Establishment method of grape genetic transformation system
Viagra seeds were treated by soaking and dark culture, and shallow culture was carried out in a bacterial environment, and Agrobacterium transformed grape plants were screened, which solved the problems of long induction time, cumbersome process and low conversion efficiency in the prior art, and achieved efficient and simple genetic transformation.
Patent Information
- Application Number
- CN202510042523.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-04-08
AI Technical Summary
The existing grape genetic transformation technology has long induction time, cumbersome process, great uncertainty, and the harm of the seed coat on the embryo, resulting in low conversion efficiency and poor consistency.
By obtaining the grape seeds with full grains for soaking treatment, and incubating them darkly on GA3-soaked gauze until cracked dew embryos were cracked. Then, in a bacterial environment, the dew embryos seeds were transferred to Agrobacterium infectious solution for shallow culture, and the Agrobacterium transformed grape plants were screened.
This method is simple to operate, avoiding the cumbersome process of cutting seeds and plant tissue culture, reducing damage to embryos, and improving the consistency and transformation efficiency of genetic transformation materials.
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Figure CN119776429A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of plant genetic engineering, and in particular to a method for establishing a grape genetic transformation system. Background Art
[0002] Grape is a fruit with high nutritional value and great economic value. With the continuous development of production, people have put forward higher requirements for grape varieties. Grape is a perennial woody plant with complex genetic background, long life cycle and highly heterozygous genome. As a result, conventional hybrid breeding is limited in grape variety improvement due to long breeding cycle and difficulty in directional improvement of grape traits.
[0003] At present, the genetic transformation of grapes is mainly carried out by introducing foreign genes into the plant genome through Agrobacterium-mediated method, pollen tube channel method, gene gun method and the latest nanoparticle-mediated method. Agrobacterium-mediated method is the most commonly used method, which has the characteristics of simple operation, high transformation efficiency and strong genetic stability. The most widely used Agrobacterium transformation method is the somatic embryogenesis system, and its main process includes the following aspects: explant embryonic callus induction and culture, embryonic callus maintenance and propagation, somatic embryo germination into seedlings, embryonic callus is a relatively stable genetic transformation material, but its induction time is long, the process is cumbersome and the uncertainty is large. Studies have found that seeds can also be used as genetic transformation materials, and seeds are easy to obtain. In the relevant technology, grape seeds are sterilized and then cut, and then pre-cultured until the abdomen shell cracks, but cutting the seed coat is more harmful to the embryo and cannot guarantee the germination rate. Summary of the invention
[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for establishing a grape genetic transformation system, which is simple to operate, avoids the tedious process of cutting seeds and plant tissue culture, avoids the damage to the embryo by cutting the seed coat, achieves the consistency of genetic transformation materials, and improves the transformation efficiency.
[0005] The present application provides a method for establishing a grape genetic transformation system, comprising:
[0006] Obtain a plurality of grape seeds with full grains, and soak each of the grape seeds in distilled water at 55° C. for 15 minutes and then leave them at room temperature for 24 hours;
[0007] Placing a plurality of soaked grape seeds on gauze in a first culture dish and culturing them in the dark for 10 to 15 days until the grape seeds crack and expose embryos, wherein the gauze is gauze soaked in 4 g / L GA3;
[0008] Prepare Agrobacterium, activate the Agrobacterium, obtain the activated Agrobacterium, and resuspend the Agrobacterium to OD 600 =0.9, and adding 100 mM acetosyringone to the 1 / 2MS liquid culture medium to obtain an Agrobacterium infection solution;
[0009] In a sterile environment, the grape seeds with cracked and exposed embryos were transferred to a second culture dish containing 20 ml of the Agrobacterium infection solution for shallow culture for 2 days;
[0010] The grape seeds that have undergone the shallow culture are cultivated to screen and obtain Agrobacterium-transformed grape plants.
