Method for nicotiana benthamiana leaf callus induction and suspension cell culturing

Abstract

The present invention provides a method for Nicotiana benthamiana leaf callus induction and suspension cell culturing, belonging to the technical field of plant tissue culturing. The present invention involves inoculating a Nicotiana benthamiana leaf onto an induction culture medium to form a callus; and inoculating the callus onto a suspension culture medium, to obtain suspension cells. Compared with other methods for creating suspension cells derived from Nicotiana benthamiana, the method of the present invention has the characteristics of simple operation and low maintenance cost. The Nicotiana benthamiana suspension cells obtained by the present invention have the characteristics of good dispersibility, rapid growth, cells appearing in chains, etc., and can be used for research in plant physiology, cytology and molecular biology.

Description

Method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing callus tissue and culturing suspended cells of Nicotiana benthamiana leaves. Background Art

[0002] Nicotiana benthamiana, native to Australia, belongs to the Solanaceae family, along with peppers, tomatoes, potatoes, and cultivated tobacco. Due to its extensive applications in plant-microbe interactions, identifying protein interactions and subcellular localization, metabolic regulation, vaccine production, and synthetic biology, Nicotiana benthamiana is considered an important model plant, alongside Arabidopsis thaliana. Nicotiana benthamiana is a key research subject in plant biology, amenable to genetic manipulation. Its suspension cells are widely used in various fields of plant physiology, plant molecular biology, and plant cell biology.

[0003] Current reports on callus induction and suspension cell culture in N. benthamiana utilize a culture medium containing a combination of cell separation agents and cell growth factors. This requires the use of two or more plant hormones, as well as the addition of functional components such as inositol and casein hydrolysate. These culture media are complex to configure and are more expensive. Studies have also shown that the addition of different hormone types and different dosages of the same hormone to the culture medium significantly affects both callus induction and suspension cell culture. There are no reports on culture media for callus induction and suspension cell culture in N. benthamiana leaves that utilize a limited number of hormones and are easily configured.

[0004] Summary of the Invention

[0005] The present invention provides a method for inducing callus tissue and culturing suspension cells from Nicotiana benthamiana leaves. The Nicotiana benthamiana suspension cells obtained by the method have the characteristics of good dispersibility, rapid growth, and cell chain-like appearance, and can be used for plant physiology, cytology, and molecular biology research.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention provides a method for inducing callus tissue from Nicotiana benthamiana leaves and culturing suspension cells. The method comprises the following steps: inoculating Nicotiana benthamiana leaves into an induction culture medium for culturing to form callus tissue; and inoculating the callus tissue into a suspension culture medium for culturing to obtain suspension cells. The induction culture medium comprises the following components: MS culture medium, 20-40 g / L sucrose, 5-10 g / L agar, 0.5-1.5 mg / L 2,4-D, 0.2-0.8 mg / L KT, and 0.1-0.5 mg / L VB1. The suspension culture medium comprises the following components: MS culture medium, 20-40 g / L sucrose, 0.1-0.5 mg / L VB1, and 0.1-0.3 mg / L 2,4-D.

[0008] Preferably, the Nicotiana benthamiana leaves are leaves of sterile Nicotiana benthamiana seedlings.

[0009] Preferably, the leaves of Nicotiana benthamiana need to be cut into pieces of 0.5 to 3 cm 2 small pieces.

[0010] Preferably, the Nicotiana benthamiana leaves are inoculated into the induction culture medium at a temperature of 25 to 30° C., a relative humidity of 55 to 65%, and cultured in the dark for 28 to 32 days.

[0011] Preferably, subculture is performed after obtaining the callus tissue, with a subculture cycle of 28 to 32 days per time and 1 to 3 subcultures.

[0012] Preferably, callus tissue with loose morphology and good growth status is selected and inoculated into the suspension culture medium.

[0013] Preferably, the culture temperature in the suspension culture medium is 25-30° C., the relative humidity is 55-65%, the rotation speed is 100-120 rpm, and the culture is carried out in the dark for 15-20 days.

