Genetic transformation method of wild soybean

Abstract

The invention belongs to the technical field of plant biology, and discloses a wild soybean genetic transformation method which comprises the following steps: S1, disinfecting wild soybean seeds; s2, the backs of the disinfected wild soybean seeds are cut once, the cut wild soybean seeds are placed on a culture medium, and imbibition seeds are obtained; s3, seed coats of the wild soybean seeds are removed, growing points of embryo tips are exposed through dissection, and explants are obtained; s4, putting the explant into an agrobacterium infection solution containing target plasmids, and carrying out vacuum infiltration in a vacuum environment; and S5, after releasing vacuum, standing and infecting in an agrobacterium infection solution for 3-16 hours. The glycine soja genetic transformation method can effectively improve the transformation efficiency of glycine soja.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for genetic transformation of wild soybean. Background Technology

[0002] Wild soybean (Glycine soja) is an annual twining herbaceous plant belonging to the genus Glycine in the family Leguminosae. It is a close ancestor of cultivated soybean (Glycine max). It mainly originated in China and is distributed throughout East Asia. It is a Class II protected wild plant in my country.

[0003] Wild soybean has significant economic and ecological value. Although cultivated soybeans are domesticated from wild soybeans, compared to cultivated soybeans, wild soybeans exhibit superior resistance to adverse conditions (such as resistance to diseases and pests, drought tolerance, and salt tolerance). Their seeds contain higher levels of nutrients and medicinal active substances (such as protein, fat, oleic acid, soy isoflavones, soy saponins, soy oligosaccharides, and soy phospholipids). Furthermore, wild soybeans possess high genetic diversity, making them a valuable germplasm resource and gene pool for crop genetic improvement and germplasm innovation. In addition, wild soybeans are an important nectar source plant, contributing to the stability of the food web, and their well-developed root system and root nodule system further aid in soil and water conservation and ecological restoration. my country has abundant wild soybean resources; however, due to ecosystem destruction and human interference, the natural population of wild soybeans is declining significantly, making the protection of wild soybean germplasm resources an important task.

[0004] Currently, the development, utilization, and protection of wild soybean resources are still in a relatively preliminary stage, largely due to a lack of analysis and research at the molecular and genetic levels. Genetic transformation technology is essential for gene function discovery, molecular mechanism analysis, and germplasm creation. To date, the establishment of genetic transformation systems for wild soybean is challenging, significantly limiting research, possibly due to its strong resistance, small seed size (generally 1.5-2 mm wide and 2-3 mm long), dense seed coat, and high seed protein content. Therefore, there is an urgent need to develop a genetic transformation technology suitable for wild soybean to provide technical support for the development and utilization of functional genes, the elucidation of important molecular mechanisms, and molecular breeding, thereby promoting the scientific research and application of wild soybean and other crops. Summary of the Invention

[0005] The purpose of this invention is to provide a genetic transformation method for wild soybean with high transformation efficiency.

[0006] The following technical solutions are used to achieve the above objectives.

[0007] This invention provides a method for genetic transformation of wild soybean, comprising the following steps:

[0008] S1. Disinfect wild soybean seeds;

[0009] S2. Make a cut on the back of the sterilized wild soybean seed, place the cut wild soybean seed on the culture medium, and obtain the seed after it has absorbed water.

[0010] S3. Remove the seed coat of wild soybean seeds and expose the growth point of the embryo tip through dissection to obtain explants;

[0011] S4. Place the explants in an Agrobacterium infection solution containing the target plasmid and perform vacuum permeation in a vacuum environment;

[0012] S5. After releasing the vacuum, let it stand in the Agrobacterium infection solution for 3h~16h for infection.

[0013] In some embodiments, the infection solution comprises a basal culture medium, dithiothreitol, zeatin, acetylsylgenone, and a penetration enhancer; the final concentration of dithiothreitol is 100 mg / L to 200 mg / L, the final concentration of zeatin is 1 mg / L to 10 mg / L, the final concentration of acetylsylgenone is 50 mg / L to 70 mg / L, and the final concentration of the penetration enhancer is 0 to 1% v / v.

