Soybean pod explosion improved gene and soybean improved breeding method

Abstract

The invention discloses a soybean pod explosion improved gene and a soybean improved breeding method. On one hand, the invention provides a soybean pod blasting improved gene, and the sequence of the soybean pod blasting improved gene is as shown in SEQ ID NO: 1. On the other hand, the invention provides an improved breeding method of the fried soybean pods, which comprises the following steps: S1, transferring the improved gene of the fried soybean pods into plasmids, constructing a recombinant vector, transferring the recombinant vector into engineering bacteria, and carrying out multiplication culture, thallus crushing and purification treatment to obtain an expanded recombinant vector; s2, the recombinant vector is electrically transferred into infection bacteria, soybean seeds are infected and cultured, and transgenic soybean seedlings are obtained; s3, after planting, screening completely unexploded plants, harvesting and seeding, and repeating for more than five generations to obtain improved seeds. The pod-cracking-resistant gene improved through genetic engineering can regulate and control soybean varieties, so that the soybean varieties have a relatively low pod-cracking rate, and meanwhile, the pod-cracking-resistant gene can be subjected to stable cross-generation inheritance.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a soybean pod-breaking improvement gene and a soybean improvement breeding method. Background Technology

[0002] Pod bursting, also known as pod splitting, refers to the spontaneous cracking of the pericarp in mature plants. It is a natural characteristic of plants, helping them disperse seeds and ensure the continuation of their lineage. Pod bursting occurs in crops of the legume family (soybeans, peas, etc.), the grass family (rice, etc.), and the cruciferous family (Arabidopsis, rapeseed, etc.). However, for agricultural production, especially large-scale agricultural production, pod bursting is a detrimental phenomenon, causing significant yield and economic losses. my country has a huge demand for soybeans, but relies heavily on imports. Increasing my country's own soybean production is crucial for food security. According to research by Chinese agricultural experts, soybean pod bursting is one of the key factors leading to reduced soybean yields in my country. Therefore, studying soybean pod bursting and cultivating soybean varieties resistant to pod bursting are of great significance for ensuring soybean production.

[0003] Factors influencing soybean pod bursting mainly include external environmental factors and internal self-driving factors. External environmental factors, such as humidity, cause soybean pods to lose water, reducing the bonding strength between pod walls and making them more prone to bursting. Internal self-driving factors are related to the soybean's genetic characteristics. During maturity, soybeans secrete various hydrolytic enzymes in the pod-pod junction area, causing some cells in that area to degrade and undergo programmed apoptosis, thereby reducing the bonding strength between pod walls.

[0004] According to existing technology, the qPDH1 gene in soybean is one of the main genes regulating the autogenic factors of soybean pod cracking. There are reports in existing technologies of improving the qPDH1 gene by studying mutant varieties of soybean with crack-resistant traits, thereby reducing the pod cracking rate. However, these existing studies have certain problems. First, in the genetically modified varieties obtained using existing technologies, the mutant gene for crack-resistant traits is not well inherited, making it difficult to stably breed crack-resistant varieties. Second, although the pod cracking rate of genetically modified soybean varieties is reduced to some extent, it is still not ideal. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems, this application proposes to develop a genetically engineered anti-pod-cracking gene that can regulate soybean varieties to achieve a lower pod-cracking rate. Furthermore, this anti-pod-cracking gene can be stably inherited across generations. This application also provides a soybean pod-cracking improvement gene and a soybean improvement breeding method.

[0006] On the one hand, this application provides a soybean pod-breaking improvement gene, which is obtained by improving the soybean qPDH1 gene, and the sequence is shown in SEQ ID NO:1.

[0007] On the other hand, this application provides a method for improving soybean pod breakage breeding, including the following steps: S1. The soybean pod-breaking improvement gene described above in this application is transferred into a plasmid to construct a recombinant vector. The recombinant vector is then transformed into engineered bacteria to obtain recombinant bacteria. The bacteria are then inoculated into a culture medium for proliferation and culture. After the bacterial cells are broken and purified, the expanded recombinant vector is obtained. S2. The expanded recombinant vector obtained in step S1 is electroporated into the infected bacteria, and the recombinant infected bacterial solution is obtained by proliferation culture. Then, soybean seeds are infected and cultured with the recombinant infected bacterial solution to obtain transgenic soybean seedlings. S3. Transplant the seedlings obtained in step S2 normally, select plants that have not broken their pods, harvest the seeds, and continue to plant for five generations or more to obtain soybean pod-breaking gene-improved seeds.