[0014] Preferably, after obtaining the suspension cells, subculture may be performed. The subculture cycle of the suspension cells is 15 to 20 days per time, and the subculture is performed 1 to 3 times.

[0015] Preferably, the conditions of the subculture are consistent with the initial culture conditions.

[0016] Preferably, the purpose of the subculture is seed preservation.

[0017] Preferably, the MS medium comprises the following components: macroelements: NH4NO3 1650mg / L, KNO3 1900mg / L, CaCl2·2H2O 440mg / L, MgSO4·7H2O 370mg / L, KH2PO4 170mg / L, trace elements: KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, iron salts: FeSO4·7H2O 27.8mg / L, Na2-EDTA·2H2O 37.3 mg / L, organic matter: inositol 100 mg / L, niacin 0.5 mg / L, glycine 2.0 mg / L.

[0018] Preferably, the preparation method of the induction culture medium comprises the following steps: adding 20-40 g / L sucrose and 5-10 g / L agar to MS culture medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 20-40 min, adding 0.5-1.5 mg / L 2,4-D, 0.2-0.8 mg / L KT and 0.1-0.5 mg / L VB1 when the culture medium is cooled to 45-55°C, mixing, pouring into a culture dish, and using after cooling and solidification.

[0019] Preferably, the preparation method of the suspension culture medium comprises the following steps: adding 20-40 g / L sucrose to MS culture medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 20-40 min, adding 0.1-0.5 mg / L VB1+0.1-0.3 mg / L 2,4-D when the culture medium is cooled to 45-55°C, mixing, and dispensing into conical flasks for later use.

[0020] The present invention provides the application of the suspension cells obtained by the culture method in the research of plant physiology, cytology and molecular biology.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention discloses a method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves. Compared to other methods for creating suspension cells from Nicotiana benthamiana, this method is simpler to operate and has lower maintenance costs. The suspension cells obtained using this method have a short growth cycle and a high growth rate. The resulting suspension cells can be applied to a variety of applications, such as research in physiology, cytology, biochemistry, developmental biology, genetics, and molecular biology, significantly shortening the growth time of experimental materials.

[0023] The induction culture medium of the present invention has a high induction rate for sterile leaves of Nicotiana benthamiana, and the callus tissue has good morphology and no browning phenomenon. After adding an appropriate amount of VB1 to the suspension culture medium, the cultured suspension cells have a good growth state, high stability, good cell dispersion and a chain shape, and clearly visible cell nuclei. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1 Effects of different hormone ratios on callus induction.

[0025] Figure 2. Morphology of callus obtained in a preferred induction medium.

[0026] Fig. 3 Morphology of suspension cells obtained in different culture media.

[0027] Figure 4. Suspension cells after vacuum filtration. DETAILED DESCRIPTION

[0028] The present invention provides a method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves, comprising the following steps: inoculating Nicotiana benthamiana leaves into an induction medium for culturing to form callus; inoculating the callus into a suspension medium for culturing to obtain suspension cells; wherein the induction medium comprises the following components: MS medium + 20-40 g / L sucrose + 5-10 g / L agar + 0.5-1.5 mg / L 2,4-D + 0.2-0.8 mg / L KT + 0.1-0.5 mg / L VB1; and the suspension medium comprises the following components: MS medium + 20-40 g / L sucrose + 0.1-0.5 mg / L VB1 + 0.1-0.3 mg / L 2,4-D. The Nicotiana benthamiana suspension cells obtained by the present invention are embryonic cells with high totipotency.

[0029] In the present invention, the induction medium comprises the following components: MS medium + 25-35 g / L sucrose + 6-9 g / L agar + 0.8-1.2 mg / L 2,4-D + 0.3-0.7 mg / L KT + 0.2-0.4 mg / L VB1; the suspension medium comprises the following components: MS medium + 25-35 g / L sucrose + 0.2-0.4 mg / L VB1 + 0.15-0.25 mg / L 2,4-D.