[0011] According to some embodiments of the present application, the preparation of Agrobacterium includes:
[0012] Select the correctly sequenced recombinant plasmid and add it to the EHA105 Agrobacterium competent cells, mix well and transfer to the electroporation cup for biological experiments, start the cell electroporator for electroporation transformation, place it in a shaker at 28°C and shake at 180rpm for 2h, then spread it on the LB solid medium containing kanamycin and rifamycin, and culture it in an incubator at 28°C for 3d to obtain the first bacterial liquid;
[0013] Picking positive monoclonal colonies from the first bacterial solution, and shaking and culturing the positive monoclonal colonies in a shaker at 28° C. and 180 rpm for 48 hours to obtain a second bacterial solution;
[0014] 1 mL of the second bacterial solution was added to 50 mL of LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifamycin, and cultured at 28°C with shaking at 180 rpm until OD 600 =1.0, and the Agrobacterium was obtained.
[0015] According to some embodiments of the present application, after obtaining the Agrobacterium, the method comprises:
[0016] The Agrobacterium was mixed with 30% glycerol in a ratio of 1:1 and stored in a -80°C refrigerator for later use.
[0017] According to some embodiments of the present application, the method for establishing the grape genetic transformation system further comprises:
[0018] Extracting DNA from leaves of the Agrobacterium-transformed grape plants and performing PCR identification;
[0019] Obtain empty plasmid DNA and untransformed grape plants;
[0020] The empty plasmid DNA was used as a positive control, and the untransformed grape plants were used as a negative control to determine the PCR identification results.
[0021] According to some embodiments of the present application, the step of extracting DNA from leaves of the Agrobacterium-transformed grape plant and performing PCR identification comprises:
[0022] The DNA of the leaves of the Agrobacterium-transformed grape plants was extracted using the DNAsecure novel plant genomic DNA extraction kit, and the DNA of the leaves of the Agrobacterium-transformed grape plants was identified by PCR using the first primer sequence PC2300GFP-R: 5'-CCGGTGGTGCAGATGAACTTCAG-3' and the second primer sequence PC2300GFP-F: 5'-ATGGTGAGCAAGGGCGAGGAGCT-3'.
[0023] According to some embodiments of the present application, the method for establishing the grape genetic transformation system further comprises:
[0024] extracting the protein of the grape plant transformed by Agrobacterium tumefaciens, and performing protein detection on the extracted protein of the grape plant transformed by Agrobacterium tumefaciens by Western blotting to obtain a protein detection result;
[0025] According to the protein detection result, determine whether a specific protein band is contained.
[0026] The beneficial effects of the present invention are embodied in that a plurality of grape seeds with full grains are obtained, and each grape seed is placed in distilled water at 55°C for immersion for 15 minutes and then allowed to stand at room temperature for 24 hours; the plurality of grape seeds that have been immersed are placed on gauze in a first culture dish, and are cultured in the dark for 10 to 15 days until the grape seeds crack and expose embryos, and the gauze is gauze immersed in 4g / L GA3; Agrobacterium is prepared, the Agrobacterium is activated, the bacteria in the activated Agrobacterium are obtained, and the bacteria are resuspended to an OD of 0.04 in a 1 / 2MS liquid culture medium. 600 =0.9, and add 100mM acetosyringone to 1 / 2MS liquid culture medium to obtain Agrobacterium infection solution; in a bacterial environment, transfer the cracked grape seeds with exposed embryos to a second culture dish containing 20ml Agrobacterium infection solution for shallow culture for 2d; cultivate the shallow cultured grape seeds to screen and obtain Agrobacterium-transformed grape plants. The present application adopts this method, which is simple to operate, avoids the tedious process of cutting seeds and plant tissue culture, avoids the damage of cutting the seed coat to the embryo, achieves the consistency of genetic transformation materials, and improves the transformation efficiency.
[0027] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
[0029] Figure 1 A schematic diagram of the genetic transformation process of embryos of immature zygotes of multiple grape seeds with full grains provided in the embodiments of the present application;
[0030] Figure 2 A schematic diagram of the PCR detection results of transgenic Chardonnay grape seeds provided in the examples of the present application;
[0031] Figure 3 A schematic diagram of the protein immunoblotting detection results of transgenic Chardonnay grape seeds provided in the examples of the present application;
[0032] Figure 4 A schematic diagram of the PCR detection results of transgenic Cabernet Sauvignon grape seeds provided in the examples of the present application;
[0033] Figure 5 This is a schematic diagram of the protein immunoblotting detection results of transgenic Cabernet Sauvignon grape seeds provided in the examples of the present application. DETAILED DESCRIPTION
[0034] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.