[0014] In some embodiments, the final concentration of the dithiothreitol is 130 mg / L to 170 mg / L, the final concentration of the zeatin is 1 mg / L to 5 mg / L, the final concentration of the acetylsylgenone is 55 mg / L to 65 mg / L, and the final concentration of the penetration enhancer is 0.01% to 0.5% v / v.

[0015] In some embodiments, the final concentration of the dithiothreitol is 148 mg / L to 152 mg / L, the final concentration of the zeatin is 1.5 mg / L to 2.5 mg / L, the final concentration of the acetylsylgenone is 58 mg / L to 62 mg / L, and the final concentration of the penetration enhancer is 0.01% to 0.05% v / v.

[0016] In some embodiments, the basal culture medium is a 1 / 2 MS solution;

[0017] And / or, the penetration enhancer is Silwet l-77.

[0018] In some embodiments, in step S2, the culture medium is 1 / 2 MS medium or MS medium, preferably 1 / 2 MS medium.

[0019] In some embodiments, in step S2, the wild soybean seeds are placed in the culture medium for 12 to 16 hours.

[0020] In some embodiments, the vacuum degree of the vacuum permeation is -0.1MPa to -0.05MPa, and the permeation time is 2 min to 10 min.

[0021] In some embodiments, the vacuum level is -0.1 MPa to -0.08 MPa, and the permeation time is 2 min to 4 min.

[0022] In some embodiments, the time for static infection treatment in step S5 is 3h~5h or 10h~16h, more preferably 14h~16h.

[0023] In some embodiments, the disinfectant used to disinfect wild soybean seeds is chlorine gas, and the disinfection time is 12h~16h.

[0024] And / or, in step S5, after the Agrobacterium infection solution has been left to stand for 3 to 16 hours, the following steps are also included:

[0025] The infected explants were sequentially co-cultured, restored, budded, screened, and rooted to obtain transgenic wild soybean lines.

[0026] Preferably, the co-culture is carried out for 2 to 5 days in a culture medium composed of MS solid medium or 1 / 2 MS solid medium supplemented with 140 mg / L to 160 mg / L dithiothreitol, 1 mg / L to 3 mg / L zeatin, and 30 mg / L to 50 mg / L acetylsyringone; and / or,

[0027] The recovery culture involves culturing the explants in a recovery medium until they turn green. The recovery medium consists of 25 mg / L to 35 mg / L FeSO4, 35 mg / L to 45 mg / L EDTA-2Na, 0.5 mg / L to 1.5 mg / L 6-BA, 140 mg / L to 160 mg / L cefotaxime sodium, 340 mg / L to 360 mg / L termethin, 2.5 g / L to 3.5 g / L B5, 25 g / L to 35 g / L sucrose, and 5 g / L to 10 g / L agar.

[0028] And / or, the bud selection includes the following steps:

[0029] The explants after recovery culture were placed in bud selection medium 1 and cultured until buds appeared. Then the buds were cut off and placed in bud selection medium 2 and cultured until the seedlings grew to about 0.5cm~2cm.

[0030] The bud selection medium 1 consists of 2.5 g / L ~ 3.5 g / L B5, 25 g / L ~ 35 g / L sucrose, 5 g / L ~ 10 g / L agar, 25 mg / L ~ 35 mg / L FeSO4, 35 mg / L ~ 45 mg / L EDTA-2Na, 1 mg / L ~ 5 mg / L 6-BA, 45 mg / L ~ 55 mg / L glutamine, 45 mg / L ~ 55 mg / L asparagine, 140 mg / L ~ 160 mg / L Cef, 340 mg / L ~ 360 mg / L Tim, and 4 mg / L ~ 8 mg / L selection agent.

