494261B.

[0051] 2. Experimental Methods Callus inoculation: Select healthy, normally growing callus tissue (Dark Purple Fritillaria or Zhejiang Fritillaria), and cut it into pieces 0.5 cm in size with a scalpel. Cut small pieces (0.5 cm in size) into shape in one cut to reduce mechanical damage caused by repeated cutting. Place the cut surfaces on different culture media (Table 2) and incubate at 21°C in the dark for 45 days.

[0052] 3. Data Statistics Browning grading: Grade 0: No browning; Grade 1: Browned area ≤ 1 / 3; Grade 2: 1 / 3 ≤ Browned area ≤ 2 / 3; Grade 3: Browned area ≥ 2 / 3; Browning rate = (Number of browned explants / Total number of explants) × 100%; Browning index = (Number of explants at each level × Level) / (Total number of explants × Highest level) × 100%; 4. Experimental Results 4.1 In the hormone-free media “5” and “5-1”, the browning of *Fritillaria purpurea* explants was the most severe, with tertiary browning being the predominant type. Figure 3 In contrast, the browning index was significantly reduced in the "P2-1" and "RP2-1" media containing the corresponding hormone toxicant. Figure 4 Since the only variable between the two formulations was the presence or absence of thiamethoxam, and other factors (such as sucrose concentration and pH) had far less impact on browning, it can be inferred that thiamethoxam plays a key regulatory role in inhibiting browning during the tissue culture of Fritillaria thunbergii. Furthermore, the callus growth was higher in the group without thiamethoxam after its addition than in the group without the hormone.

[0053] 4.2 The browning index of “P2-2 live” is 52% ( Figure 2 Compared to "P2-2", the addition of activated charcoal exacerbated the browning of the dark purple fritillary bulb and slowed down its proliferation rate due to the absorption of nutrients. This may be because the activated charcoal adsorbed the hormone toxicity ...

[0054] 4.3 Comparing “P2-1” and “P2-2”, increasing the pH value can help reduce browning, and the slightly acidic environment plays an auxiliary role in resisting browning.

[0055] 4.4 Comparing “P2-2”, “RP2-1”, and “RP2-2”, reducing the sucrose concentration can reduce browning, and sucrose also plays an auxiliary role in controlling the browning of *Fritillaria purpurea*. However, too low a sucrose content affects the proliferation rate. Considering both proliferation rate and anti-browning effect, a sucrose concentration of 25 g / L was selected as the optimal culture condition. Figure 4 ).

[0056] 4.5 Compared with existing anti-browning methods (W1, W2, W3, W4), the browning rate of the *Fritillaria purpurea* callus in the formulation of Example 1 ("RP2-1") was significantly lower than that of the reported methods, and the proliferation rate was also higher than that of the existing methods, making it more suitable for *Fritillaria purpurea*.

[0057] 4.6 The same experimental method was validated in Fritillaria thunbergii callus tissue. Compared with the control group, "RP2-1" could significantly control the browning of Fritillaria thunbergii. Figure 6 And promotes the growth of Fritillaria thunbergii ( ) Figure 5 ).

[0058] 4.7 Activated carbon can control the browning of Fritillaria thunbergii and slow down the growth rate of callus tissue.

[0059] 4.8 Although activated carbon can control the browning of Fritillaria thunbergii, it cannot control the browning of Fritillaria purpurea. This suggests that the browning mechanisms of Fritillaria purpurea and Fritillaria thunbergii may differ, but toxicazine can control the browning of both simultaneously.

[0060] In summary, this invention provides a fritillaria tissue culture medium, its preparation method, and the methods and applications of fritillaria tissue culture. This invention is the first to discover that a low concentration (1 mg / L) of thiamethoxam can achieve highly efficient anti-browning of fritillaria callus tissue; furthermore, it establishes a ternary synergistic system of "thiamethoxam-low sucrose-high pH," which, compared with existing technologies, not only significantly improves anti-browning efficiency but also simultaneously promotes healthy callus proliferation, resolving the contradiction between "inhibiting browning" and "promoting growth" in traditional fritillaria tissue culture methods. This invention is applicable to various fritillaria species, including *Fritillaria purpurea*, *Fritillaria cirrhosa*, and *Fritillaria thunbergii*, and possesses universality and industrialization potential.

Claims

1. A fritillaria tissue culture medium, characterized in that, 4.74 g / L MS, 1 mg / L cyproconazole, 25 g / L sucrose, 8 g / L agar; the pH of the fritillaria tissue culture medium was 6.

5.

2. A method for preparing the Fritillaria cirrhosa tissue culture medium according to claim 1, characterized in that, The method includes the following steps: mixing MS, chlordane, sucrose, agar and water, adjusting the pH with a pH adjuster, and sterilizing to obtain the final product.

3. The method according to claim 2, characterized in that, The sterilization temperature is 110~130℃, the pressure is 0.1~0.2Mpa, and the time is 10~30min.

4. A method for fritillaria tissue culture, characterized in that, The method for culturing Fritillaria tissue includes the following steps: inoculating Fritillaria tissue into the Fritillaria tissue culture medium of claim 1 and culturing it in the dark at 15-30°C for 5-50 days.

5. The method for fritillaria tissue culture according to claim 4, characterized in that, The method for culturing fritillaria tissue involves culturing it in the dark at 20-25°C for 7-45 days.

6. The method for fritillaria tissue culture according to claim 4, characterized in that, The fritillaria bulbs are selected from Fritillaria thunbergii, Fritillaria thunbergii, and Fritillaria cirrhosa; the tissue is callus tissue.

7. The use of the Fritillaria tissue culture medium formulation of claim 1 or the Fritillaria tissue culture method of any one of claims 4 to 6 in the anti-browning of easily browning plants.

8. The use of the Fritillaria tissue culture medium formulation of claim 1 or the Fritillaria tissue culture method of any one of claims 4 to 6 in the in vitro propagation, germplasm preservation or genetic transformation of Fritillaria.

9. The use according to claim 8, characterized in that, The fritillaria bulbs are selected from dark purple fritillaria, Zhejiang fritillaria, and rolled leaf fritillaria.

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