[0035] In the description of the present application, it should be understood that descriptions involving orientation, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.
[0036] In the description of this application, if there is a description of first or second, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.
[0037] In the description of this application, unless otherwise clearly defined, terms such as setting, installing, connecting, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.
[0038] Grape is a fruit with high nutritional value and great economic value. With the continuous development of production, people have put forward higher requirements for grape varieties. Grape is a perennial woody plant with complex genetic background, long life cycle and highly heterozygous genome. As a result, conventional hybrid breeding is limited in grape variety improvement due to long breeding cycle and difficulty in directional improvement of grape traits.
[0039] At present, the genetic transformation of grapes is mainly carried out by introducing foreign genes into the plant genome through Agrobacterium-mediated method, pollen tube channel method, gene gun method and the latest nanoparticle-mediated method. Agrobacterium-mediated method is the most commonly used method, which has the characteristics of simple operation, high transformation efficiency and strong genetic stability. The most widely used Agrobacterium transformation method is the somatic embryogenesis system, and its main process includes the following aspects: explant embryonic callus induction and culture, embryonic callus maintenance and propagation, somatic embryo germination into seedlings, embryonic callus is a relatively stable genetic transformation material, but its induction time is long, the process is cumbersome and the uncertainty is large. Studies have found that seeds can also be used as genetic transformation materials, and seeds are easy to obtain. In the relevant technology, grape seeds are sterilized and then cut, and then pre-cultured until the abdomen shell cracks, but cutting the seed coat is more harmful to the embryo and cannot guarantee the germination rate.
[0040] In order to solve the above problems, the present application proposes a method for establishing a grape genetic transformation system. The embodiments of the present application are further described below in conjunction with the accompanying drawings.
[0041] Embodiment 1:
[0042] Reference Figure 1 A method for establishing a grape genetic transformation system comprises the following steps:
[0043] Step S100, obtaining a plurality of grape seeds with full grains, and soaking each grape seed in 55° C. distilled water for 15 minutes and then leaving it at room temperature for 24 hours.
[0044] In this step, the grape seeds are Chardonnay seeds.
[0045] Step S200, placing the soaked grape seeds on the gauze in the first culture dish, and culturing them in the dark for 10 to 15 days until the grape seeds crack and expose their embryos.
[0046] In this step, the gauze is gauze soaked in 4 g / L GA3. Specifically, the soaked seeds are placed on a 90 cm first culture dish of gauze soaked in 4 g / L GA3 and cultured in the dark for 10-15 days until the seeds crack and the embryos are exposed.
[0047] It should be noted that GA3, or gibberellin, is a plant hormone that is often used to promote seed germination, drought resistance, disease resistance, etc.; the mechanism by which GA3 promotes seed germination is to activate various growth substances in the seeds, increase their content, and shorten the seed germination cycle.
[0048] Step S300, prepare Agrobacterium, activate Agrobacterium, obtain the activated Agrobacterium, and resuspend the Agrobacterium in 1 / 2MS liquid culture medium to an OD of 600 =0.9, and add 100mM acetosyringone into 1 / 2MS liquid culture medium to obtain Agrobacterium infection solution.
[0049] Step S400: In a sterile environment, the grape seeds with cracked and exposed embryos are transferred to a second culture dish containing 20 ml of Agrobacterium infection solution for shallow culture for 2 days.
[0050] In this step, in a sterile environment laboratory (i.e., not operating in a clean bench), the Chardonnay grape seeds with cracked and exposed embryos were clamped into the infection solution, and the Chardonnay grape seeds with cracked and exposed embryos were placed on a second 90 cm culture dish containing 20 ml of infection solution and cultured shallowly for 2 days. At the same time, seeds that were not infected with Agrobacterium were set as negative controls.
[0051] Step S500, cultivating the shallow cultured grape seeds and screening to obtain Agrobacterium-transformed grape plants.
