A method for screening the optimal plant callus culture medium formula

Abstract

The present invention provides a method for screening an optimal plant callus culture medium formula, and belongs to the technical field of plant tissue culture. The present invention realizes efficient screening of the optimal culture medium formula for callus proliferation or rich in active ingredients by adopting a cell culture plate in combination with a response surface method, and solves the problems in the prior art that the culture medium dosage is large, the operation is cumbersome, the culture needs to occupy a large space, and it is not conducive to large-scale screening. The method provided by the present invention not only reduces the dosage of the culture medium and reduces the cost, but also improves the screening efficiency by reducing the necessary number of tests, and realizes high-throughput screening. In addition, the present invention also optimizes the culture medium formula, quickly finds the optimal conditions for promoting callus proliferation or being rich in active ingredients, and provides an economical, efficient and flexible technical means for plant cell culture and active ingredient research.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for screening an optimal plant callus culture medium formula. Background Art

[0002] Plant tissue culture refers to the technology of propagating and regenerating plant cells, tissues or organs on a specific culture medium under a sterile environment. By controlling the culture conditions (such as temperature, light and nutrients), the division, differentiation and growth of plant cells can be promoted, thereby rapidly propagating plants or cultivating specific plant tissues and organs. This technology is widely used in plant breeding, genetic improvement, medicinal plant production, and plant disease prevention and control.

[0003] In the process of plant tissue culture, suitable culture medium is crucial, which directly determines the growth and development of the culture. The culture medium not only provides the basic nutrients required for plant cell division and differentiation, but also regulates the proliferation, regeneration and organ formation of tissues by adding different types of plant hormones. An inappropriate culture medium may lead to growth stagnation, deformity or death, while a carefully screened and optimized culture medium can maximize cell activity, accelerate the culture cycle, and improve survival rate and quality. Therefore, the selection and optimization of the culture medium is the core element for the success of plant tissue culture. In the prior art, the screening of culture medium for plant tissue culture, especially callus proliferation, is still in the form of culture dishes or culture bottles.

[0004] Chinese patent CN108148799A discloses a method for efficiently screening rice callus culture medium, comprising the following steps: ① callus induction; ② callus subculture; ③ after inducing callus bud differentiation, the differentiated callus is placed on a rooting medium and cultured into seedlings, and finally the callus induction rate and seedling rate are counted.

[0005] However, the existing methods require a large amount of culture medium, are cumbersome to operate, require a large space for cultivation, and are not conducive to large-scale screening. Summary of the invention

[0006] The object of the present invention is to provide a method for screening an optimal plant callus culture medium formula, which has the advantages of small culture medium dosage, simple operation, high efficiency and suitability for screening large quantities of culture medium formulas.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for screening an optimal plant callus culture medium formula, comprising the following steps:

[0009] By setting different basic culture medium types, plant hormone types and plant hormone concentrations, a variety of culture media to be screened are obtained, and each culture medium to be screened is added to different wells of a cell culture plate;

[0010] Inoculating the plant callus suspension into each culture medium to be screened, culturing, and obtaining cultured plant callus;

[0011] The proliferation coefficient of each culture medium to be screened is calculated by the weight of the plant callus suspension before culture and the weight of the plant callus after culture;

[0012] The calculation formula of the proliferation coefficient is:

[0013] Proliferation coefficient = weight of plant callus after culture / weight of plant callus suspension before culture;

[0014] The type of basic culture medium was used as the selection index to compare the values ​​among the groups and obtain the culture medium with the highest proliferation coefficient to be screened. The data of the culture medium with the highest proliferation coefficient to be screened were subjected to response surface analysis to obtain the optimal plant callus culture medium formula.

[0015] Preferably, the cell culture plate has 48 or more wells.

[0016] Preferably, the response surface analysis includes the following steps:

[0017] Fit the data with a quadratic multiple regression equation to obtain a quadratic multiple regression equation;

[0018] According to the quadratic multiple regression equation, the effects of variables on callus proliferation were analyzed by observing the shapes of response surface plots and contour plots.

[0019] Preferably, the method for preparing the plant callus suspension comprises the following steps:

[0020] The plant callus tissue is mixed with water or a subculture medium, ground, and subcultured to obtain a plant callus tissue suspension.

[0021] Preferably, the number of generations of subculture is 2 to 5.

[0022] Preferably, the number of types of the culture medium to be screened is equal to the number of wells of the cell culture plate.

[0023] Preferably, the cell concentration in the plant callus suspension is 50-500 g / L.

