Callus induction culture medium for genetic transformation, culture medium combination and genetic transformation method

Abstract

The invention belongs to the technical field of plant genetic transformation, and particularly relates to a callus induction culture medium for genetic transformation, a culture medium combination and a genetic transformation method. The culture medium composition comprises a callus induction culture medium, a recovery culture medium, a screening culture medium, a differential culture medium and a rooting culture medium, wherein each 1L of the callus induction culture medium is prepared from the following components: 5-8g of tryptone, 5-8g of yeast, 100-120mg of sodium chloride, 0.8-1g of glutamic acid, 250-300mg of monopotassium phosphate, 100-150mg of magnesium sulfate, 0.8-1mu g of biotin, 10-20g of mannitol and the balance of water. By optimizing the culture medium formula and the transformation condition, the induction efficiency, the regeneration efficiency, the transformation efficiency and the plant regeneration capacity of the wheat are improved, and the method can be widely applied to the technical field of agricultural biology, especially in research, development and production of transgenic crops, and is beneficial to rapidly cultivating new varieties with excellent characters.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic transformation, and in particular relates to a callus induction medium, a medium combination and a genetic transformation method for genetic transformation. Background Art

[0002] Plant genetic transformation technology is an important technology in modern biotechnology. It can introduce exogenous genes into plant genomes to improve crop agronomic traits or produce pharmaceutical proteins. However, existing transformation methods have problems such as low conversion rate, complex operation, and long cycle. The previous transformation methods were all patented by Japan Tobacco, and the transformation efficiency in Fielder was about 50% (Hanane Aadel, Sripada M. Udupa, Rabha Abdelwahd, et al. Agrobacterium-Mediated Genetic Transformation of Bread Wheat (Triticum aestivum L.) Using Immature Embryos. Romanian Agricultural Research. 2021; 38(0): 99-107. . ), in 2023, Ye Xingguo's team from the Crop Science Institute of the Chinese Academy of Agricultural Sciences used TaWOX5 to significantly improve the efficiency of wheat genetic transformation, and the transformation efficiency of easy-to-transform varieties such as Fielder reached 75-96% (Ke Wang, Lei Shi, Xiaona Liang, et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nature Plants. 2022; 8(2): 110-117.). Although the above varieties are easy to transform under laboratory conditions, they are not varieties that are widely promoted in production, and their transformation results lack application value and persuasiveness in actual agricultural production.

[0003] Moreover, the previous wheat embryonic callus genetic transformation methods still have the problem of low induction rate, the main reason for which is the unreasonable composition of the induction medium. Summary of the invention

[0004] The present invention provides a callus induction medium, a medium combination and a genetic transformation method for genetic transformation. By optimizing the medium formula and transformation conditions, the induction efficiency, regeneration efficiency, transformation efficiency and plant regeneration ability are significantly improved, which is of great significance to the research and application in the field of plant biotechnology.

[0005] The present invention first provides a callus induction medium for genetic transformation, wherein each 1L of the callus induction medium is made of the following components:

[0006] Trypsin 5-8g, yeast 5-8g, sodium chloride 100-120mg, glutamic acid 0.8-1g, potassium dihydrogen phosphate 250-300mg, magnesium sulfate 100-150mg, biotin 0.8-1μg, mannitol 10-20g, make up to 1L with water.

[0007] Callus often turns brown during tissue culture, which seriously affects its quality and greatly reduces the efficiency of genetic transformation. If the browning of callus can be effectively inhibited, the genetic transformation efficiency of wheat immature embryo callus can be significantly improved. Previous studies have reported that mannitol, as an osmotic pressure regulator, has the effect of inhibiting callus browning in tissue culture. In order to clarify the specific role of mannitol in wheat callus induction culture, we added different concentrations of mannitol to the basal induction medium and observed the induction rate of immature embryo callus. The results showed that the induction rate of wheat embryonic callus first increased and then decreased within the mannitol concentration range of 0-25g / L. The induction rate was higher at 10-20g / L, especially at 15g / L, the induction rate reached 73%, which can lay a foundation for improving the efficiency of wheat genetic transformation.

