An Agrobacterium-mediated genetic transformation method for the seed embryos of Lonicera maackii

Abstract

The present invention provides a method for genetic transformation of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens. The method includes: (1) obtaining Lonicera maackii (Rupr.) Maxim. seed embryos; (2) immersing the Lonicera maackii (Rupr.) Maxim. seed embryos in an infection solution and performing infection by vacuum infiltration to obtain the infected Lonicera maackii (Rupr.) Maxim. seed embryos; the infection solution is obtained by the following method: Agrobacterium tumefaciens is cultured in an infection culture solution, the obtained culture is centrifuged to obtain a precipitate; the precipitate is resuspended in an infection suspension to obtain the infection solution; the Agrobacterium tumefaciens contains a target gene; (3) performing a first culture on the infected Lonicera maackii (Rupr.) Maxim. seed embryos under light-shielded conditions, and then performing a second culture in a gauze to obtain Lonicera maackii (Rupr.) Maxim. containing the target gene. Thus, Lonicera maackii (Rupr.) Maxim. containing the target gene can be obtained conveniently and quickly, and the function of the target gene can be studied.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic transformation, and particularly relates to a method for genetic transformation of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens. Background Art

[0002] Lonicera maackii (Rupr.) Maxim. is a plant of the genus Lonicera in the family Caprifoliaceae. It is a common tree species in China and one of the four major ornamental tree species in landscaping. The whole plant of Lonicera maackii (Rupr.) Maxim. can be used medicinally. The leaves have antipyretic, immunity-enhancing and antibacterial effects, the roots have bactericidal and antimalarial effects, and the flowers can be used as a substitute for honeysuckle in medicine. The active ingredients of Lonicera maackii (Rupr.) Maxim. include chlorogenic acid, terpenoids, volatile oils and flavonoids, etc.

[0003] As an important life stage of higher plants, seeds are the young bodies of plants, possessing numerous physiological and biochemical processes in plants and being ideal materials for studying plant growth and development patterns. In addition, seeds are also of great significance for the study of plant phylogeny and plant domestication. So far, there is still a lack of genetic transformation methods for Lonicera maackii (Rupr.) Maxim., which limits the exploration and utilization of its genetic resources. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related technologies to some extent. The present invention has invented a method for genetic transformation using Lonicera maackii (Rupr.) Maxim. seed embryos, providing an effective tool for future research on its gene functions.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for genetic transformation of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens, comprising:

[0007] (1) Obtaining Lonicera maackii (Rupr.) Maxim. seed embryos;

[0008] (2) Immersing the Lonicera maackii (Rupr.) Maxim. seed embryos in an infection solution and performing infection by vacuum infiltration to obtain infected Lonicera maackii (Rupr.) Maxim. seed embryos;

[0009] The infection solution is obtained by the following method:

[0010] Agrobacterium tumefaciens is cultured in an infection culture solution, and the obtained culture is centrifuged to obtain a precipitate;

[0011] The precipitate is resuspended in an infection suspension to obtain the infection solution;

[0012] The Agrobacterium tumefaciens contains a target gene.

[0013] (3) Performing a first culture on the infected Lonicera maackii (Rupr.) Maxim. seed embryos under light-shielded conditions, and then performing a second culture in a gauze to obtain Lonicera maackii (Rupr.) Maxim. containing the target gene.

[0014] According to an embodiment of the present invention, the above-mentioned Agrobacterium-mediated genetic transformation method of Lonicera maackii seeds can further include the following technical features:

[0015] According to an embodiment of the present invention, step (2) further includes:

[0016] (2-1) Immerse the Lonicera maackii seeds in the infection solution, and perform the first vacuum infiltration treatment under a vacuum pressure less than -0.08 MPa and at room temperature for 30 minutes;

[0017] (2-2) Treat the Lonicera maackii seeds after the first vacuum infiltration treatment at room temperature and normal pressure for 5-10 minutes;

[0018] (2-3) Continue to perform the second vacuum infiltration treatment under a vacuum pressure less than -0.08 MPa and at room temperature for 5 minutes to obtain the infected Lonicera maackii seeds.