[0030] In the present invention, the preparation method of the induction culture medium includes the following steps: adding 20-40 g / L sucrose and 5-10 g / L agar to MS culture medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L potassium hydroxide, sterilizing with wet heat at 121°C and 101 MPa for 20-40 min, adding 0.5-1.5 mg / L 2,4-D, 0.2-0.8 mg / L KT and 0.1-0.5 mg / L VB1 when the culture medium is cooled to 45-55°C, mixing, pouring into a culture dish, and using after cooling and solidification.

[0031] In the present invention, the preparation method of the suspension culture medium includes the following steps: adding 20-40 g / L sucrose to MS culture medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L potassium hydroxide, sterilizing with wet heat at 121°C and 101 MPa for 20-40 min, adding 0.1-0.5 mg / L VB1+0.1-0.3 mg / L 2,4-D when the culture medium is cooled to 45-55°C, mixing, and dispensing into conical flasks for later use.

[0032] In the present invention, the MS culture medium formula is: macroelements: NH4NO3 1650mg / L, KNO3 1900mg / L, CaCl2·2H2O 440mg / L, MgSO4·7H2O 370mg / L, KH2PO4 170mg / L, trace elements: KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, iron salts: FeSO4·7H2O 27.8mg / L, Na2-EDTA·2H2O 37.3 mg / L, organic substances: inositol 100 mg / L, nicotinic acid 0.5 mg / L, glycine 2.0 mg / L. The MS culture medium of the present invention can be prepared according to conventional preparation methods.

[0033] The leaves of the tobacco of the present invention are leaves of sterile seedlings of tobacco of the present invention. The leaves of the tobacco of the present invention need to be cut into 0.5-3 cm 2 Small pieces, preferably cut into 0.8 to 1.5 m 2 small pieces.

[0034] In the present invention, the sheared Nicotiana benthamiana leaves are inoculated into an induction culture medium and placed in an incubator to induce the formation of callus tissue. The culture temperature of the Nicotiana benthamiana leaves inoculated into the induction culture medium of the present invention is 25 to 30°C, the relative humidity is 55 to 65%, and the culture is dark for 28 to 32 days. In order to prevent insufficient nutrient supply, the present invention can also perform subculture, the culture medium used for subculture remains unchanged, the subculture cycle is 28 to 32 days / time, and the subculture is 1 to 3 times. The purpose of the subculture in the present invention is to retain the well-grown Nicotiana benthamiana callus tissue

[0035] In the present invention, callus tissue with loose morphology and good growth status is selected, gently kneaded and dispersed in a clean bench, and then inoculated with 0.5-3 cm 3 The suspension culture medium is then added to a volume and placed in a shaker for culture. The culture temperature in the suspension culture medium is 25-30° C., the relative humidity is 55-65%, the rotation speed is 100-120 rpm, and the culture is carried out in the dark for 15-20 days. To prevent insufficient nutrient supply, the present invention can also perform subculture, wherein the culture medium used for subculture remains unchanged, the subculture cycle is 15-20 days / time, and the subculture is performed 1-3 times. The purpose of the subculture in the present invention is to retain well-grown Nicotiana benthamiana suspension cells.

[0036] The present invention also provides applications of suspension cells obtained by the culture method in plant physiology, cytology, and molecular biology research. The suspension cells described herein are embryonic cells with high omnipotence and can be widely used in biochemistry, developmental biology, and genetics research. They can also be directly used for protoplast isolation, culture, hybridization, gene transfer, and secondary metabolite production. They can also be used to screen for desired mutants at the cellular level in a short period of time.

[0037] In the present invention, unless otherwise specified, all raw materials and reagents are commercially available products well known to those skilled in the art.

[0038] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Culture medium preparation

[0041] The preparation method of the induction culture medium includes the following steps: adding 30 g / L sucrose and 8 g / L agar to MS culture medium, adjusting the pH to 5.9±0.1 with 1 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 30 minutes, adding 1 mg / L 2,4-D, 0.5 mg / L KT and 0.3 mg / L VB1 when the culture medium is cooled to 50°C, mixing, pouring into a culture dish, and using after cooling and solidification.