[0008] Secondly, the present invention provides a culture medium combination for genetic transformation, including the callus induction culture medium, and also includes a recovery culture medium, a screening culture medium, a differentiation culture medium and a rooting culture medium.

[0009] Furthermore, each 1L of the recovery medium is prepared by mixing equal volumes of the mixed solution A and agarose;

[0010] Each 1L of the mixed solution A is composed of:

[0011] MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2,4-D 2.5mg / ml 0.3-0.5ml, make up to 1L with water.

[0012] Furthermore, each 1L of the screening medium is prepared by mixing equal volumes of mixed solution B and agarose:

[0013] The composition of each 1L of mixed solution B is:

[0014] MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2.5mg / ml 2,4-D 0.3-0.5ml, l00mg / ml hygromycin 0.3-0.5ml, make up to 1L with water.

[0015] Furthermore, each 1L of the differentiation medium is prepared by mixing equal volumes of mixed solution C and agarose:

[0016] Each 1L of the mixed solution C is composed of:

[0017] MS10-12g, MES2-3g, glucose 20-22g, AS 1.8-2.2ml with a concentration of 100mM, AgNO3 silver nitrate 1.8-2.2ml with a concentration of 5mM, CuSO4·5H2O with a concentration of 5mM

[0018] 1.8-2.2ml, 6-BA with a concentration of 2mg / ml 0.3-0.5ml, TDZ thidiazuron with a concentration of 2mg / ml 0.8-1ml, kinetin 0.8-1mg, NAA 0.8-1mg, Timentin with a concentration of 160mg / ml 0.8-1ml, and make up to 1L with water.

[0019] Furthermore, each 1L of the rooting medium is prepared by mixing equal volumes of the mixed solution D and agarose:

[0020] The composition of each 1L of the mixed solution D is:

[0021] MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2.5mg / ml 2,4-D 0.3-0.5ml, 2mg / ml 6-BA 0.4ml, make up to 1L with water.

[0022] Again, the present invention provides the use of the above-mentioned culture medium combination in plant genetic transformation.

[0023] Furthermore, the plant includes wheat.

[0024] Furthermore, the wheat is Zhengmai 7698.

[0025] Finally, the present invention provides a wheat genetic transformation method, which is carried out using the above-mentioned culture medium combination, and the method comprises the following steps:

[0026] S1. Pre-cultivating and activating Agrobacterium;

[0027] S2, stripping and disinfecting the wheat grain embryos, and co-culturing with the activated Agrobacterium in a callus induction medium for 2-3 days to obtain infected embryos;

[0028] S3, excise the embryonic axis and transfer to recovery medium, with the wound surface in contact with the medium, and culture in the dark at 24-26°C for 6-7 days;

[0029] S4, transfer the material to the screening medium, with the wound surface in contact with the medium, and culture in the dark at 24-26°C for 14-15 days, then transfer to a new screening medium and culture under the same conditions for 14-15 days;

[0030] S5, replacing the screening medium with differentiation medium and transferring the cells to culture under light conditions;

[0031] When materials from S6 and S5 began to produce green clusters, they were transferred to rooting medium and cultured at 24-26°C in light conditions until root development and regeneration shoots appeared;

[0032] S7, rooting and acclimation.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Optimization of culture medium formula: Based on the culture medium reported previously, the present invention significantly improves the transformation efficiency of Zhengmai 7698 by adding a specific formula, such as mannitol and Zeatin (a cytokinin) and other nutrients. These improved culture medium formulas can better promote the transformation and regeneration of wheat embryos, and the transformation efficiency can reach 73%-81.3%. This increase in efficiency not only reduces the variation and non-target effects during the transformation process, but also speeds up the transformation process, improves the quality and quantity of transformants, and provides a solid foundation for subsequent breeding and research work.