[0019] According to an embodiment of the present invention, the infection culture medium includes:

[0020] Yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 45-55 mg / L, rifampicin 45-55 mg / L, 1 mM 2-morpholinoethanesulfonic acid, and 0.02 mM acetosyringone. Each concentration is the working concentration.

[0021] According to an embodiment of the present invention, the infection suspension includes:

[0022] Magnesium chloride hexahydrate 2.033 g / L, 10 mM 2-morpholinoethanesulfonic acid, and 0.2 mM acetosyringone. Each concentration is the working concentration.

[0023] According to an embodiment of the present invention, the Agrobacterium is inoculated into the infection culture medium and cultured at 25-35 °C and 180-200 rpm for 15-18 hours, and then the infection culture medium is continuously added and cultured at 25-35 °C and 180-200 rpm to obtain a culture, and the OD600 of the culture is 0.5-0.8;

[0024] Centrifuge the culture at 8000-1000 rpm for 40-60 s, discard the supernatant, and obtain a precipitate;

[0025] The precipitate is resuspended in the infection suspension to obtain the infection solution.

[0026] According to an embodiment of the present invention, the Agrobacterium contains a vector, the vector contains a target gene, and the vector is a plasmid.

[0027] According to an embodiment of the present invention, the time of the first cultivation is 20 to 30 hours.

[0028] According to an embodiment of the present invention, the second cultivation is carried out under the conditions of 20 to 25 °C, with 16 hours of light and 8 hours of dark treatment, and the cultivation time is 5 to 10 days.

[0029] According to an embodiment of the present invention, the target gene is derived from at least one of plants, animals, and microorganisms. For example, it can be Lonicera maackii, sugar beet, or other plants.

[0030] According to an embodiment of the present invention, it includes a reporter gene, and the reporter gene is the Ruby gene.

[0031] The second aspect of the present invention provides a transgenic plant, which is Lonicera maackii, and the transgenic plant is obtained by using the method for genetic transformation of Lonicera maackii seed embryos mediated by Agrobacterium in the first aspect.

[0032] The beneficial effects obtained by the present invention are at least:

[0033] The present invention provides a method for genetic transformation of Lonicera maackii seed embryos mediated by Agrobacterium. Through this method, the genes of Lonicera maackii can be conveniently and quickly studied. The method provided by the present invention provides a convenient method for the gene regulation mechanism, gene function verification, protein interaction, etc. of Lonicera maackii, provides a theoretical basis for the genetic improvement and good variety breeding of Lonicera maackii, especially plays an important role in the functional research of forest tree seeds, and has high practical application value.

[0034] It has multiple advantages, including:

[0035] 1. Shorten the cycle of obtaining transgenic Lonicera maackii: There is no need to wait for the transgenic plants to mature and bear seeds to obtain stably transformed plants, overcoming the limitation of the long growth cycle of Lonicera maackii seeds in the traditional method.

[0036] 2. Simple and rapid operation: The seeds do not need to be pre-cultured, and the tissue culture step can be selectively omitted after transformation.

[0037] 3. Avoid tissue regeneration: The seed embryo has the ability of morphogenesis, providing a new way for transgenic operation of plants with difficult tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a flow chart of the transformation of Lonicera maackii seed embryos provided in Example 1 of the present invention.

[0039] Figure 2 It is a transformation result diagram of Lonicera maackii seed embryos provided in Example 1 of the present invention (scale = 1 mm, A1 - A3 are wild-type seed embryos; B1 - B3 are seed embryos transformed with the 35S::Ruby vector).

[0040] Figure 3 Electrophoretogram of PCR identification of Ruby gene expression in the seed embryo according to Embodiment 1 of the present invention (1-6 are the seed embryos showing red after infection, and 7-10 are wild-type seed embryos). Specific embodiments

[0041] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0042] Currently, there is still a lack of a genetic transformation method for Lonicera maackii, which thus limits the exploration and utilization of the genetic resources of Lonicera maackii. The present invention provides an Agrobacterium-mediated genetic transformation method for the seed embryos of Lonicera maackii, providing an effective tool for the study of the gene functions of Lonicera maackii.