[0031] The bud selection medium 2 consists of 2.5 g / L ~ 3.5 g / L B5, 25 g / L ~ 35 g / L sucrose, 5 g / L ~ 10 g / L agar, 25 mg / L ~ 35 mg / L FeSO4, 35 mg / L ~ 45 mg / L EDTA-2Na, 0.08 mg / L ~ 0.15 mg / L LIAAA, 0.5 mg / L ~ 1.5 mg / L Zeatin, 0.1 ~ 1 mg / L gibberellin, 140 mg / L ~ 160 mg / L Cef, 340 mg / L ~ 360 mg / L mg / L LTiMo, 45 mg / L ~ 55 mg / L LCln, 45 mg / L ~ 55 mg / L Aln, 90 mg / L ~ 110 mg / L pyroglutamic acid, and 2 mg / L ~ 8 mg / L selection agent.

[0032] And / or, the rooting propagation involves culturing seedlings in a rooting medium, which is composed of a solid medium supplemented with 90 mg / L~110 mg / L Cef, 90 mg / L~110 mg / L Tim, and 0.5 mg / L~1.5 mg / L 3-indolebutyric acid. Preferably, the solid medium is B5 solid medium or MS solid medium.

[0033] In some embodiments, the screening agent includes, but is not limited to, glufosinate, kanamycin, hygromycin B, glyphosate, etc., and in this embodiment, glufosinate is preferred.

[0034] In this invention, we discovered that the characteristics of wild soybean seeds, such as strong resistance, small seed size, dense seed coat, and high seed protein content, make it difficult to establish a genetic transformation system. Furthermore, various transformation methods have failed to improve the genetic transformation efficiency of wild soybean. Ultimately, we developed a genetic transformation method that effectively adapts to the specific structure and physiological characteristics of wild soybean seeds. This method, through a series of organically combined operations, including using appropriate inoculum and inoculum time on pretreated wild soybean seeds, and vacuum permeation pretreatment, effectively overcomes the adverse effects of the structure and physiological characteristics of wild soybean seeds on the transformation process, thereby significantly improving the transformation efficiency of wild soybean. Attached Figure Description

[0035] Figure 1 These are morphological diagrams of soybean seeds and wild soybean seeds.

[0036] Figure 2 These are morphological images of soybean seeds and wild soybean seeds before and after soaking in a culture medium; among them, Figure 2 (a) Cultivated soybeans placed on 1 / 2 MS medium for 0 h. Figure 2 (b) Cultivated soybeans placed on 1 / 2 MS medium for 16 h. Figure 2 (c) Wild soybeans from group A that were placed on 1 / 2 MS medium for 0 h. Figure 2 (d) shows the state of Group A wild soybeans after 16 hours on 1 / 2 MS medium.

[0037] Figure 3 This is a diagram illustrating the genetic transformation process of group B wild soybean; among which, Figure 3 (a) shows the state of group B wild soybeans after 16 hours on 1 / 2 MS medium. Figure 3 (b) shows the morphology of group B wild soybean after the seed coat has been removed, and the morphology of the exposed growth point of the embryo tip. Figure 3 (c) shows the explant status after co-culturing with wild soybean in group B. Figure 3 (d) shows the growth of germinated explants obliquely inserted into the recovery medium; Figure 3 (e) shows the growth of shoots that emerge from explants after being transferred to shoot selection medium 1. Figure 3 (f) Growth of explant buds in bud selection medium 2, Figure (3g) shows the rooting of resistant seedlings after being transplanted into rooting medium, and Figure (3h) shows the growth of transgenic seedlings after being transplanted into soil and growing to pod formation.

[0038] Figure 4 This is a diagram showing the PCR results of the transformant leaves. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0040] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0042] Unless otherwise specified, the culture medium in the embodiments of the present invention is prepared using conventional techniques known to those skilled in the art.

[0043] In the genetic transformation method of wild soybean of the present invention, a series of operations are carried out, including first culturing with 1 / 2MS medium to replace water, and pretreatment by making a cut on the back of the seed to allow the wild soybean seed to absorb water and soften the seed coat. Then, the appropriate concentration of the infection solution, vacuum treatment before infection and infection time are organically combined to overcome the adverse effects of the structure and physiological characteristics of wild soybean seed on the transformation process and effectively improve the transformation efficiency of wild soybean.