[0052] In this step, under bacterial conditions, the shallow cultured grape seeds were cultured on the 20th day in a 20 ml Agrobacterium infection solution (OD 600 =0.9) in a 90 cm second culture dish, the germination rate after 2 days of shallow culture reached 77.78% (as shown in Tables 1 and 2). On the 35th day, 14 well-growing and robust Agrobacterium-transformed grape plants were selected for identification of transgenic grape plants.
[0053] For example, the following Table 1 shows the effects of the infection time and concentration of the Agrobacterium infection solution on the genetic transformation of Chardonnay seeds with cracked and exposed embryos, and Table 2 shows the effects of the infection time and volume of the Agrobacterium infection solution d on the genetic transformation of Chardonnay seeds with cracked and exposed embryos. Table 1 is as follows:
[0054]
[0055]
[0056] Table 2 is as follows:
[0057]
[0058] It should be noted that the method for establishing the grape genetic transformation system set forth in the present application is simple to operate and avoids the tedious process of cutting seeds and plant tissue culture; it has high repeatability and universality, a unified, simple, and easily repeatable method for processing materials, and a high transformation efficiency; more than 90% of the seedlings after screening are transgenic plants.
[0059] In the present application, a plurality of grape seeds with full grains are obtained, and each grape seed is immersed in 55° C. distilled water for 15 minutes and then left to stand at room temperature for 24 hours; the immersed grape seeds are placed on gauze in a first culture dish, and cultured in the dark for 10 to 15 days until the grape seeds crack and expose embryos, and the gauze is gauze immersed in 4 g / L GA3; Agrobacterium is prepared, the Agrobacterium is activated, the activated Agrobacterium cells are obtained, and the cells are resuspended to OD 0. 600 =0.9, and add 100mM acetosyringone to 1 / 2MS liquid culture medium to obtain Agrobacterium infection solution; in a bacterial environment, transfer the cracked grape seeds with exposed embryos to a second culture dish containing 20ml Agrobacterium infection solution for shallow culture for 2d; cultivate the shallow cultured grape seeds to screen and obtain Agrobacterium-transformed grape plants. The present application adopts this method, which is simple to operate, avoids the tedious process of cutting seeds and plant tissue culture, avoids the damage of cutting the seed coat to the embryo, achieves the consistency of genetic transformation materials, and improves the transformation efficiency.
[0060] It is understood that the preparation of Agrobacterium in step S300 includes but is not limited to the following steps:
[0061] Step S310, select the correctly sequenced recombinant plasmid and add it to the EHA105 Agrobacterium competent cells, mix well and transfer to the electroporation cup for biological experiments, start the cell electroporator for electroporation transformation, place it in a shaker at 28°C and shake at 180rpm for 2h, then spread it on the LB solid culture medium containing kanamycin and rifamycin, and culture it in an incubator at 28°C for 3d to obtain the first bacterial liquid.
[0062] In this step, the cell electroporator is an Eppendorf 4308 electroporator. The Eppendorf 4308 electroporator is started for electroporation, and the transformation parameters are set as follows: 1.8 KV, 5 ms.
[0063] It should be noted that kanamycin (Kanamycin, abbreviated as Kan) and rifamycin (RifamycinS, abbreviated as Rif) are used to screen the transformed plants.
[0064] Step S320, picking positive monoclonal colonies from the first bacterial solution, and culturing the positive monoclonal colonies in a shaker at 28° C. and 180 rpm for 48 hours to obtain a second bacterial solution.
[0065] Step S330: 1 mL of the second bacterial solution was added to 50 mL of LB liquid culture medium containing 50 mg / L kanamycin and 25 mg / L rifamycin, and cultured at 28°C with shaking at 180 rpm until OD 600 =1.0, and Agrobacterium is obtained.
[0066] In this step, the LB liquid medium is: 10 g / L sodium chloride + 10 g / L tryptone + 5 g / L yeast extract; the LB solid medium is: 10 g / L sodium chloride + 10 g / L tryptone + 5 g / L yeast extract + 15 g / L agar.