[0035] 2. Variety selection of transformation materials: In the past, most wheat transformant materials were model wheat varieties such as Fielder. Although these varieties are easy to transform under laboratory conditions, they are not varieties that are widely promoted in production. The present invention uses Zhengmai 7698, which is a wheat variety that is planted on a large scale in production. Therefore, its transformation results have higher application value and persuasiveness in actual agricultural production. This transformation technology for actual planted varieties can better meet the actual needs of agricultural production and help accelerate the commercial application of excellent traits.

[0036] These innovations not only improve the efficiency and practicality of wheat transformation, but also provide new technical paths for wheat genetic improvement and molecular breeding. Through the application of these technologies, new varieties with excellent traits can be bred more quickly, thereby improving wheat yield and quality to meet the growing demand for food. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure is a flow chart of genetic transformation of the present invention. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0039] Explanation of symbols:

[0040] 2,4-D: 2,4-dichlorophenoxyacetic acid.

[0041] 6-BA:6-benzylaminopurine,6-benzylaminopurine.

[0042] AS: Acetosyringone, 3',5'-dimethoxy-4'-hydroxyacetophenone, acetosyringone. MS: Murashige and Skoog medium, MS medium.

[0043] MES: 2-(N-morpholino)ethanesulfonic acid. resting: restoring medium.

[0044] CIM: Callus Induction Medium, callus induction medium.

[0045] S1: Callus Selection Medium1, Callus Selection Medium.

[0046] S2: Callus Selection Medium 2, second screening medium.

[0047] CDM:Callus Differentiation Medium, differentiation medium.

[0048] CRM: Callus Rooting Medium, rooting medium.

[0049] KT:Kinetin.

[0050] NAA:1-Naphthylacetic acid, naphthylacetic acid.

[0051] OD:Optical Density, optical density.

[0052] TDZ:Thidiazuron.

[0053] ZT:Zeatin.

[0054] Agarose: agarose.

[0055] biotin: biotin.

[0056] DMSO: dimethyl sulfoxide.

[0057] WIM: resuspension buffer.

[0058] The culture medium formulas involved in the following examples are as follows:

[0059] 2,4-D (2.5mg / ml) preparation method

[0060] 125mg 2,4-D + 50ml anhydrous ethanol, filter into a 1.5ml EP tube, quickly divide into packages to prevent evaporation, and store at -20℃.

[0061] ZT (1mg / ml) preparation method:

[0062] Dissolve 50 mg zeatin + 100 μl 1M NaOH, dilute to 50 ml with ddH2O, filter into a 1.5 ml EP tube, and store at -20°C.

[0063] AgNO3 (5μM) preparation method:

[0064] 42.5mg AgNO3+50ml ddH2O, after dissolution, filter into a 1.5ml EP tube and store at -20℃.

[0065] Note: Explosive items, keep away from fire and static electricity.

[0066] Preparation method of CuSO4·5H2O (5mM):

[0067] Dissolve 125 mg CuSO4·5H2O+100 ml ddH2O, filter into a 1.5 ml EP tube, and store at -20°C.

[0068] Biotin (1mM) preparation method:

[0069] 10mg biotin + 41ml ddH2O, dissolve, filter into 1.5ml EP tube, and store at -20℃.

[0070] AS (100mM) preparation method:

[0071] Dissolve 500 mg of 3'5'-dimethoxy-4'-hydroxyacetophenone in 25 ml of DMSO, filter into a 1.5 ml EP tube, and store at -20°C.

[0072] WIM (PH = 5.8) was prepared according to the composition shown in Table 1:

[0073] Table 1 WIM composition

[0074] WIM (PH = 5.8) 100mL 1L MS 0.044g 0.44g MES 0.05g 0.5g Glucose 1g 10g

[0075] Adjust the pH to 5.8 with 1M NaOH, filter and sterilize, and store at 4°C. The above operations were completed in a clean bench.