[0024] Preferably, the plant is Korean ginseng.

[0025] Preferably, the basic culture medium types include 1 / 2MS culture medium, MS culture medium, WPM culture medium, RM culture medium, KC culture medium and VW culture medium;

[0026] The plant growth regulators include 6-BA, 2,4-D and KT;

[0027] The concentration of the plant growth regulator is a concentration point value between 0.01 and 5 mg / L.

[0028] Preferably, the quadratic multiple regression equation is: proliferation coefficient Y=4.23-0.1513A-04271B+0.3336C+0.8253AB-0.5888AC+0.25BC-1.31A2-0.4695B2-0.6165C2.

[0029] Beneficial effects of the present invention:

[0030] The present invention realizes efficient screening of the optimal culture medium formula for callus proliferation by adopting a cell culture plate combined with a response surface method. The method provided by the present invention not only reduces the amount of culture medium used and reduces the cost, but also improves the screening efficiency by reducing the necessary number of tests. At the same time, the use of the cell culture plate saves space, so that a cell culture plate can simultaneously screen a variety of different culture medium formulas, realizing high-throughput screening. In addition, the present invention also optimizes the culture medium formula, quickly finds the optimal conditions that promote callus proliferation or are rich in active ingredients, and provides an economical, efficient and flexible technical means for plant cell culture and active ingredient research. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The effect of the interaction between 6-BA (A) and 2,4-D (B) on the proliferation coefficient of Korean ginseng callus;

[0032] Figure 2 The effect of the interaction between 6-BA (A) and KT (C) on the proliferation coefficient of Korean ginseng callus;

[0033] Figure 3 The interactive effect of 2,4-D (B) and KT (C) on the proliferation coefficient of Korean ginseng callus;

[0034] Figure 4 This is a photograph of the present invention using a cell culture plate for culture medium screening. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

[0037] Establishment of callus induction and suspension culture system of Korean ginseng

[0038] Cut the root of Korean ginseng as an explant, put it in a clean beaker, add an appropriate amount of detergent and stir thoroughly until foam appears, gently scrub the surface of the explant with a brush, and rinse with running water for 30 to 40 minutes. In the clean bench, put the rinsed explant into a sterilized small beaker, soak and disinfect it in 75% alcohol for 50 seconds, rinse it with sterile ultrapure water 3 times, soak and disinfect it in 2% NaClO solution (mixed with 1 to 3 drops of Tween 20) for 6 minutes, and rinse it with sterile ultrapure water 5 times. Use sterile filter paper to absorb the water of the aseptically treated Korean ginseng explant, and cut it into small sections of 1 to 2 cm with a sterile scalpel, place it in the induction culture medium, and culture it under dark conditions at 25±2℃. The culture medium is replaced every 20 days. After 3 generations of culture, block Korean ginseng callus tissue can be obtained.

[0039] The induction medium comprises 4.41 g / L MS medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), and a NaOH aqueous solution, the pH value of which is adjusted to 5.8, and the medium is sterilized by high pressure at 121° C. for 15 min. When the temperature of the medium drops to 55-65° C., filtered and sterilized plant hormones are added to a final concentration of: 1.0 mg / L 6-BA (6-Benzylaminopurine) and 2.0 mg / L IBA (3-Indole butyric acid).

[0040] The induced blocky Korean ginseng callus tissue was cut into small particles and gently ground into a suspension in sterile water or subculture medium, and then transferred to a 250 mL Erlenmeyer flask containing 100 mL of subculture medium. The Erlenmeyer flask was placed in a shaker, and the temperature was controlled at 25±2°C and the rotation speed was 120 rpm. Fresh subculture medium was replaced every 20 days of culture. After 3 generations of culture, a Korean ginseng callus suspension was obtained.

[0041] The subculture solution comprises 4.41 g / L MS culture medium powder (Coolaber, PM1011), 30 g / L sucrose (Coolaber, CS10581), and a NaOH aqueous solution with the pH value adjusted to 5.8, and sterilized by high pressure at 121° C. for 15 min; when the culture medium temperature drops to 55-65° C., filter-sterilized plant hormones are added to make the final concentrations: 1.0 mg / L 6-BA, 2.0 mg / L IBA, and 1 mg / L ZT (Zeatin).