[0042] The preparation method of the suspension culture medium includes the following steps: adding 30 g / L sucrose to the MS culture medium, adjusting the pH to 5.9±0.1 with 8 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 30 minutes, adding 0.3 mg / L VB1+0.2 mg / L 2,4-D when the culture medium is cooled to 50°C, mixing, and dispensing into Erlenmeyer flasks for later use, wherein 50 mL of the suspension culture medium is added to a 250 mL Erlenmeyer flask.

[0043] 2. A method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves comprises the following steps:

[0044] (1) Cut the leaves of the sterile seedlings of Nicotiana benthamiana into 1 cm pieces on a clean bench. 2 The small pieces were inoculated into the induction medium and placed in an incubator at 27°C, relative humidity 60%, dark culture for 30 days to induce callus formation. In addition, the obtained callus was selected to form a 0.5 cm 3 , transferred to a new induction medium with the same formula for subculture, and subcultured every 30 days for seed preservation.

[0045] (2) Select callus tissue with loose morphology and good growth condition, gently knead and disperse it in a clean bench, and then inoculate 1cm 3 Transfer the volume of the suspension culture medium to a shaker and culture in the dark at 27°C, 60% relative humidity, and 110 rpm for 18 days to obtain suspension cells. Alternatively, transfer the obtained suspension cells to a new suspension culture medium of the same formulation and subculture them every 18 days for seed preservation.

[0046] Example 2

[0047] 1. Culture medium preparation

[0048] The preparation method of the induction culture medium includes the following steps: adding 35 g / L sucrose and 9 g / L agar to MS culture medium, adjusting the pH to 5.9±0.1 with 1.5 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 33 minutes, adding 1.2 mg / L 2,4-D, 0.7 mg / L KT and 0.4 mg / L VB1 when the culture medium is cooled to 50°C, mixing, pouring into a culture dish, and using after cooling and solidification.

[0049] The preparation method of the suspension culture medium includes the following steps: adding 35 g / L sucrose to the MS culture medium, adjusting the pH to 5.9±0.1 with 9 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 30 minutes, adding 0.4 mg / L VB1+0.25 mg / L 2,4-D when the culture medium is cooled to 50°C, mixing, and dispensing into Erlenmeyer flasks for later use, wherein 50 mL of the suspension culture medium is added to a 250 mL Erlenmeyer flask.

[0050] 2. A method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves comprises the following steps:

[0051] (1) Cut the leaves of the sterile seedlings of Nicotiana benthamiana into 1.5 cm pieces on a clean bench. 2 The small pieces were inoculated into the induction medium and placed in an incubator at 29°C, relative humidity 65%, dark culture for 32 days to induce callus formation. In addition, the obtained callus was selected to form a 0.5 cm 3 , transferred to a new induction medium with the same formula for subculture, and subcultured every 30 days for seed preservation.

[0052] (2) Select callus tissue with loose morphology and good growth condition, gently knead and disperse it in a clean workbench, and then inoculate 2cm 3 Transfer the volume of the suspension culture medium to a shaker and culture in the dark at 29°C, 65% relative humidity, and 110 rpm for 20 days to obtain suspension cells. Alternatively, transfer the obtained suspension cells to a new suspension culture medium of the same formulation and subculture every 18 days for seed preservation.

[0053] Example 3

[0054] 1. Culture medium preparation

[0055] The preparation method of the induction culture medium includes the following steps: adding 25 g / L sucrose and 6 g / L agar to MS culture medium, adjusting the pH to 5.9±0.1 with 0.5 mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101 MPa for 35 minutes, adding 0.5 mg / L 2,4-D, 0.2 mg / L KT and 0.1 mg / L VB1 when the culture medium is cooled to 50°C, mixing, pouring into a culture dish, and using after cooling and solidification.

[0056] The preparation method of the suspension culture medium includes the following steps: adding 25g / L sucrose to MS culture medium, adjusting the pH to 5.9±0.1 with 6mol / L potassium hydroxide, sterilizing by wet heat at 121°C and 101MPa for 35min, adding 0.1mg / L VB1+0.1mg / L 2,4-D when the culture medium is cooled to 50°C, mixing, and dispensing into Erlenmeyer flasks for later use, wherein 50mL of suspension culture medium is added to a 250mL Erlenmeyer flask.