[0076] 100×BCI Vitamin, prepared according to the composition shown in Table 2:

[0077] Table 2 100×BCI Vitamin composition

[0078] 100×BCI Vitamin 100ml 1L Thiamine HCl 10mg(0.01g) 100mg Myoinositol 3.5g 35g Proline 7g 70g

[0079] Recovery medium was prepared according to the composition shown in Table 3:

[0080] Table 3 Composition of recovery medium

[0081] Resting (PH = 5.8) 250ml 1L MS 2.5g 10g Casein hydrolysate 0.5g 2g Maltose 15g 60g 100×BCI vitamins 5ml 20ml <![CDATA[CuSO4·5H2O(5mM)]]> 0.5ml 2ml Timentin (160mg / ml) 0.5ml 2ml 2,4-D (2.5 mg / ml) 0.1ml 0.4ml

[0082] The above mixed solution was made up to 500 ml, adjusted to pH = 5.8, filtered to sterilize, mixed with 500 ml of Agarose solution and poured into a dish.

[0083] Callus induction medium (PH=7) was prepared according to the composition shown in Table 4:

[0084] Table 4 Callus induction medium composition

[0085] CIM(PH=7) 100ml 1L Tryptone 0.5g 5g Yeast 0.5g 5g NaClSodium Chloride 10mg 100mg Glutamic acid 0.1g 1g <![CDATA[Potassium dihydrogen phosphate KH2PO4]]> 25mg 250mg <![CDATA[Magnesium sulfate]]> 10mg 100mg Biotin 0.1 μg 1 μg

[0086] Adjust the pH to 7 with 1M NaOH, filter and sterilize, and store at 4°C. The above operations should be completed in a clean room as much as possible.

[0087] The first and second screening culture media are prepared according to the composition shown in Table 5:

[0088] Table 5 Callus one screen, two screen medium composition

[0089] S1, S2 (PH = 5.8) 250ml 1L MS 2.5g 10g Casein hydrolysate 0.5g 2g Maltose 15g 60g 100xBCI vitamins 5ml 20ml <![CDATA[CuSO4·5H2O(5mM) Copper sulfate]]> 0.5ml 2ml Timentin (160mg / ml) 0.5ml 2ml 2,4-D (2.5 mg / ml) 0.1ml 0.4ml chlortetracycline hygromycin (l00mg / ml) 0.1ml 0.4ml

[0090] The above mixed solution was made up to 250 ml, adjusted to pH = 5.8, filtered to sterilize, mixed with 250 ml of Agarose solution and poured into a dish.

[0091] Differentiation medium (PH = 5.8) was prepared according to the composition shown in Table 6:

[0092] Table 6 Differentiation medium composition

[0093] CDM (PH = 5.8) 250ml 1L MS 0.22g 0.88g MES 0.25g 1g Glucose 5g 20g AS(100mM) 0.5ml 2ml <![CDATA[AgNO3 Silver nitrate (5 mM)]]> 0.5ml 2ml <![CDATA[CuSO4·5H2O Copper sulfate (5 mM)]]> 0.5ml 2ml 6-BA (2mg / ml) 0.1ml 0.4ml TDZ Thidiazuron (2mg / ml) 0.1ml 0.8ml Kinetin 0.2mg 0.8mg NAA naphthyl acetate 0.2mg 0.8mg Timentin (160mg / ml) 0.25ml 1ml

[0094] The above mixed solution was made up to 250 ml, adjusted to pH = 5.8, filtered to sterilize, mixed with 250 ml of Agarose solution and poured into a dish.

[0095] Rooting medium (PH = 5.8) was prepared according to the composition shown in Table 7:

[0096] Table 7 Rooting medium composition

[0097]

[0098]

[0099] The above mixed solution was made up to 250 ml, adjusted to pH = 5.8, filtered to sterilize, mixed with 250 ml of Agarose solution and poured into a dish.