[0043] To this end, the present invention provides an Agrobacterium-mediated genetic transformation method for the seed embryos of Lonicera maackii, including:[[]]

[0044] (1) Obtaining the seed embryos of Lonicera maackii;

[0045] (2) Immersing the seed embryos of Lonicera maackii in an infection solution and performing infection by the vacuum infiltration method to obtain the infected seed embryos of Lonicera maackii;

[0046] The infection solution is obtained by the following method:

[0047] Agrobacterium is cultured in an infection culture solution, and the obtained culture is centrifuged to obtain a precipitate;

[0048] The precipitate is resuspended in an infection suspension to obtain the infection solution;

[0049] The Agrobacterium contains a target gene.

[0050] (3) Performing a first culture on the infected seed embryos of Lonicera maackii under light-shielded conditions, and then performing a second culture in a gauze to obtain Lonicera maackii containing the target gene.

[0051] The mentioned seed embryos of Lonicera maackii are seed embryos with complete structure and good activity.

[0052] According to the specific embodiments, step (2) further includes:

[0053] (2-1) Immersing the seed embryos of Lonicera maackii in an infection solution and performing a first vacuum infiltration treatment under a vacuum pressure less than -0.08 MPa and at room temperature for 30 minutes;

[0054] (2-2) Treat the Lonicera maackii seed embryos after the first vacuum infiltration at normal temperature and pressure for 5 - 10 minutes;

[0055] (2-3) Continue the second vacuum infiltration under a vacuum pressure less than -0.08 MPa and at normal temperature for 5 minutes to obtain the Lonicera maackii seed embryos after infection.

[0056] The mentioned vacuum infiltration treatment is a technique that uses a vacuum environment to infiltrate liquids or gases into the interior of solid materials. Through vacuum infiltration treatment, the Agrobacterium can be better adsorbed and infiltrated into the infected solid materials through the pressure difference to improve the infection efficiency; moreover, when the pressure of the vacuum infiltration treatment is less than -0.08 MPa, the infection efficiency can be improved. Specifically in the present invention, setting two vacuum infiltration treatments can avoid the damage caused to the Lonicera maackii seed embryos by long-term vacuum treatment, and at the same time further improve the infection efficiency of the Lonicera maackii embryos. The mentioned vacuum infiltration treatment time starts counting from when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08 MPa, and the treatment under the mentioned normal temperature and pressure conditions starts counting from when the pressure shown on the vacuum gauge of the vacuum tank is the atmospheric pressure.

[0057] The mentioned infection culture medium includes: 5 g / L of yeast extract, 10 g / L of peptone, 10 g / L of sodium chloride, 45 - 55 mg / L of spectinomycin, 45 - 55 mg / L of rifampicin, 1 mM of 2-(N-morpholino)ethanesulfonic acid, and 0.02 mM of acetosyringone. The infection culture medium composed of these substances can better promote the infection effect of Agrobacterium.

[0058] According to the embodiments of the present invention, the infection suspension includes:

[0059] 2.033 g / L of magnesium chloride hexahydrate, 10 mM of 2-(N-morpholino)ethanesulfonic acid, and 0.2 mM of acetosyringone.

[0060] During the experiment, the Agrobacterium can be first inoculated into the infection culture medium and cultured at 25 - 35°C and 180 - 200 rpm for 15 - 18 hours, and then continue to add the infection culture medium and culture at 25 - 35°C and 180 - 200 rpm to obtain a culture, and the OD600 of the culture is 0.5 - 0.8 (for example, 0.6);

[0061] Centrifuge the culture at 8000 - 1000 rpm for 40 - 60 s, discard the supernatant to obtain a precipitate;

[0062] Resuspend the precipitate in the infection suspension to obtain the infection solution.