[0042] Batch screening of optimal culture medium for callus proliferation using 96-well cell culture plates combined with response surface methodology

[0043] Experimental design: For specific plant types, the key culture medium type or growth regulator type that affects the proliferation of plant callus is determined by single-factor experimental analysis or consulting reported literature. The present invention takes Korean ginseng callus as an example, and uses six culture media such as 1 / 2MS, MS, WPM, RM, KC, and VW, and three plant growth regulators (hormones) such as 6-BA, 2,4-D, and KT, and combines the response surface experimental design and analysis method to screen the optimal culture medium formula for callus proliferation. The specific experimental design is as follows: According to the response surface experimental design, a screening culture medium containing different types or different plant growth regulators is prepared, and 200 μL of different screening culture media (a total of 96 types) are added to the sterile cell culture plate. After the culture medium solidifies, 50 μL of the obtained suspension culture cells (cell weight 0.01g) are inoculated on the culture medium, 25±2°C, dark culture for 20 days, weigh the weight of Korean ginseng callus on each culture medium, and calculate the proliferation coefficient (callus proliferation coefficient = callus fresh weight after 20 days of inoculation / callus fresh weight before inoculation). The specific experimental design and results are shown in Table 1.

[0044] Table 1 96-well culture dish screening medium formula

[0045]

[0046]

[0047]

[0048]

[0049] The 1 / 2MS medium in the preparation of the 96-well callus tissue culture medium includes: 2.3g / L 1 / 2MS culture medium powder (Coolaber, PM1061), 30g / L sucrose (Coolaber, CS10581), 7.5g / L agar (Coolaber, CA1331), the pH value of the solution after mixing is 5.8, and it is sterilized by high pressure at 121°C for 20min; when the temperature of the culture medium drops to 55-65°C, the corresponding hormone is added according to the response surface experimental design.

[0050] The MS culture medium in the preparation of the 96-well callus tissue culture medium comprises: 4.41 g / L MS culture medium powder (Coolaber, PM1011), 2.3 g / L 1 / 2MS culture medium powder (Coolaber, PM1061), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). After mixing, the pH value of the solution is adjusted to 5.8, and the solution is sterilized at 121° C. for 20 minutes. When the temperature of the culture medium drops to 55-65° C., the corresponding hormone is added according to the response surface experimental design.

[0051] The WPM medium in the preparation of the 96-well callus culture medium comprises: 2.14 g / L WPM culture medium powder (Solarbio, LA6881), 0.56 g / L calcium nitrate powder (Shanghai Test, 80029018), 30 g / L sucrose (Coolaber, CS10581), 7.5 g / L agar (Coolaber, CA1331), the pH value of the solution after mixing is 5.8, and the solution is sterilized at 116° C. for 30 minutes; when the temperature of the culture medium drops to 55-65° C., the corresponding hormone is added according to the response surface experimental design.

[0052] The RM medium in the preparation of the 96-well callus culture medium contains 32.74 g / L RM medium powder (CHINOOK, CN260638), the pH value of the solution is adjusted to 5.8 after mixing, and it is sterilized at 121° C. for 20 minutes; when the temperature of the culture medium drops to 55-65° C., the corresponding hormone is added according to the response surface experimental design.

[0053] The KC medium in the preparation of the 96-well callus culture medium contains 29 g / L KC medium powder (REBIO, T214), the pH value of the solution is adjusted to 5.1±0.2 after mixing, and the solution is sterilized at 115° C. for 30 min; when the temperature of the culture medium drops to 55-65° C., the corresponding hormone is added according to the response surface experimental design.

[0054] The VW culture medium in the preparation of the 96-well callus culture medium comprises: 1.63 g / L VW culture medium powder (CHINOOK, CN260637), 30 g / L sucrose (Coolaber, CS10581), and 7.5 g / L agar (Coolaber, CA1331). After mixing, the pH value of the solution is adjusted to 5.8, and the solution is sterilized at 121° C. for 20 min. When the temperature of the culture medium drops to 55-65° C., the corresponding hormone is added according to the response surface experimental design.

[0055] Result analysis: According to the experimental results, the maximum proliferation coefficients of the six culture media were: MS (4.368) > VW (3.050) > 1 / 2MS (2.988) > WPM (2.566) > RM (2.512) > KC (2.104), indicating that MS medium is superior to the other five culture media in the proliferation culture of Korean ginseng callus. Subsequent analyses were performed based on the response surface analysis of the data from MS medium.

[0056] Design-Expert software was used to fit the data to a quadratic multiple regression equation, and the results are shown in Table 2.