[0044] Specifically, due to the characteristics of wild soybean seeds, such as strong resistance, small seed size, dense seed coat, and high seed protein content, experiments during pretreatment revealed that conventional water soaking to induce seed imbibition could lead to easy embryo rot or affect explant acquisition and subsequent transfection. Conversely, directly placing intact wild soybean seeds in the culture medium prevented imbibition, leaving the seed coat hard and difficult to peel, hindering explant acquisition. Given these issues, we found that using 1 / 2 MS medium instead of water culture and making a slit along the back of the seed were necessary to achieve imbibition and soften the seed coat, which was beneficial for subsequent transfection.

[0045] When wild soybean seeds were treated with the aforementioned pretreatment to successfully obtain explants with exposed embryonic growth points, it was further discovered that the concentration of the infection solution, pre-infection vacuum treatment, and infection time all affected the transformation results of wild soybean during the complex process of infection and culture. It is necessary to organically combine the appropriate concentration of the infection solution, pre-infection vacuum treatment, and infection time with this pretreatment to effectively overcome the adverse effects of the structure and physiological characteristics of wild soybean seeds on the transformation process and effectively improve the transformation efficiency of wild soybean.

[0046] Terminology: The overnight processing time described in this invention is 12h to 16h.

[0047] The present invention will be further described in detail below with reference to specific embodiments.

[0048] Example 1

[0049] This embodiment provides a method for genetic transformation of wild soybean, which specifically includes the following steps:

[0050] 1. Genetic transformation

[0051] 1.1 The plasmid was converted into Agrobacterium using existing conventional methods:

[0052] 1 μL of pTF101 plasmid was mixed with 50 μL of EHA105 competent cells and added to an electrode cup. Electroporation was performed using a GenePulser Xcell™ electroporation system (Bio-Rad, USA). The electroporated mixture was added to 200 μL of LB liquid medium and incubated at 28°C (200 rpm) for 2.5 h. 50 μL of the bacterial culture was plated onto LB solid medium plates containing 50 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kan) and incubated upside down at 28°C for 3 days. Single colonies were picked and cultured overnight in 5 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. The bacterial culture was then stored at -80°C for later use.

[0053] 1.2 Wild soybean conversion

[0054] 1.2.1 Seed sterilization

[0055] After placing wild soybean seeds in a petri dish, leave the dish and lid open and place it in a desiccator within a fume hood. Place a beaker containing sodium hypochlorite (NaClO) solution in the desiccator, and slowly add HCl along the beaker wall to the NaClO solution at a ratio of 3.5 mL concentrated hydrochloric acid (HCl) / 100 mL NaClO. Then quickly seal the desiccator to allow the released chlorine gas to sterilize. After overnight treatment, open the desiccator to allow the chlorine gas to fully evaporate, then remove the petri dish, seal it, and store it for later use.

[0056] The disinfection results showed that the chlorine treatment for wild soybean seeds was effective because the seed coat of wild soybeans is dense and water cannot easily penetrate the seed. This method is not only convenient and labor-saving, but also allows for large-scale production. Furthermore, the contamination rate of the disinfected seeds was 0%, and the seed viability was not affected.

[0057] 1.2.2 Explant preparation

[0058] Wild soybeans and cultivated soybeans are not the same species. Generally speaking, wild soybeans are small in size, with a dense and hard seed coat, and differ greatly in morphology from cultivated soybeans. Figure 1 . Figure 1 As shown on the left, the yellow and larger one is William 82, a cultivated soybean variety; Figure 1 As shown on the right, the small, dark brown explant is wild soybean DA056. When obtaining seed explants for Agrobacterium infection, the seed coat needs to be removed and the seeds dissected. Before removing the seed coat, cultivated soybeans and wild soybeans were grouped and treated as follows:

[0059] Cultivated soybean group: Seeds were placed on 1 / 2 MS medium and treated in the dark overnight.