[0068] In some embodiments, 1 mL of the second bacterial solution was added to 50 mL of YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L rifamycin, and cultured at 28°C with shaking at 180 rpm until OD 600 =1.0, Agrobacterium is obtained, but it is not limited to the embodiment of the present application; YEP is the full name of Yeast Extract Peptone, which is a standard liquid culture medium.
[0069] It is understood that after obtaining Agrobacterium in step S330, the following steps are included but not limited to:
[0070] Step S331, mix Agrobacterium and 30% glycerol in a ratio of 1:1 and store in a -80°C refrigerator for later use.
[0071] It is understood that the method for establishing the grape genetic transformation system also includes but is not limited to the following steps:
[0072] Step S600, extracting DNA from leaves of grape plants transformed with Agrobacterium and performing PCR identification;
[0073] Step S700, obtaining empty plasmid DNA and untransformed grape plants;
[0074] Step S800, using the empty plasmid DNA as a positive control and the untransformed grape plant as a negative control to determine the PCR identification result.
[0075] It should be noted that PCR testing, or polymerase chain reaction testing, is a molecular biology technique based on the natural mechanism of double-stranded DNA replication. Through a series of carefully designed reaction steps, it can amplify trace amounts of DNA fragments to sufficient quantities in a short period of time for subsequent research and testing.
[0076] Exemplarily, PCR identification is performed on DNA from leaves of Agrobacterium-transformed grape plants, PC2300 empty plasmid DNA is used as a positive control, and untransformed Chardonnay plants are used as a negative control, such as Figure 2 As shown, the test results showed that all 14 Chardonnay plants had the target band, which indicated that the PC2300 empty vector had been successfully inserted into the grape genome.
[0077] It is understood that after obtaining Agrobacterium in step S600, the following steps are included but not limited to:
[0078] Step S610, using the DNAsecure new plant genomic DNA extraction kit to extract DNA from leaves of grape plants transformed with Agrobacterium, and using the first primer sequence PC2300GFP-R:5'
[0079] PCR identification of DNA from leaves of Agrobacterium-transformed grape plants was performed using the primer sequence PC2300GFP-F: 5′-ATGGTGAGCAAGGGCGAGGAGCT-3′.
[0080] In this step, DNA from grape plant leaves was extracted using DNAsecure Plant Kit (TIANGEN) as a template, and PC2300GFP-R:5'
[0081] -CCGGTGGTGCAGATGAACTTCAG-3' and PC2300GFP-F:5'
[0082] -ATGGTGAGCAAGGGCGAGGAGCT-3' was used for PCR identification, PC2300 empty plasmid DNA was used as a positive control, and untransformed 'Chardonnay' plants were used as a negative control. Figure 2 As shown, the test results showed that all 14 grape strains had the target band, which indicated that the PC2300 empty vector had been successfully inserted into the Chardonnay genome and screened using solid-liquid co-culture.
[0083] It is understood that the method for establishing the grape genetic transformation system also includes but is not limited to the following steps:
[0084] Step S910, extracting proteins from grape plants transformed by Agrobacterium, and performing protein detection on the extracted proteins from grape plants transformed by Agrobacterium through a Western blotting experiment to obtain protein detection results;
[0085] Step S920: Determine whether a specific protein band is contained according to the protein detection result.
[0086] For example, Figure 3 As shown, in order to further identify the transgenic plants, 8 of the 14 Chardonnay seedlings were selected, their proteins were extracted and tested by Western blotting, and specific protein bands were successfully detected.
[0087] It should be noted that based on the above results, it is preliminarily judged that under non-sterile conditions, the transformation of Chardonnay seeds as the recipient material was carried out in a 20 ml Agrobacterium infection solution (OD 600 =0.9) in a 90 cm second culture dish for shallow culture for 2 days, and the conversion rate can reach 50.00%.
[0088] In some embodiments, the grape seeds are Cabernet Sauvignon seeds, and the specific variety of grapes is not limited to the embodiments of the present application.