[0008] Optionally, in step S1, the plasmid is selected as pEGCas9PM4-B plasmid, the sequence of which is shown in SEQ ID NO:2.

[0009] Optionally, in step S1, the engineered bacteria are selected from strain DB3.1.

[0010] Further optionally, step S1 specifically includes the following steps: S1-1. Mix the recombinant vector with Escherichia coli DB3.1 cell lysate and incubate on ice for at least 5 minutes to obtain the mixture. S1-2. The mixture obtained in step S1-1 is subjected to electroporation using an electroporator, and then LB culture medium at 37°C is added. The mixture is then incubated at 37°C for more than 2 hours to obtain bacterial culture. S1-3. Take the bacterial culture obtained in step S1-2 and inoculate it into LB medium. Incubate at 37°C for at least 24 hours. After single colonies grow, pick cells from the colonies, extract plasmids, and sequence them. Select colonies with correct sequencing results and inoculate them again into LB medium. Incubate at 37°C in a shaker until OD (Organic Degree) is reached. 600 The value was 3.0~6.0, and the proliferation culture was completed.

[0011] Optionally, in step S2, the infecting bacterium is Agrobacterium EHA105.

[0012] Optionally, in step S2, the proliferation culture to obtain the recombinant infectious bacterial solution includes the following steps: Sa, the expanded recombinant vector, was electrotransformed into EHA105 Agrobacterium-mediated peptide-sensing cells, plated on LB solid medium with rifampicin and kanamycin, and induced in a constant temperature incubator at 28°C for more than 24 hours. After single colonies grew, cells were picked from the colonies, plasmids were extracted, and colonies with correct sequencing results were selected after sequencing identification. Sb: Collect colonies with correct sequencing results obtained in step Sa, inoculate them into LB solid medium containing rifampicin and kanamycin, and incubate at 28°C until OD. 600 The value is between 3.0 and 6.0. Sc. Collect bacterial colony cells, inoculate them into the infection medium, resuspend them, and place them in a sterile centrifuge tube. Vortex the centrifuge tube until the bacterial colony cells are evenly dispersed in the suspension to obtain the recombinant infection solution.

[0013] Optionally, in step S2, the infection culture includes the following steps: S2-1. Select disease-free soybean seeds, sterilize them to obtain sterile soybean seeds, sow them on germination medium, and culture them in the dark at 24~26℃ for 16~36 hours. After removing contaminated and diseased seeds, germinated soybean seeds are obtained. S2-2. Dissect the germinated soybean seeds obtained in step S2-1, trim the hypocotyl, remove the cotyledons, retain two protofolies and the stem tip, and slightly damage the stem tip to obtain explants. S2-3. Inoculate the explants obtained in step S2-2 with recombinant infectious bacterial solution. After infection, discard the recombinant infectious bacterial solution and inoculate onto co-culture medium. Culture in the dark at 24-26℃ for 3-5 days to obtain co-cultured explants. S2-4. Trim the hypocotyls of the co-cultured explants obtained in step S2-3 and transplant them into a recovery medium without selection agent. Culture them for 4-7 days at 24-26ºC under a 16 / 8 hour light / dark cycle. Then transplant the explants into a regeneration medium containing selection agent and antibiotics and continue to culture them for 14-21 days under the same conditions to obtain regenerated explants. S2-5. Remove the necrotic tissue and cotyledons from the regenerated explants obtained in step S2-4, transplant them into elongation medium, and continue to culture them under the same culture conditions as in step S2-4. Trim the necrotic tissue every 21 days until the regenerated explants grow into transplantable seedlings. Select positive seedlings to complete the infection culture.

[0014] Optionally, in steps S2-4, the light intensity of the illumination is 60~80µEm. -2 S -1 .

[0015] Further optionally, in steps S2-5, the screening of positive seedlings is carried out by extracting the qPDH1 gene from a small amount of leaf tissue and then identifying it using Sanger sequencing.

[0016] In summary, the present invention has at least one of the following beneficial technical effects: 1. Based on the soybean qPDH1 gene, this application obtained a soybean pod-breaking improvement gene by knocking out and modifying some gene segments. After being transferred into soybean, this gene can be inherited relatively stably and can effectively improve the soybean pod-breaking problem and significantly reduce the pod-breaking rate.