[0060] Wild soybean group A: Wild soybeans were placed on 1 / 2 MS medium and treated in the dark overnight.

[0061] Wild soybean group B: Make a cut (greater than 1 mm) on the back of the sterilized seed (the back of the seed is the raised side of the seed surface, opposite to the hilum) with a scalpel, place it on 1 / 2 MS medium, and treat it in the dark overnight.

[0062] The formulation of 1 / 2MS medium is as follows: containing 2.2 g / L Murashige & Skoog Basal Medium with Vitamins (MS) (PhytoTech Labs, US, catalog number M519), 30 g / L sucrose, 7 g / L agar, and pH 5.8.

[0063] The results are as follows Figure 2 and Figure 3 As shown.

[0064] Figure 2 (a) Cultivated soybeans placed on 1 / 2 MS medium for 0 h. Figure 2 (b) Cultivated soybeans placed on 1 / 2 MS medium for 16 hours, such as Figure 2 (a) and Figure 2 As shown in (b), soaking the cultivated soybeans directly in 1 / 2 MS medium overnight allowed the seeds to absorb water and soften the seed coat, which then made it easy to peel off the thin seed coat and obtain soybean explants.

[0065] Figure 2 (c) Wild soybean A group, incubated on 1 / 2 MS medium for 0 h. Figure 2 (d) shows the state of wild soybean A group after 16 hours on 1 / 2MS medium. It can be seen that wild soybean seeds cannot absorb water, the seed coat remains hard and difficult to peel off, and wild soybean explants cannot be dissected and obtained.

[0066] Figure 3 (a) This shows the state of wild soybean group B after 16 hours on 1 / 2 MS medium. It can be seen that the wild soybean successfully absorbs water and the seed coat softens. Furthermore, experiments revealed that this step should not be done using conventional methods—soaking in water or similar solutions—as this can easily cause the seed embryo to rot, or affect explant acquisition and subsequent transfection. We found that using 1 / 2 MS medium instead of water culture, and making a slit along the back of the seed, is necessary to achieve water absorption and soften the seed coat, which is beneficial for subsequent transfection.

[0067] Next, the sucked-up wild soybean was dissected under a microscope using forceps: the seed coat of the sucked-up seed was removed, and one cotyledon and two true leaves were removed from the seed. Figure 3 (b) above), exposing the growth point of the embryonic tip ( Figure 3 (b) (see the image below), the remaining seed portion is a wild soybean explant.

[0068] 1.2.3 Agrobacterium infection and co-culture

[0069] Agrobacterium containing the target plasmid was streaked onto a plate. After 2 days, single colonies were collected and plated or shaken. On the second day, Agrobacterium was collected and the cells were resuspended in the infection solution, and the OD600 value of the infection solution was adjusted to approximately 0.5 for later use. The infection solution was formulated as follows: 150 mg / L of 1,4-Dithiothreitol (DTT), 2 mg / L of Zeatin, 60 mg / L of Acetosyringone (AS), and 0.01% v / v of Silwet L-77 in 1 / 2 MS solution.

[0070] After placing the soybean explants in the infection solution, they were subjected to vacuum permeation treatment at -0.08 MPa for 3 minutes, followed by slow release. The infection solution containing the explants was then left to stand in the dark for 15 hours.

[0071] After infection, the explants were removed, dried on filter paper, and then placed on co-culture medium and treated in the dark for 4 days. Figure 3 (c)).

[0072] The co-culture medium was formulated as follows: 1 / 2 MS solid medium (2.2 g / L MS, 30 g / L sucrose, 7 g / L agar) containing 150 mg / L DTT, 2 mg / L Zeatin, and 40 mg / L LAs.

[0073] 1.2.3 Sprout Induction and Screening

[0074] After co-culturing, the explants were obliquely inserted into the recovery medium. Figure 3 (d)) ), restored the explant to green.