[0063] Among them, the Agrobacterium contains a vector, the vector contains a target gene, and the vector is a plasmid. Agrobacterium-mediated genetic transformation is a technique widely used in plant genetic engineering, which uses the Ti plasmid of Agrobacterium to introduce foreign genes (target genes) into plant genes. In this article, the available Agrobacterium can be Agrobacterium tumefaciens, such as strains GV3101, LBA4404 or EHA105, etc., which can carry and transfer foreign genes. Then, the target gene is first inserted into a vector suitable for Agrobacterium-mediated transformation, such as pCAMBIA1301 or other vectors suitable for plant gene transformation. These vectors usually contain necessary marker genes (such as GUS or GFP) as well as necessary promoters and terminators.

[0064] According to the specific embodiment, the time of the first culture is 20 to 30 hours.

[0065] According to the specific embodiment, the second culture is carried out under the conditions of 20 to 25 °C, with 16 hours of light and 8 hours of dark treatment, and the culture time is 5 to 10 days.

[0066] According to the specific embodiment, a reporter gene is also included, and the reporter gene is the RUBY gene. These reporter genes can be used as screening markers to help screen out successfully transformed plants, and methods such as PCR and Southern hybridization are used to verify whether the foreign gene has been successfully integrated into the plant genome. Then, the screened transgenic plants can be regenerated on an appropriate medium. After obtaining the regenerated plants, the stability and expression of the transgenic plants are further verified by molecular biology methods.

[0067] The technical solution of the present invention will be described below through specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as a limitation on the protection scope of the present invention. Unless otherwise specified, the reagents used in the examples can be obtained through commercial purchase.

[0068] Example 1

[0069] Refer to as Figure 1 shown, Example 1 provides a method for transforming the seeds of Lonicera maackii, which specifically includes:

[0070] (1) Streak the Agrobacterium strain containing the 35S::Ruby (Ruby is the reporter gene) plasmid on the LB resistance activation medium, suspend and culture the monoclonal colonies in the infection culture solution, collect the Agrobacterium, and after resuspending in the infection suspension, obtain an infection solution with an OD600 of 0.6 - 0.8.

[0071] 1) Streak the Agrobacterium strain containing the 35S::Ruby plasmid on the LB resistant activation medium and incubate it in an incubator at 28 °C for 2 - 3 days.

[0072] 2) Pick a single colony from step 1) and inoculate it into 1 mL of infection culture medium. Shake the culture at 28 °C and 180 - 200 rpm for 16 hours for activation.

[0073] 3) Add fresh infection culture medium to the infection culture medium in step 2) to make it 5 mL. Shake the culture at 28 °C and 180 - 200 rpm for 3 hours to make the OD600 of the infection culture medium approximately 0.6.

[0074] 4) Take 1 mL of the infection culture medium in step 3) into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm at room temperature for 45 seconds, discard the supernatant, add 1 mL of infection suspension to resuspend the precipitate, and let it stand for 3 hours after resuspension.

[0075] Among them, the formula of the LB resistant activation medium is: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar 5 g / L, spectinomycin 50 mg / L, rifampicin 50 mg / L.

[0076] The formula of the infection culture medium is: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 50 mg / L, rifampicin 50 mg / L, 1 mM 2 - morpholinoethanesulfonic acid, 0.02 mM acetosyringone.

[0077] The formula of the infection suspension is: magnesium chloride hexahydrate 2.033 g / L, 10 mM 2 - morpholinoethanesulfonic acid, 0.2 mM acetosyringone. The infection suspension is prepared freshly before use.

[0078] (2) Obtain the Lonicera maackii (Rupr.) Maxim. embryo through the following steps.

[0079] 1) Soak the Lonicera maackii (Rupr.) Maxim. seeds in distilled water for 24 hours.

[0080] 2) Under a stereomicroscope, use a scalpel soaked in the infection solution to carefully cut the seed coat along the hilum. Then use the back of the scalpel to squeeze the cotyledons of the seed to slightly expose the embryo, and carefully pick out the embryo with complete structure and good activity. The dissected embryo should be immediately immersed in the infection solution.