[0017] 2. The soybean pod-breaking improvement breeding method of this application involves a specific step in which the soybean pod-breaking improvement gene of this application is transferred into soybean seeds through infection to obtain seedlings. After planting and screening, the seedlings are obtained after five generations. The soybean pod-breaking improvement seeds obtained by this application have an extremely low pod-breaking rate after planting, and the pod-breaking resistance characteristic can be inherited relatively stably. Attached Figure Description

[0018] Figure 1 This is a diagram showing the results of the planting experiment in this application; Figure 2 This is a diagram showing the results of the genetic attribute test in this application. Detailed Implementation

[0019] The present application will be further described in detail below with reference to the embodiments.

[0020] This application provides a soybean pod-breaking improvement gene, which is obtained by modifying the soybean qPDH1 gene, and its sequence is shown in SEQ ID NO:1.

[0021] Specifically, this application is based on the qPDH1 gene extracted from Zhongdou 40. After knocking out some gene fragments and modifying some gene fragments, the soybean pod-breaking improvement gene of this application was obtained.

[0022] The qPDH1 gene extracted from soybean 40 used in this application has the specific sequence shown in SEQ ID NO:3.

[0023] This application uses the above-mentioned genes to improve soybean breeding, which can obtain improved soybean seeds with relatively stable genetic characteristics and extremely low pod breakage rate.

[0024] Specifically, the soybean pod-breaking improvement breeding method of this application includes the following steps: S1. The soybean pod-breaking improvement gene described above in this application is transferred into a plasmid to construct a recombinant vector. The recombinant vector is then transformed into engineered bacteria to obtain recombinant bacteria. The bacteria are then inoculated into a culture medium for proliferation and culture. After the bacterial cells are broken and purified, the expanded recombinant vector is obtained. S2. The expanded recombinant vector obtained in step S1 is electroporated into the infected bacteria, and the recombinant infected bacterial solution is obtained by proliferation culture. Then, soybean seeds are infected and cultured with the recombinant infected bacterial solution to obtain transgenic soybean seedlings. S3. Transplant the seedlings obtained in step S2 normally, select plants that have not broken their pods, harvest the seeds, and continue to plant for five generations or more to obtain soybean pod-breaking gene-improved seeds.

[0025] The following are embodiments of this application.

[0026] The main raw materials and reagents used in the embodiments of this application are all commercially available.

[0027] Example 1: Construction of a recombinant plasmid containing a soybean pod-breaking improvement gene The plasmid construction in this embodiment includes the following steps: S1-1. The soybean pod-breaking improvement gene of this application was transformed into the pEGCas9PM4-B plasmid to obtain a recombinant vector; 5 μL of the recombinant vector was mixed with 50 μL of Escherichia coli DB3.1 cell broth and treated in an ice bath for 5 min to obtain a mixture. S1-2. Using an electroporator, the mixture obtained in step S1-1 is subjected to electroporation with voltage of 1800V, resistance of 200Ω, and capacitance of 25μF. Then, it is added to LB culture medium at 37℃ and incubated in a 37℃ incubator for 2 hours to obtain bacterial culture. S1-3. Take the bacterial culture obtained in step S1-2 and inoculate it into LB medium. Incubate at 37°C for at least 24 hours. After single colonies grow, pick cells from the colonies, extract plasmids, and sequence them. Select colonies with correct sequencing results and inoculate them again into LB medium. Incubate at 37°C in a shaker until OD (Organic Degree) is reached. 600 If the value is 3.0 or higher, complete the proliferation culture; S1-4. After the bacterial culture was proliferated, the bacterial solution was collected and centrifuged at 8000 r / min for 15 min, then resuspended in phosphate buffer, and crushed using a pressure crusher. The bacterial solution was then collected and centrifuged again at 8000 r / min for 15 min using a low-temperature refrigerated centrifuge to obtain the lysate. The lysate was then extracted and purified by plasmid extraction to obtain a recombinant plasmid containing the soybean pod-breaking improvement gene.