[0075] The recovery medium was formulated as follows: 28 mg / L FeSO4, 38 mg / L EDTA-2Na, 1 mg / L 6-benzylaminopurine (6-BA), 150 mg / L cefotaxime sodium (Cef), 350 mg / L Timentin (Tim), 3.1 g / L Gamborg B-5 Basal Medium (B5) (PhytoTechLabs, US, catalog number G398), 30 g / L sucrose, and 7 g / L agar.

[0076] Next, the explants were transferred to bud selection medium 1 to induce budding ( Figure 3 (e)).

[0077] The formula for the bud selection medium 1 is: 3.1 g / L B5, 30 g / L sucrose, 7 g / L agar, 28 mg / L FeSO4, 38 mg / L EDTA-2Na, 2 mg / L 6-BA, 50 mg / L glutamine (Gln), 50 mg / L asparagine (Asn), 150 mg / L Cef, 350 mg / L Tim, and the selection agent [6 mg / L phosphinothricin (PTT)] in a solid medium.

[0078] After the buds emerge, cut them off and place them in bud selection medium 2 for selection. Figure 3(f)), wait until the resistant seedlings grow to about 1cm.

[0079] The formula for bud selection medium 2 is as follows: 3.1 g / L B5, 30 g / L sucrose, 7 g / L agar, 28 mg / L FeSO4, 38 mg / L EDTA-2Na, 0.1 mg / L indole-3-acetic acid (IAA), 1 mg / L Zeatin, 0.5 mg / L Gibberellin A3 (GA3), 150 mg / L Cef, 350 mg / L Tim, 50 mg / L Gln, 50 mg / L Aln, 100 mg / L Pyroglutamic acid (Pyr), and a selection agent (4 mg / L PPT) in a solid medium.

[0080] All the above cultivation steps were carried out at a temperature of 25℃ ± 2℃ and a photoperiod of day:night = 16h:8h.

[0081] 1.2.4 Rooting and Propagation

[0082] Once the resistant seedlings have grown to about 1 cm, they are transferred to a rooting medium to root (Figure (3g)).

[0083] The rooting medium was formulated as follows: B5 solid medium containing 100 mg / L Cef, 100 mg / L Tim, and 1 mg / L 3-indolebutyric acid (IBA), 3.1 g / L B5, 20 g / L sucrose, 7 g / L agar, 28 mg / L FeSO4, and 38 mg / L EDTA-2Na. The tissue culture room temperature was 25℃ ± 2℃, and the photoperiod was day:night = 16 h:8 h.

[0084] After PCR identification, the transgenic seedlings will be transplanted into the soil to grow until harvest (Figure (3h)).

[0085] 2. Identification of transgenic lines

[0086] DNA extraction and PCR identification

[0087] Leaves of the transformed organisms obtained from the transformation test were collected, and genomic DNA was extracted using the NuClean Plant Genomic DNA Kit (Kangwei Century, China). Then, according to the instructions, PCR amplification of the plant genome was performed using a 2×Taq PCR StarMix (Dye) (GenStar, China). Bar_T_F and Bar_T_R amplified the pTF101 resistance marker gene fragment, with a size of ~0.7kb (representative PCR results are shown in the figure). Figure 4 ).

[0088] Table 1 Primer sequences used for PCR detection

[0089]

[0090] Example 2

[0091] This embodiment describes the genetic transformation of wild soybean using the method described in Example 1. The difference lies in:

[0092] In step (1.2.3 Agrobacterium infection and co-culture), wild soybeans were infected with infection solutions containing different As concentrations for 4 hours. The transformation results are shown in Table 2.

[0093] Table 2. Genetic transformation results after 4 hours of infection with As-containing solutions of different concentrations.

[0094]

[0095] As shown in Table 2, when wild soybeans were infected with an As concentration of 40-80 mg / L, explants were obtained in the early stages of tissue culture, and the germination of the explants was good. However, after a period of selection, most of the resistant seedlings died, and only a few seedlings survived in the As concentration treatments of 40-60 mg / L. PCR analysis of the transgenic status of the resistant seedlings showed negative results in all cases. Therefore, although the germination induction and selection methods used in this example are suitable, the infection treatment needs further optimization.