[0057] 2. A method for inducing callus and culturing suspension cells from Nicotiana benthamiana leaves comprises the following steps:

[0058] (1) Cut the leaves of the sterile seedlings of Nicotiana benthamiana into 0.5 cm pieces on a clean bench. 2 The small pieces were inoculated into the induction medium and placed in an incubator at 25°C, 55% relative humidity, dark culture for 25 days to induce callus formation. 3 , transferred to a new induction medium with the same formula for subculture, and subcultured every 30 days for seed preservation.

[0059] (2) Select callus tissue with loose morphology and good growth condition, gently knead and disperse it in a clean workbench, and then inoculate 0.5cm 3 Transfer the volume of the suspension culture medium to a shaker and culture in the dark at 25°C, 55% relative humidity, and 110 rpm for 15 days to obtain suspension cells. Alternatively, transfer the obtained suspension cells to a new suspension culture medium of the same formulation and subculture every 18 days for seed preservation.

[0060] Comparative Example 1

[0061] The difference from Example 1 is that 0.5 mg / L 2,4-D and 0.2 mg / L KT are added to the induction medium, and no subculture step is required. The other steps are the same.

[0062] Comparative Example 2

[0063] The difference from Example 1 is that 0.5 mg / L KT and 1 mg / L NAA were added to the induction medium, and no subculture step was required. The other steps were the same.

[0064] Comparative Example 3

[0065] The difference from Example 1 is that 0.2 mg / L 6-BA and 2 mg / L NAA were added to the induction medium, and no subculture step was required. The other steps were the same.

[0066] Comparative Example 4

[0067] The difference from Example 1 is that VB1 is not added to the suspension culture medium, and no subculture step is required. The other steps are the same.

[0068] Example 4

[0069] (1) The calli growing at the leaf cuts in the induction medium in Example 1 and Comparative Examples 1-3 were observed and photographed for the induced calli grown to 30 days. The results are shown in FIG1 .

[0070] As shown in Figure 1, the callus formation process revealed that the white, loose callus growing at the leaf incision in Example 1 was in good growth condition and could be used for subsequent cell suspension processing. The results showed that the induction medium prepared with MS medium + 30 g / L sucrose + 8 g / L agar + 1 mg / L 2,4-D + 0.5 mg / L KT + 0.3 mg / L VB1 produced the best callus growth.

[0071] Calli obtained from leaves induced in the preferred medium were transferred to a new induction medium of the same formulation and subcultured twice (30 days each time) and the cell status was observed. The results are shown in Figure 2. As shown in Figure 2, the results indicate that calli with loose morphology and good growth status can be obtained in the preferred medium.

[0072] (2) Microscopic observation of the suspension cells obtained in Example 1 and Comparative Example 4 was performed, and the results are shown in Figure 3. As shown in Figure 3, the suspension cells from Nicotiana benthamiana leaves cultured in the suspension medium supplemented with VB1 exhibited good growth, good cell dispersion, and a chain-like shape, with clearly visible nuclei. However, the suspension cells cultured in the suspension medium without VB1 exhibited poor growth, with a rounded appearance and uneven dispersion. The resulting suspension cells were embryonic and highly totipotent.

[0073] (3) The suspension cells obtained after culturing for 18 days in Example 1 were vacuum filtered to remove the culture medium until the cells appeared loose and anhydrous by visual observation. The biomass was weighed on a balance, and the biomass reached 41.8 g / 100 mL after 18 days of culture. The results are shown in FIG4 .

[0074] In summary, the culture method of the present invention can induce callus tissue with good growth status and high induction rate. The obtained Nicotiana benthamiana suspension cells grow fast, and the biomass can reach 41.8g / 100mL in an 18-day culture cycle, which greatly shortens the experimental period. The suspension cells obtained by the method of the present invention are stable in morphology, have high uniformity in cell size and dispersion, can be evenly exposed to the surrounding environment, and have good experimental repeatability. At the same time, the suspension cells obtained by the present invention are embryonic cells with high totipotency and can be used for plant regeneration; they can be used for transgenic materials, have no plant structural characteristics, are easy to carry out transgenic operations, and can save time in inducing callus tissue.