[0081] (3) Infect the Lonicera maackii (Rupr.) Maxim. embryo obtained in step (2) with the infection solution obtained in step (1) by vacuum infiltration method.

[0082] 1) Open the lid of the centrifuge tube containing the infection solution and the seed embryo soaked in the infection solution, place it in a vacuum tank, and use a vacuum pump to evacuate. Start timing when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08 MPa, and perform vacuum infiltration treatment at room temperature for 30 minutes.

[0083] 2) Slowly release the air to restore the pressure in the vacuum tank to atmospheric pressure, then open the vacuum tank and take out the centrifuge tube. Start timing when the pressure shown on the vacuum gauge of the vacuum tank is atmospheric pressure, and perform treatment at room temperature and atmospheric pressure for 5 minutes. During this period, gently shake the centrifuge tube to suspend the seed embryo.

[0084] 3) Open the lid of the centrifuge tube containing the infection solution and the seed embryo soaked in the infection solution again, place it in a vacuum tank, and use a vacuum pump to evacuate. Start timing when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08 MPa, and perform vacuum infiltration treatment at room temperature for 5 minutes.

[0085] 4) Wrap the centrifuge tube containing a small amount of resuspension solution and the seed embryo treated by vacuum infiltration with tin foil, and culture it in the dark at 28 °C for 24 hours.

[0086] (4) Continue to culture the Lonicera maackii (Rupr.) Maxim. seed embryo after infection in step (3) in a moist gauze, and use the PCR method for transformation verification.

[0087] 1) Cut the gauze into a size of 8 cm × 8 cm, place 5 layers of gauze flat in a square petri dish, and completely moisten it with sterile distilled water.

[0088] 2) Use a plastic pipette or pipettor (cut off the tip of the pipette tip) to suck and evenly transfer the seed embryo after dark culture to the moist gauze in the petri dish.

[0089] 3) Cover the petri dish and place it in an illumination incubator at 22 °C with 16 hours of light and 8 hours of darkness. After about 2 to 7 days, some seed embryos will show a red phenotype.

[0090] 4) Randomly select 6 seed embryos showing a red phenotype and 4 wild-type seed embryos, extract DNA roughly with Edwards reagent, and perform PCR identification. Bands of the Ruby gene were amplified in all 6 red seed embryos. The transformation result diagram of the Lonicera maackii (Rupr.) Maxim. seed embryo is as shown in Figure 2 shown, and the Ruby gene expression result is as shown in Figure 3 shown. The results of Example 1 show that by the method provided by the present invention, the target gene Ruby can be successfully transferred into the Lonicera maackii (Rupr.) Maxim. seed embryo, and a genetic transformation system of the Lonicera maackii (Rupr.) Maxim. seed embryo is successfully constructed.

[0091] Comparative Example 1

[0092] The difference between Comparative Example 1 and Example 1 is that in step (3), the centrifuge tube containing the infection solution and the seed embryo soaked in the infection solution was opened and placed in a vacuum chamber, and a vacuum was pumped using a vacuum pump. When the pressure shown on the vacuum gauge of the vacuum chamber was less than -0.08 MPa, timing started, and vacuum infiltration treatment was carried out at room temperature for 20 - 30 minutes. The result was found that: the seed embryos of Lonicera maackii after treatment did not show red color.

[0093] The results showed that only performing one vacuum infiltration treatment, without recovery and without performing vacuum infiltration treatment again, could not effectively transfer the target gene into the seed embryos of Lonicera maackii.

[0094] Comparative Example 2

[0095] The difference between Comparative Example 2 and Example 1 is that in step (3), the centrifuge tube containing the infection solution and the seed embryo soaked in the infection solution was opened and placed in a vacuum chamber, and a vacuum was pumped using a vacuum pump. When the pressure shown on the vacuum gauge of the vacuum chamber was less than -0.08 MPa, timing started, and vacuum infiltration treatment was carried out at room temperature for 35 minutes. The result was found that: the seed embryos of Lonicera maackii died.