[0089] For example, refer to Figures 4 to 5 The same transformation method as above was used. Cabernet Sauvignon seeds were selected as grape seeds. Agrobacterium EHA105 was used as the bioengineering strain. The plant expression vector PC2300 for screening the reporter gene GFP was used as an empty vector. The target vector was successfully transferred into the 'Cabernet Sauvignon' grape plant. On the 35th day after transformation, protein and genomic DNA were extracted from the grape plant, and detected by Western blotting and PCR amplification experiments, and specific protein bands and PCR bands were successfully detected.
[0090] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
[0091] The above is a preferred embodiment of the present application. 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 application. These improvements and modifications are also considered to be within the scope of protection of the present application.
[0092] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.
Claims
1. A method for establishing a grape genetic transformation system, characterized in that: include: Obtain a plurality of grape seeds with full grains, and soak each of the grape seeds in distilled water at 55° C. for 15 minutes and then leave them at room temperature for 24 hours; Placing a plurality of soaked grape seeds on gauze in a first culture dish and culturing them in the dark for 10 to 15 days until the grape seeds crack and expose embryos, wherein the gauze is gauze soaked in 4 g / L GA3; Prepare Agrobacterium, activate the Agrobacterium, obtain the activated Agrobacterium, and resuspend the Agrobacterium to OD 600 =0.9, and adding 100 mM acetosyringone to the 1 / 2MS liquid culture medium to obtain an Agrobacterium infection solution; In a sterile environment, the grape seeds with cracked and exposed embryos were transferred to a second culture dish containing 20 ml of the Agrobacterium infection solution for shallow culture for 2 days; The grape seeds that have undergone the shallow culture are cultivated to screen and obtain Agrobacterium-transformed grape plants.
2. The method for establishing a grape genetic transformation system according to claim 1, characterized in that: In the preparation of Agrobacterium, the method comprises: Select the correctly sequenced recombinant plasmid and add it to the EHA105 Agrobacterium competent cells, mix well and transfer to the electroporation cup for biological experiments, start the cell electroporator for electroporation transformation, place it in a shaker at 28°C and shake at 180rpm for 2h, then spread it on the LB solid medium containing kanamycin and rifamycin, and culture it in an incubator at 28°C for 3d to obtain the first bacterial liquid; Picking positive monoclonal colonies from the first bacterial solution, and shaking and culturing the positive monoclonal colonies in a shaker at 28° C. and 180 rpm for 48 hours to obtain a second bacterial solution; 1 mL of the second bacterial solution was added to 50 mL of LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifamycin, and cultured at 28°C with shaking at 180 rpm until OD 600 =1.0, and the Agrobacterium was obtained.
3. The method for establishing a grape genetic transformation system according to claim 2, characterized in that: After obtaining the Agrobacterium, the method comprises: The Agrobacterium was mixed with 30% glycerol in a ratio of 1:1 and stored in a -80°C refrigerator for later use.
4. The method for establishing a grape genetic transformation system according to claim 1, characterized in that: The method for establishing the grape genetic transformation system also includes: Extracting DNA from leaves of the Agrobacterium-transformed grape plants and performing PCR identification; Obtain empty plasmid DNA and untransformed grape plants; The empty plasmid DNA was used as a positive control, and the untransformed grape plants were used as a negative control to determine the PCR identification results.
5. The method for establishing a grape genetic transformation system according to claim 4, characterized in that: The step of extracting DNA from leaves of the Agrobacterium-transformed grape plant and performing PCR identification comprises: The DNA of the leaves of the Agrobacterium-transformed grape plants was extracted using the DNAsecure novel plant genomic DNA extraction kit, and the DNA of the leaves of the Agrobacterium-transformed grape plants was identified by PCR using the first primer sequence PC2300GFP-R: 5'-CCGGTGGTGCAGATGAACTTCAG-3' and the second primer sequence PC2300GFP-F: 5'-ATGGTGAGCAAGGGCGAGGAGCT-3'.
6. The method for establishing a grape genetic transformation system according to claim 4, characterized in that: The method for establishing the grape genetic transformation system also includes: extracting the protein of the grape plant transformed by Agrobacterium tumefaciens, and performing protein detection on the extracted protein of the grape plant transformed by Agrobacterium tumefaciens by Western blotting to obtain a protein detection result; According to the protein detection result, determine whether a specific protein band is contained.