[0075] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for inducing callus of Nicotiana benthamiana leaves and culturing suspension cells, characterized in that, It includes the following steps: Inoculate Nicotiana benthamiana leaves into an induction medium for culture to form callus; inoculate the callus into a suspension medium for culture to obtain suspension cells. Among them, the induction medium includes the following components: MS medium + 20 - 40 g / L sucrose + 5 - 10 g / L agar + 0.5 - 1.5 mg / L 2,4-D + 0.2 - 0.8 mg / L KT + 0.1 - 0.5 mg / L VB1. The suspension medium includes the following components: MS medium + 20 - 40 g / L sucrose + 0.1 - 0.5 mg / L VB1 + 0.1 - 0.3 mg / L 2,4-D.

2. The cultivation method according to claim 1, characterized in that, The Nicotiana benthamiana leaves are leaves of Nicotiana benthamiana sterile seedlings.

3. The culturing method according to claim 1, characterized in that The Nicotiana benthamiana leaves need to be cut into small pieces of 0.5-3 cm 2 in size.

4. The culturing method according to claim 1, wherein The culture temperature for inoculating Nicotiana benthamiana leaves into the induction medium is 25 - 30 °C, the relative humidity is 55 - 65%, and it is cultured in the dark for 28 - 32 days.

5. The culturing method according to claim 1, wherein, After obtaining the callus, subculture can be carried out. The subculture cycle is 28 - 32 days per time, and the subculture is carried out 1 - 3 times.

6. The cultivation method according to claim 1, wherein Select callus with loose morphology and good growth state and inoculate it into the suspension medium.

7. The cultivation method according to claim 1, wherein The culture temperature in the suspension medium is 25 - 30 °C, the relative humidity is 55 - 65%, the rotation speed is 100 - 120 rpm, and it is cultured in the dark for 15 - 20 days.

8. The cultivation method according to claim 1, characterized in that After obtaining the suspension cells, subculture can be carried out. The subculture cycle of the suspension cells is 15 - 20 days per time, and the subculture is carried out 1 - 3 times.

9. The cultivation method according to claim 5 or 8, characterized in that, The conditions for the subculture are the same as the initial culture conditions.

10. The culturing method according to claim 5 or 8, characterized in that, The purpose of the subculture is to preserve the strain.

11. The culturing method according to claim 1, wherein, The MS medium includes the following components: Macronutrients: NH4NO3 1650 mg / L, KNO3 1900 mg / L, CaCl2·2H2O 440 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L; Micronutrients: KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L; Iron salts: FeSO4·7H2O 27.8 mg / L, Na2-EDTA·2H2O 37.3 mg / L; Organic substances: inositol 100 mg / L, nicotinic acid 0.5 mg / L, glycine 2.0 mg / L.

12. The culture method according to claim 1, characterized in that the preparation method of the induction medium comprises the following steps: adding 20-40 g / L of sucrose and 5-10 g / L of agar to the MS medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L of potassium hydroxide, performing moist heat sterilization at 121°C and 101 MPa for 20-40 min, adding 0.5-1.5 mg / L of 2,4-D, 0.2-0.8 mg / L of KT and 0.1-0.5 mg / L of VB1 and mixing evenly when the medium cools to 45-55°C, pouring it into a petri dish, and using it after cooling and solidifying.

13. The culturing method according to claim 1, wherein The preparation method of the suspension medium comprises the following steps: adding 20-40 g / L of sucrose to the MS medium, adjusting the pH to 5.9±0.1 with 0.5-1.5 mol / L of potassium hydroxide, performing moist heat sterilization at 121°C and 101 MPa for 20-40 min, adding 0.1-0.5 mg / L of VB1 + 0.1-0.3 mg / L of 2,4-D and mixing evenly when the medium cools to 45-55°C, and subpackaging it into a triangular flask for standby.

14. Application of the suspension cells obtained by the culture method according to any one of claims 1-13 in plant physiology, cytology and molecular biology research.

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