[0028] Example 2: Preparation of recombinant infectious bacterial suspension The preparation of the recombinant infectious bacterial solution in this embodiment includes the following steps: Sa. The recombinant vector obtained in Example 1 was electrotransformed into EHA105 Agrobacterium competent peptide cells according to the electrotransformation steps described in the EHA105 Agrobacterium electrotransformation manual. The cells were plated on LB solid medium containing rifampicin and kanamycin and induced in a constant temperature incubator at 28°C for more than 24 hours. After single colonies grew, cells were picked from the colonies, plasmids were extracted, and colonies with correct sequencing results were selected after sequencing identification. Sb: Collect colonies with correct sequencing results obtained in step Sa, inoculate them into LB solid medium containing rifampicin and kanamycin, and incubate at 28°C until OD. 600 Version 3.0 and above; Sc. Collect bacterial colony cells, inoculate them into the infection medium for Agrobacterium tumefaciens EHA105, resuspend them and place them in a sterile centrifuge tube. Vortex the centrifuge tube until the bacterial colony cells are evenly dispersed in the suspension to obtain the recombinant infection solution. The inoculation amount in step Sc should be such that the final recombinant bacterial suspension, when measured with a spectrophotometer, has an absorbance value (A660) of 0.60 ± 0.15.

[0029] Example 3 Infection culture of soybean seeds The infection culture in this embodiment includes the following steps: S2-1. Select disease-free medium soybean seeds (40), sterilize them overnight with chlorine, and allow the chlorine to evaporate after 3 weeks to obtain sterile soybean seeds. Sow the sterilized seeds on a germination medium and culture them in the dark at 24-26℃ for 28 hours. After removing contaminated and diseased seeds, germinated soybean seeds are obtained. S2-2. Dissect the germinated soybean seeds obtained in step S2-1. Hold the seed with tweezers and cut off the elongated hypocotyl with a scalpel, leaving about 2 mm. Discard the seed coat to expose the cotyledons. Fix the seed with tweezers and remove one cotyledon with a scalpel to expose the stem tip with two protocolumnar leaves. Under a dissecting microscope, use the blunt end of a scalpel to remove the two protocolumnar leaves to expose the meristematic tissue at the apex. Gently damage the stem tip with the sharp end of a blade to obtain the explant. S2-3. The explants obtained in step S2-2 are inoculated with 50 ml of the recombinant bacterial solution from Example 2 at a rate of 80 explants per ml. The explants are inoculated with the recombinant bacterial solution overnight. The recombinant bacterial solution is then removed and discarded. The explants are then inoculated onto a co-culture medium and cultured in the dark at 24-26°C for 4 days to obtain co-cultured explants. S2-4. Trim the elongated hypocotyls of the co-cultured explants obtained in step S2-3, and transplant them into recovery medium without selection agent. Incubate at 24-26ºC for 60-80 µM. -2 S -1Under light intensity and a 16 / 8 hour light / dark cycle, the explants were cultured for 7 days. Then, the explants were transplanted into a regeneration medium containing selection agents and antibiotics and cultured for another 21 days under the same conditions to obtain regenerated explants. S2-5. Remove the necrotic tissue and cotyledons from the regenerated explants obtained in step S2-4, transplant them into elongation medium, and continue to culture them under the same culture conditions as in step S2-4. Trim the necrotic tissue every 21 days until the regenerated explants grow into transplantable seedlings. Take a small amount of leaf tissue from each seedling, extract the qPDH1 gene from the tissue, and identify it using Sanger sequencing. Select positive seedlings to obtain seedlings that have completed infection culture.

[0030] Example 4: Improved Breeding The improved breeding in this embodiment adopts the following steps: The seedlings obtained in Example 3 are transplanted into a sterile experimental field with a row spacing of 45cm, a plant spacing of 12cm, and a planting depth of 3.5cm; after normal planting, the soybeans are harvested 12 days after full maturity, and the plants without pod bursting are selected for seed collection; the obtained seeds are bred normally, and then planted with a row spacing of 45cm, a plant spacing of 12cm, and a planting depth of 3.5cm, and after planting, the soybeans are harvested 12 days after full maturity, and the plants without pod bursting are selected for seed collection. After repeating five generations, improved soybean seeds are obtained.

[0031] Comparative Example 1 This comparative example uses the pEGCas9PM4-B-qPDH1 vector (publication number CN115927452A, invention title: A method for improving soybean pod breakage using gene editing) as the recombinant vector. Seedlings were cultivated following the steps in Examples 1-3 of this application. After normal planting, the soybeans were harvested. Since a certain pod breakage rate still exists after planting, plants with a pod breakage rate of less than 5% were selected for seed collection.

[0032] Comparative Example 2 The seeds of the Zhongdou 40 blast-resistant pod mutant were selected as comparative example 2 of this application. The qPDH1 gene of the mutant seeds is shown in the sequence SEQ ID NO:4.