[0100] Test methods

[0101] See also Figure 1 The genetic transformation method of the present invention comprises the following steps:

[0102] Preparation one day before transformation:

[0103] 1. Bacterial culture:

[0104] In a clean bench, 800 μl of Agrobacterium bacterial solution was added to 10 ml of CIM medium (pH=7, without antibiotics).

[0105] Incubate at 28°C, 200 rpm for 16 hours.

[0106] 2. Culture medium preparation:

[0107] Prepare WIM medium and (CIM + different concentrations of mannitol) medium.

[0108] A 100 mM AS (acetosyringone) solution was prepared.

[0109] Operations on the day of conversion:

[0110] 1. Resuspend Agrobacterium:

[0111] Mix 800 μl of Agrobacterium EHA105 (containing the target gene Rht-D1b) bacterial solution with WIM and adjust the OD to 0.5.

[0112] Centrifuge at 3000 rpm for 10 minutes in a centrifuge preheated to 28°C and remove the supernatant.

[0113] Resuspend the cells in WIM to an OD value of 0.5, and add a total of 500 μl WIM in multiple times.

[0114] Add AS to a final concentration of 100 μM, protect from light, and incubate at room temperature at 80 rpm for 4-6 hours.

[0115] 2. Processing of plant materials:

[0116] A 0.05% biotanning agent (Silwet L-77) solution was prepared by adding 5 μl of Silwet L-77 to 10 ml of WIM.

[0117] The kernels were peeled from the middle of the ear of Zhengmai 7698 14 days after flowering, disinfected with 70% ethanol for 1 minute in a clean bench, rinsed once with sterile water, treated with 10% NaClO for 10 minutes, gently shaken, and rinsed 3 times with sterile water.

[0118] Under a microscope, the seed coat was torn with forceps to remove the embryos, which were placed in WIM containing 0.05% silwet L-77, with about 100 embryos in each tube. The WIM was then aspirated and fresh WIM was added.

[0119] Centrifuge at 3000 rpm for 10 minutes in a 4°C precooled centrifuge and discard the supernatant.

[0120] 3. Infection and co-cultivation:

[0121] Add 1 ml of activated Agrobacterium to each EP tube, mix by inversion for 30 seconds, let stand at room temperature for 20 minutes, centrifuge at 7000 rpm for 10 minutes in a centrifuge precooled at 4°C, discard the supernatant, and place the embryos on sterile filter paper to absorb the bacterial solution.

[0122] The young embryos were inoculated with the scutellum plane facing upward on CIM medium, with about 30 embryos placed in each dish, and cultured in the dark at 25°C for 2 days.

[0123] Example 1

[0124] CIM callus induction medium, with 0 g of mannitol added, and other ingredients were the same.

[0125] Example 2

[0126] CIM callus induction medium, with 5 g of mannitol added, and other ingredients were the same.

[0127] Example 3

[0128] CIM callus induction medium, with 10 g of mannitol added, and other ingredients were the same.

[0129] Example 4

[0130] CIM callus induction medium, with 15 g of mannitol added, and other ingredients were the same.

[0131] Example 5

[0132] CIM callus induction medium, with 20 g of mannitol added, and other ingredients were the same.

[0133] Example 6

[0134] CIM callus induction medium, with 25 g of mannitol added, and other ingredients were the same.

[0135] The embryonic callus induction rates corresponding to the above 6 concentrations of mannitol are shown in Table 8. Finally, the concentration of Example 4 was selected, and the embryonic callus induction rate reached 73% by adding 15 g of mannitol.

[0136] Table 8 Effect of mannitol on callus induction rate

[0137] method Mannitol (g / L) Embryogenic callus induction rate (%) Example 1 0 14 Example 2 5 19 Example 3 10 47 Example 4 15 73 Example 5 20 52 Example 6 25 22

[0138] Subsequent processing:

[0139] 1. Recovery:

[0140] After 2 days, the infected embryos were taken out from the incubator, and the embryo axes were cut off on CIM medium using a No. 15 surgical blade and forceps under a microscope, and transferred to resting medium with the wound surface in contact with the medium. The embryos were cultured in the dark at 25°C for 7 days.