[0096] The results showed that only performing one vacuum infiltration treatment, without recovery and without performing vacuum infiltration treatment again; even if the time of this vacuum infiltration treatment was extended, the target gene could not be successfully introduced into the seed embryos of Lonicera maackii, and it would also cause the death of the seed embryos of Lonicera maackii, which might be related to the relatively delicate nature of the seed embryos.

[0097] Comparative Example 3

[0098] The difference between Comparative Example 3 and Example 1 is that in step (4), the seed embryos treated by vacuum infiltration were directly cultured continuously in a moist gauze. The result was found that: the seed embryos of Lonicera maackii after treatment did not show red color. The results showed that without the first culture, only culturing in the gauze could not effectively introduce the target gene into the seed embryos of Lonicera maackii.

[0099] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An Agrobacterium-mediated genetic transformation method for the seeds of Lonicera maackii (Rupr.) Maxim., characterized in that, Comprising: (1) Obtaining the seeds embryos of Lonicera maackii (Rupr.) Maxim.; (2) Immersing the seeds embryos of Lonicera maackii (Rupr.) Maxim. into an infection solution, and performing infection by the vacuum infiltration method to obtain the infected seeds embryos of Lonicera maackii (Rupr.) Maxim.; The infection solution is obtained by the following method: Agrobacterium is cultured in an infection culture solution, and the obtained culture is centrifuged to obtain a precipitate; The precipitate is resuspended in an infection suspension to obtain the infection solution; The Agrobacterium contains a target gene; (3) Culturing the infected seeds embryos of Lonicera maackii (Rupr.) Maxim. for the first time under light avoidance conditions, and then performing the second culture in a gauze to obtain Lonicera maackii (Rupr.) Maxim. containing the target gene; Among them, step (2) further includes: (2-1) Immersing the seeds embryos of Lonicera maackii (Rupr.) Maxim. into the infection solution, and performing the first vacuum infiltration treatment under a vacuum pressure less than -0.08 MPa and normal temperature conditions for 30 minutes; (2-2) Treating the seeds embryos of Lonicera maackii (Rupr.) Maxim. after the first vacuum infiltration treatment under normal temperature and normal pressure conditions for 5 - 10 minutes; (2-3) Continuing to perform the second vacuum infiltration treatment under a vacuum pressure less than -0.08 MPa and normal temperature conditions for 5 minutes to obtain the infected seeds embryos of Lonicera maackii (Rupr.) Maxim.

2. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, wherein The infection culture solution includes: Yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 50 mg / L, rifampicin 50 mg / L, 1 mM 2-morpholinoethanesulfonic acid, and 0.02 mM acetosyringone; The infection suspension includes: Magnesium chloride hexahydrate 2.033 g / L, 10 mM 2-morpholinoethanesulfonic acid, and 0.2 mM acetosyringone.

3. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, wherein The Agrobacterium is inoculated into the infection culture solution and cultured at 25 - 35 °C and 180 - 200 rpm for 15 - 18 hours, and then the infection culture solution is continuously added and cultured at 25 - 35 °C and 180 - 200 rpm to obtain a culture, and the OD600 of the culture is 0.5 - 0.8; Centrifuging the culture at 8000 - 1000 rpm for 40 - 60 s, discarding the supernatant to obtain a precipitate; The precipitate is resuspended in the infection suspension to obtain the infection solution.

4. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The Agrobacterium contains a vector, the vector contains a target gene, and the vector is a plasmid.

5. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The time of the first culture is 20 - 30 hours.

6. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The second culture is carried out under the conditions of 20 - 25 °C, 16 hours of light, and 8 hours of dark treatment, and the culture time is 5 - 10 days.

7. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, characterized in that, The target gene comes from plants, animals or microorganisms.

8. The genetic transformation method of Lonicera maackii (Rupr.) Maxim. seed embryos mediated by Agrobacterium tumefaciens according to claim 1, characterized in that Comprising: A reporter gene, and the reporter gene is the Ruby gene.

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