[0033] Planting experiment: Using Zhongdou 40 soybean as a control example, the qPDH1 gene of the selected Zhongdou 40 soybean is shown in sequence SEQ ID NO:3. Soybean seeds obtained in Example 4 of this application without five generations of repeated breeding and screening were used as examples in this application. Along with soybean seeds from Comparative Examples 1 and 2, 10,000 plants were planted each using normal breeding methods, with a row spacing of 45 cm, a plant spacing of 12 cm, and a planting depth of 3.5 cm. After normal planting and harvesting, the pod breakage rate was calculated. The results are shown in Table 1 and... Figure 1 As shown.

[0034] Table 1. Results of Pod Breakage Rate Testing in Examples and Comparative Examples Pod breakage rate (%) Example 0.9 Comparative Example 1 4.7 Comparative Example 2 8.9 Comparison Example 17.2 Genetic attribute experiment Using Zhongdou 40 soybean as a control example, the qPDH1 gene of the selected Zhongdou 40 soybean is shown in sequence SEQ ID NO:3. Soybean seeds obtained without five generations of repeated breeding and screening in Example 4 of this application were used as examples in this application. Cross-generational breeding was carried out according to the method of Example 4, with 10,000 plants planted each time, and the proportion of plants without pod bursting in each generation was calculated. Using soybean seeds from Comparative Examples 1 and 2, normal breeding was carried out, followed by planting at a row spacing of 45 cm, a plant spacing of 12 cm, and a planting depth of 3.5 cm, with 10,000 plants planted in each generation; five generations of cross-generational breeding were carried out, and the plants with the lowest pod bursting rate in each generation were selected for seed collection, and the proportion of plants without pod bursting in each generation was calculated. The results are shown in Table 2 and... Figure 2 As shown.

[0035] Table 2. Results of cross-generational breeding tests in the examples and comparative examples. generation(%) Second generation (%) Third generation (%) Fourth generation (%) Five Dynasties (%) Example 96.4 97.2 98.3 98.8 99.4 Comparative Example 1 65.8 54.2 27.3 11.2 7.9 Comparative Example 2 52.8 21.6 9.8 2.9 0 Comparison Example 7.9 7.2 8.3 7.1 7.7 Through Table 1 and Figure 1 The data shows that the soybean pod-breaking improvement gene of this application, after inoculation and seedling cultivation, resulted in an extremely low pod-breaking rate of less than 1% in the first generation of seedlings. In contrast, the gene improvement method in Comparative Example 1 yielded soybeans with a significantly lower pod-breaking rate than the control, but still above 4%. The mutant soybeans in Comparative Example 2, although exhibiting some resistance to pod-breaking and with a significantly lower pod-breaking rate than the control, still had a pod-breaking rate above 8%. Therefore, the soybean pod-breaking improvement gene of this application can significantly reduce the pod-breaking rate of soybeans.

[0036] Through Table 2 and Figure 2 The data shows that the soybean pod-breaking resistance gene of this application, after infection and seedling cultivation, exhibits stable pod-breaking resistance in each generation during cross-generational breeding. Furthermore, after five generations of breeding, the proportion of seeds without pod-breaking resistance in the fifth generation reaches over 99%. In contrast, the soybean seeds of Comparative Examples 1 and 2 show extremely unstable inheritance of pod-breaking resistance; this resistance gradually diminishes with cross-generational breeding, and the proportion of seeds without pod-breaking resistance decreases with each generation. Therefore, the soybean pod-breaking resistance gene of this application, after improved breeding, results in soybean seeds with highly stable pod-breaking resistance.

[0037] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A soybean pod-breaking improvement gene, characterized in that, The soybean pod-breaking improvement gene was obtained by modifying the soybean qPDH1 gene, and its sequence is shown in SEQ ID NO:

1.

2. A method for improving soybean pod-breaking breeding, characterized in that, Includes the following steps: S1. The soybean pod-breaking improvement gene described in claim 1 is transferred into a plasmid to construct a recombinant vector. The recombinant vector is then transformed into engineered bacteria to obtain recombinant bacteria. The bacteria are then inoculated into a culture medium for proliferation and culture. After the bacterial cells are broken and purified, the expanded recombinant vector is obtained. S2. The expanded recombinant vector obtained in step S1 is electroporated into the infected bacteria, and the recombinant infected bacterial solution is obtained by proliferation culture. Then, soybean seeds are infected and cultured with the recombinant infected bacterial solution to obtain transgenic soybean seedlings. S3. Transplant the seedlings obtained in step S2 normally, select plants that have not broken their pods, harvest the seeds, and continue to plant for five generations or more to obtain soybean pod-breaking gene-improved seeds.