[0057] Table 2 Analysis of variance of regression model

[0058]

[0059]

[0060] As shown in Table 2, F = 31.71, P = 0.0002 < 0.01, indicating that the regression model is extremely significant; F = 3.81, P = 0.1507 > 0.05, indicating that the model is reliable and there is no lack of fit factor. In summary, the model has a good fitting degree and can be used for screening the optimal culture medium for Korean ginseng callus proliferation;

[0061] The quadratic multiple regression equation is:

[0062] Y=4.23-0.1513A-04271B+0.3336C+0.8253AB-0.5888AC+0.25BC-1.31A2-0.4695B2-0.6165C2.

[0063] According to the regression equation, the effects of 6-BA, 2,4-D and KT on the proliferation of Korean ginseng callus can be analyzed by observing the shapes of the response surface graph and contour graph. The response surface graph and contour graph can effectively display the interaction relationship between independent variables, and their influence on the response value can be judged by observing the steepness of the slope of the surface graph. Figures 1 to 3The results showed that the interaction between 6-BA, 2,4-D and KT had a significant effect on the proliferation of Korean ginseng callus. The optimal medium for Korean ginseng callus proliferation was obtained by response surface optimization: MS medium + 1.72357 mg / L 6-BA + 0.72696 mg / L 2,4-D + 0.07 mg / L KT. The predicted proliferation coefficient under this culture condition was 4.40158. In order to facilitate practical operation, the medium formula was revised to: MS medium + 1.724 mg / L 6-BA + 0.727 mg / L 2,4-D + 0.07 mg / L KT. Under this culture condition, the proliferation coefficients obtained in 5 parallel experiments were: 4.414, 4.378, 4.380, 4.407, 4.386, with an average value of 4.393, and the error between the theoretical value and the theoretical value was 0.2%, which was close to the predicted value. This shows that the response surface method can be used to screen the optimal medium formula for Korean ginseng callus proliferation.

[0064] It can be seen from the above examples that the method provided by the present invention for batch screening of the optimal culture medium formula for callus proliferation using a 96-well cell culture plate combined with response surface methodology is feasible, scientific, economical and efficient.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for screening an optimal plant callus culture medium formula, characterized in that: The following steps are involved: By setting different basic culture medium types, plant hormone types and plant hormone concentrations, a variety of culture media to be screened are obtained, and each culture medium to be screened is added to different wells of a cell culture plate; Inoculating the plant callus suspension into each culture medium to be screened, culturing, and obtaining cultured plant callus; The proliferation coefficient of each culture medium to be screened is calculated by the weight of the plant callus suspension before culture and the weight of the plant callus after culture; The calculation formula of the proliferation coefficient is: Proliferation coefficient = weight of plant callus after culture / weight of plant callus suspension before culture; The type of basic culture medium was used as the selection index to compare the values ​​among the groups and obtain the culture medium with the highest proliferation coefficient to be screened. The data of the culture medium with the highest proliferation coefficient to be screened were subjected to response surface analysis to obtain the optimal plant callus culture medium formula.

2. The screening method according to claim 1, characterized in that The cell culture plate has more than 48 wells.

3. The screening method according to claim 1, characterized in that The response surface analysis includes the following steps: Fit the data with a quadratic multiple regression equation to obtain a quadratic multiple regression equation; According to the quadratic multiple regression equation, the effects of variables on callus proliferation were analyzed by observing the shapes of response surface plots and contour plots.

4. The screening method according to claim 1, characterized in that The preparation method of the plant callus suspension comprises the following steps: The plant callus tissue is mixed with water or a subculture medium, ground, and subcultured to obtain a plant callus tissue suspension.

5. The screening method according to claim 4, characterized in that The number of generations of the subculture is 2 to 5.

6. The screening method according to claim 1, characterized in that The number of types of the culture medium to be screened is equal to the number of wells in the cell culture plate.

7. The screening method according to claim 1, characterized in that The cell concentration in the plant callus suspension is 50-500 g / L.

8. The screening method according to claim 3, characterized in that The plant is Korean ginseng.

9. The screening method according to claim 8, characterized in that The basic culture medium types include 1 / 2MS culture medium, MS culture medium, WPM culture medium, RM culture medium, KC culture medium and VW culture medium; The plant growth regulators include 6-BA, 2,4-D and KT; The concentration of the plant growth regulator is a concentration point value between 0.01 and 5 mg / L.

10. The screening method according to claim 8, characterized in that The quadratic multiple regression equation is: proliferation coefficient Y=4.23-0.1513A-04271B+0.3336C+0.8253AB-0.5888AC+0.25BC-1.31A2-0.4695B2-0.6165C2.

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