[0141] 2. Screening:

[0142] First screening: transfer the material to S1 medium, with the wound surface in contact with the medium, and culture in the dark at 25°C for 15 days;

[0143] Second screening: Transfer the material to S2 medium and culture under the same conditions for 15 days.

[0144] 3. Regeneration:

[0145] The medium was changed to CDM and cultured under 24-hour light conditions.

[0146] Zeatin, as a cytokinin, can promote cell division. In the process of callus differentiation, cell division is the basis for the formation of different tissues and organs. It can stimulate callus cells to enter a division state, increase the number of cells, and provide sufficient cell basis for subsequent differentiation. At the same time, Zeatin has a significant promoting effect on bud differentiation, can activate the expression of genes related to the formation of bud meristems, induce callus cells to differentiate in the direction of buds, and provide necessary signals and material support for the development of buds by regulating the physiological state and metabolic process of cells. In addition, Zeatin can also interact with other plant hormones to jointly regulate the differentiation of callus. For example, it can work synergistically with auxin to adjust the ratio of roots and buds, so that the callus can differentiate into a more complete plant structure. It can also affect the synthesis and signal transduction of hormones such as ethylene, reducing the impact of adverse factors on callus differentiation.

[0147] Embodiment 7:

[0148] CDM differentiation medium, add 0ml Zeatin (1mg / ml), other components are the same.

[0149] Embodiment 8:

[0150] CDM differentiation medium, add 1 ml of Zeatin (1 mg / ml), and the other components are the same.

[0151] Embodiment 9:

[0152] CDM differentiation medium, add 2 ml Zeatin (1 mg / ml), other components are the same.

[0153] Embodiment 10:

[0154] CDM differentiation medium, add 3 ml Zeatin (1 mg / ml), other components are the same.

[0155] Embodiment 11:

[0156] CDM differentiation medium, add 4 ml Zeatin (1 mg / ml), other components are the same.

[0157] Embodiment 12:

[0158] CDM differentiation medium, add 5 ml Zeatin (1 mg / ml), other components are the same.

[0159] The embryonic callus induction rates corresponding to the above 6 concentrations of mannitol are shown in Table 9. Finally, the concentration of Example 10 was selected. When 3 ml of Zeatin (1 mg / ml) was added, that is, when the final concentration was 3 mg / L, the callus differentiation and regeneration rate could be increased to 81.3%.

[0160] Table 10 Effect of Zeatin on callus differentiation and regeneration rate

[0161] method Zeatin(mg / L) Callus differentiation and regeneration rate (%) Example 7 0 1.4 Example 8 1 9.5 Example 9 2 22.4 Example 10 3 81.3 Embodiment 11 4 57.7 Example 12 5 27.1

[0162] 4. Rooting and training:

[0163] When green clusters begin to form, they are transferred to CRM medium and cultured at 25°C in the light until the root system develops to 1-2 cm roots and the regenerated shoots continue to grow to 2-3 cm.

[0164] When the plants are relatively strong, they are transferred to resting medium for seedling cultivation. After 14 days, the roots are rinsed clean and transplanted into soil.

[0165] 5. Molecular detection and screening of mature plants:

[0166] Use conventional molecular detection techniques to conduct molecular detection of resistant individuals and screen transformed transgenic strains.

[0167] In 2022-2024, the inventor used Zhengmai 7698 grains as materials and adopted the genetic transformation method of the present invention for transformation. 120 sets of parallel experiments were conducted. After RT-PCR and sequencing detection, the transformation efficiency obtained was 73%-81.3%.

[0168] The above steps provide a systematic process of Agrobacterium-mediated plant transformation, including key steps such as preliminary preparation, treatment, cultivation, screening and plant regeneration. Each step must strictly follow the aseptic operation technique to ensure transformation efficiency and healthy plant growth.