3. The soybean pod-breaking improvement breeding method according to claim 2, characterized in that, In step S1, the plasmid used is pEGCas9PM4-B, and its sequence is shown in SEQ ID NO:

2.

4. The soybean pod-breaking improvement breeding method according to claim 2, characterized in that, In step S2, the engineered bacteria used is strain DB3.

1.

5. The soybean pod-breaking improvement breeding method according to claim 2, characterized in that, Step S1 specifically includes the following steps: S1-1. Mix the recombinant vector with Escherichia coli DB3.1 cell lysate and incubate on ice for at least 5 minutes to obtain the mixture. S1-2. The mixture obtained in step S1-1 is subjected to electroporation using an electroporator, and then LB culture medium at 37°C is added. The mixture is then incubated at 37°C for more than 2 hours to obtain bacterial culture. S1-3. Take the bacterial culture obtained in step S1-2 and inoculate it into LB medium. Incubate at 37°C for at least 24 hours. After single colonies grow, pick cells from the colonies, extract plasmids, and sequence them. Select colonies with correct sequencing results and inoculate them again into LB medium. Incubate at 37°C in a shaker until OD (Organic Degree) is reached. 600 The value was 3.0~6.0, and the proliferation culture was completed.

6. The soybean pod-breaking improvement breeding method according to claim 2, characterized in that, In step S2, the infecting bacterium is Agrobacterium tumefaciens EHA105.

7. The soybean pod-breaking improvement breeding method according to claim 2, characterized in that, In step S2, the proliferation culture to obtain the recombinant infectious bacterial solution includes the following steps: Sa, the expanded recombinant vector, was electrotransformed into EHA105 Agrobacterium-mediated peptide-sensing cells, plated on LB solid medium with rifampicin and kanamycin, and induced in a constant temperature incubator at 28°C for more than 24 hours. After single colonies grew, cells were picked from the colonies, plasmids were extracted, and colonies with correct sequencing results were selected after sequencing identification. Sb: Collect colonies with correct sequencing results obtained in step Sa, inoculate them into LB solid medium containing rifampicin and kanamycin, and incubate at 28°C until OD. 600 The value is between 3.0 and 6.

0. Sc. Collect bacterial colony cells, inoculate them into the infection medium, resuspend them, and place them in a sterile centrifuge tube. Vortex the centrifuge tube until the bacterial colony cells are evenly dispersed in the suspension to obtain the recombinant infection solution.

8. The soybean pod-breaking improvement breeding method according to claim 7, characterized in that, In step S2, the infection culture includes the following steps: S2-1. Select disease-free soybean seeds, sterilize them to obtain sterile soybean seeds, sow them on germination medium, and culture them in the dark at 24~26℃ for 16~36 hours. After removing contaminated and diseased seeds, germinated soybean seeds are obtained. S2-2. Dissect the germinated soybean seeds obtained in step S2-1, trim the hypocotyl, remove the cotyledons, retain two protofolies and the stem tip, and slightly damage the stem tip to obtain explants. S2-3. Inoculate the explants obtained in step S2-2 with recombinant infectious bacterial solution. After infection, discard the recombinant infectious bacterial solution and inoculate onto co-culture medium. Culture in the dark at 24-26℃ for 3-5 days to obtain co-cultured explants. S2-4. Trim the hypocotyls of the co-cultured explants obtained in step S2-3 and transplant them into a recovery medium without selection agent. Culture them for 4-7 days at 24-26ºC under a 16 / 8 hour light / dark cycle. Then transplant the explants into a regeneration medium containing selection agent and antibiotics and continue to culture them for 14-21 days under the same conditions to obtain regenerated explants. S2-5. Remove the necrotic tissue and cotyledons from the regenerated explants obtained in step S2-4, transplant them into elongation medium, and continue to culture them under the same culture conditions as in step S2-4. Trim the necrotic tissue every 21 days until the regenerated explants grow into transplantable seedlings. Select positive seedlings to complete the infection culture.

9. The method for improving soybean pod breaking and breeding according to claim 8, characterized in that, In steps S2-4, the light intensity of the illumination is 60~80µEm. -2 S -1 .

10. The soybean pod-breaking improvement breeding method according to claim 8, characterized in that, In steps S2-5, the positive seedlings are selected by extracting the qPDH1 gene from a small amount of leaf tissue and then identifying it using Sanger sequencing.

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