[0096] Example 3

[0097] This embodiment describes the genetic transformation of wild soybean using the method described in Example 1. The difference lies in:

[0098] In step (1.2.3 Agrobacterium infection and co-culture), during the infection process, explants were first treated with infection solutions containing different As concentrations in a -0.08 MPa vacuum for 3 min, followed by infection for different durations. The transformation results are shown in Table 3.

[0099] Table 3 Results of transformation using optimized infection conditions

[0100]

[0101] Table 3 shows that the concentration of the infection solution, the vacuum treatment before infection, and the infection time all affect the transformation results of wild soybean. When vacuum permeation treatment was applied to the explants during the infection process, it was difficult to obtain transgenic wild soybean seedlings even with changes in the As concentration when the treatment duration was 4 hours. Transgenic wild soybean seedlings were only obtained at an As concentration of 60 mg / L, with a transformation rate of 2.78%. However, by using vacuum permeation treatment on the explants during the infection process, controlling the As concentration of the infection solution to 60 mg / L, and extending the infection time to 15 hours, the transformation efficiency of wild soybean reached 3.45%, which is 24% higher than the 4-hour infection treatment. Therefore, the optimal method for Agrobacterium tumefaciens infection of soybean is to immerse the explants in an infection solution with an As concentration of 60 mg / L, perform vacuum permeation treatment, and then infect for 14-16 hours. This treatment can overcome the adverse effects of soybean seed structure and physiological characteristics on the transformation process and effectively improve the transformation efficiency of soybean.

[0102] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for genetic transformation of wild soybean, characterized in that, Includes the following steps: S1. Disinfect wild soybean seeds; S2. Make a cut on the back of the sterilized wild soybean seed, place the cut wild soybean seed on the culture medium, and obtain the seed after it has absorbed water. S3. Remove the seed coat of wild soybean seeds and expose the growth point of the embryo tip through dissection to obtain explants; S4. Place the explants in an Agrobacterium infection solution containing the target plasmid and perform vacuum permeation in a vacuum environment; S5. After releasing the vacuum, let it stand in the Agrobacterium infection solution for 3h~16h for infection.

2. The method for genetic transformation of wild soybean as described in claim 1, characterized in that, The infection solution consists of a basic culture medium, dithiothreitol, zeatin, acetylsylgenone, and a penetration enhancer; the final concentration of dithiothreitol is 100 mg / L to 200 mg / L, the final concentration of zeatin is 1 mg / L to 10 mg / L, the final concentration of acetylsylgenone is 50 mg / L to 70 mg / L, and the final concentration of the penetration enhancer is 0 to 1% v / v.

3. The method for genetic transformation of wild soybean as described in claim 2, characterized in that, The final concentration of the dithiothreitol is 130 mg / L to 170 mg / L, the final concentration of the zeatin is 1 mg / L to 5 mg / L, the final concentration of the acetylsylgenone is 55 mg / L to 65 mg / L, and the final concentration of the penetration enhancer is 0.01% to 0.5% v / v; preferably, the final concentration of the dithiothreitol is 148 mg / L to 152 mg / L, the final concentration of the zeatin is 1.5 mg / L to 2.5 mg / L, the final concentration of the acetylsylgenone is 58 mg / L to 62 mg / L, and the final concentration of the penetration enhancer is 0.01% to 0.05% v / v.

4. The method for genetic transformation of wild soybean as described in claim 2, characterized in that, The basic culture medium was a 1 / 2 MS solution; And / or, the penetration enhancer is Silwet l-77.

5. The method for genetic transformation of wild soybean as described in any one of claims 1-4, characterized in that, In step S2, the culture medium is either 1 / 2 MS medium or MS medium, preferably 1 / 2 MS medium.

6. The method for genetic transformation of wild soybean as described in claim 5, characterized in that, In step S2, the wild soybean seeds are placed in the culture medium for 12-16 hours.