[0169] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0170] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0171] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A callus induction medium for genetic transformation, characterized in that: Each 1L of the callus induction medium is made of the following components: Trypsin 5-8g, yeast 5-8g, sodium chloride 100-120mg, glutamic acid 0.8-1g, potassium dihydrogen phosphate 250-300mg, magnesium sulfate 100-150mg, biotin 0.8-1μg, mannitol 10-20g, make up to 1L with water.

2. A culture medium combination for genetic transformation, characterized in that: The method comprises the callus induction medium according to claim 1, and further comprises a recovery medium, a screening medium, a callus differentiation medium and a rooting medium.

3. The culture medium combination according to claim 2, characterized in that: Each 1L of the recovery medium is prepared by mixing equal volumes of mixed solution A and agarose; Each 1L of the mixed solution A is composed of: MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2,4-D 2.5mg / ml 0.3-0.5ml, make up to 1L with water.

4. The culture medium combination according to claim 3, characterized in that: Each 1L of the screening medium is prepared by mixing equal volumes of mixed solution B and agarose: The composition of each 1L of the mixed solution B is: MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2.5mg / ml 2,4-D 0.3-0.5ml, l00mg / ml hygromycin 0.3-0.5ml, make up to 1L with water.

5. The culture medium combination according to claim 4, characterized in that: Each 1L of the differentiation medium is prepared by mixing equal volumes of mixed solution C and agarose: Each 1L of the mixed solution C is composed of: MS10-12g, MES2-3g, glucose 20-22g, AS 1.8-2.2ml with a concentration of 100mM, AgNO3 silver nitrate 1.8-2.2ml with a concentration of 5mM, CuSO4·5H2O with a concentration of 5mM 1.8-2.2ml, 6-BA with a concentration of 2mg / ml 0.3-0.5ml, TDZ thidiazuron with a concentration of 2mg / ml 0.8-1ml, kinetin 0.8-1mg, NAA 0.8-1mg, Timentin with a concentration of 160mg / ml 0.8-1ml, and make up to 1L with water.

6. The culture medium combination according to claim 5, characterized in that: Each 1L of the rooting medium is prepared by mixing equal volumes of mixed solution D and agarose: The composition of each 1L of the mixed solution D is: MS10-12g, hydrolyzed casein 2-3g, maltose 55-65g, 100×BCI vitamins 20-22ml, 5mM CuSO4·5H2O 1.8-2.2ml, 160mg / ml Timentin 1.8-2.2ml, 2.5mg / ml 2,4-D 0.3-0.5ml, 2mg / ml 6-BA 0.4ml, make up to 1L with water.

7. Use of the culture medium combination according to any one of claims 2 to 6 in plant genetic transformation.

8. The use according to claim 7, characterized in that: The plants include wheat.

9. The use according to claim 8, characterized in that: The wheat mentioned is Zhengmai 7698.

10. A method for genetic transformation of wheat, characterized in that: The method is carried out using the culture medium combination according to any one of claims 2 to 6, and comprises the following steps: S1. Pre-cultivating and activating Agrobacterium; S2, stripping and disinfecting the wheat grain embryos, and co-culturing with the activated Agrobacterium in a callus induction medium for 2-3 days to obtain infected embryos; S3, excise the embryonic axis and transfer to recovery medium, with the wound surface in contact with the medium, and culture in the dark at 24-26°C for 6-7 days; S4, transfer the material to the screening medium, with the wound surface in contact with the medium, and culture in the dark at 24-26°C for 14-15 days, then transfer to a new screening medium and culture under the same conditions for 14-15 days; S5, replacing the screening medium with differentiation medium and transferring the cells to culture under light conditions; When materials from S6 and S5 began to produce green clusters, they were transferred to rooting medium and cultured at 24-26°C in light conditions until roots developed and regenerated shoots appeared; S7, rooting and acclimation.

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