7. The method for genetic transformation of wild soybean as described in any one of claims 1-4, characterized in that, In step S5, the time for static infiltration treatment is 3h~5h or 10h~16h, preferably 14h~16h.

8. The method for genetic transformation of wild soybean as described in any one of claims 1-4, characterized in that, The vacuum degree of the vacuum infiltration is -0.1MPa to -0.05MPa, and the infiltration time is 2min to 10min.

9. The method for genetic transformation of wild soybean as described in claim 8, characterized in that, The vacuum level is -0.1MPa to -0.08MPa, and the permeation time is 2min to 4min.

10. The method for genetic transformation of wild soybean as described in any one of claims 1-4, characterized in that, The disinfectant used to disinfect wild soybean seeds is chlorine gas, and the disinfection time is 12h~16h; And / or, in step S5, after the Agrobacterium infection solution has been left to stand for 3 to 16 hours, the following steps are also included: The infected explants were sequentially co-cultured, restored, budded, screened, and rooted to obtain transgenic wild soybean lines. Preferably, the co-culture is carried out for 2 to 5 days in a culture medium composed of MS solid medium or 1 / 2 MS solid medium supplemented with 140 mg / L to 160 mg / L dithiothreitol, 1 mg / L to 3 mg / L zeatin, and 30 mg / L to 50 mg / L acetylsyringone; and / or, The recovery culture involves culturing the explants in a recovery medium until they turn green. The recovery medium consists of 25 mg / L to 35 mg / L FeSO4, 35 mg / L to 45 mg / L EDTA-2Na, 0.5 mg / L to 1.5 mg / L 6-BA, 140 mg / L to 160 mg / L cefotaxime sodium, 340 mg / L to 360 mg / L termethin, 2.5 g / L to 3.5 g / L B5, 25 g / L to 35 g / L sucrose, and 5 g / L to 10 g / L agar. And / or, the bud selection includes the following steps: The explants after recovery culture were placed in bud selection medium 1 and cultured until buds appeared. Then the buds were cut off and placed in bud selection medium 2 and cultured until the seedlings grew to about 0.5cm~2cm. The bud selection medium 1 is composed of 2.5 g / L ~ 3.5 g / L B5, 25 g / L ~ 35 g / L sucrose, 5 g / L ~ 10 g / L agar, 25 mg / L ~ 35 mg / L FeSO4, 35 mg / L ~ 45 mg / L EDTA-2Na, 1 mg / L ~ 5 mg / L 6-BA, 45 mg / L ~ 55 mg / L glutamine, 45 mg / L ~ 55 mg / L asparagine, 140 mg / L ~ 160 mg / L Cef, 340 mg / L ~ 360 mg / L Tim, and 4 mg / L ~ 8 mg / L selection agent. The bud selection medium 2 consists of 2.5 g / L ~ 3.5 g / L B5, 25 g / L ~ 35 g / L sucrose, 5 g / L ~ 10 g / L agar, 25 mg / L ~ 35 mg / L FeSO4, 35 mg / L ~ 45 mg / L EDTA-2Na, 0.08 mg / L ~ 0.15 mg / L LIAAA, 0.5 mg / L ~ 1.5 mg / L Zeatin, 0.1 ~ 1 mg / L gibberellin, 140 mg / L ~ 160 mg / L Cef, 340 mg / L ~ 360 mg / L Tim, 45 mg / L ~ 55 mg / L LCln, 45 mg / L ~ 55 mg / L Aln, 90 mg / L ~ 110 mg / L pyroglutamic acid, and 2 mg / L ~ 8 mg / L selection agent. And / or, the rooting propagation involves culturing seedlings in a rooting medium, which is composed of a solid medium supplemented with 90 mg / L~110 mg / L Cef, 90 mg / L~110 mg / L Tim, and 0.5 mg / L~1.5 mg / L 3-indolebutyric acid. Preferably, the solid medium is B5 solid medium or MS solid medium.

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