Agrobacterium rhizogenes-mediated high-efficiency genetic transformation method of soybean cotyledon and the rhizobacterium used

Abstract

The application belongs to the field of biology and modern agricultural technology, and discloses a rooting aid invasion agent, and a formula thereof is as follows: B5 culture medium 3.0-3.5 g / L, sucrose 28-32 g / L, MES 3.8-4.0 g / L, acetyl-syringone 38-42 mg / L, 6-BA 1.5-1.8 mg / L, gibberellin 0.02-0.03 mg / L, and surfactant Silwet L-77 100 ml / L, and pH is 5.4. The application also discloses a soybean hairy root induction and transformation method mediated by Agrobacterium rhizogenes by using the rooting aid invasion agent, and the method comprises the following steps: disinfection and germination of soybean seeds; resuspension of Agrobacterium rhizogenes with a target expression plasmid in the rooting aid invasion agent to obtain an Agrobacterium rhizogenes aid invasion liquid; infection; co-culture; and screening of positive roots.

Description

Technical Field

[0001] This invention belongs to the fields of biology and modern agricultural technology, and specifically relates to a method for inducing and transforming soybean hairy roots mediated by Agrobacterium rhizogenes and the root-infecting agent used. Background Technology

[0002] Soybeans (Glycine max) are a high-quality source of plant protein and can be processed into feed or high-quality, economical vegetable oil, with huge demand from my country's livestock and catering industries. A key aspect of increasing soybean self-sufficiency is increasing investment in science and technology, securing a leading position in high-tech soybean breeding, and improving the efficiency of soybean cultivation in my country. Genetic transformation methods for soybeans are of great significance for molecular research on soybean-related physiological and biological functions and for the breeding of resistant new varieties. In the field of agricultural breeding, gene editing technology is a powerful tool for turning the tide in the seed industry. The notice "National Transgenic Soybean Variety Approval Standards (Trial)" issued by the Ministry of Agriculture and Rural Affairs in June 2022 signifies that soybean transgenic editing breeding and commercialization are about to usher in a period of rapid development. Currently, soybean transformation efficiency remains very low, far from meeting the needs of large-scale transformation screening applications in high-throughput molecular breeding.

[0003] Agrobacterium rhizogenes can be used to rapidly and easily transform soybeans into transgenic hairy roots. Agrobacterium rhizogenes is a Gram-negative soil bacterium belonging to the genus *Agrobacterium* in the family Rhizobiaceae. Similar to *Agrobacterium tummefaciens*, Agrobacterium rhizogenes transfers its endogenous T-DNA from an extrachromosomal replicon (called a root-inducing plasmid) into the plant's genomic DNA. Agrobacterium rhizogenes contains a root-inducing pRi2659 agrobacterium-based Ri plasmid, which contains the root locus (rol) genes rolA, rolB, rolC, and rolD in the T-DNA region, capable of inducing hairy roots from the damaged surface after explant infection. This method can rapidly produce a large number of transgenic soybean roots in a short time, allowing for related molecular detection and biological experiments. Furthermore, the produced soybean hairy roots can be further induced to differentiate, generating new callus tissue, thus yielding heritable transformant lines. This mechanism allows for the overexpression or RNAi of target genes in a shorter time compared to soybean genetic transformation.

[0004] Currently, Agrobacterium rhizogenes-mediated soybean transformation technology can be divided into three types: 1. Aerial root transformation method, where the transformation site is the soybean hypocotyl. This method has a low proportion of positive roots and a high false positive rate; 2. Aseptic tissue culture method, where the transformation site is the soybean cotyledon. The current drawbacks of this method are: cumbersome operation, low efficiency in producing positive roots, and easy contamination of explants; therefore, there is room for optimization. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for efficient genetic transformation of soybean cotyledons mediated by Agrobacterium rhizogenes and the rooting agent used therein.

[0006] This invention addresses the shortcomings of Agrobacterium-mediated soybean transformation by making technical improvements to overcome these shortcomings. The technical problems solved are: shortening the hairy root transformation cycle and improving the efficiency of positive root production; reducing operational steps and minimizing human contamination.

[0007] To solve the above-mentioned technical problems, the present invention provides a root-infection aid with the following formula: B5 medium 3.0-3.5 g / L, sucrose 28-32 g / L, MES (sodium MES) 3.8-4.0 g / L, acetylsylgenin 38-42 mg / L, 6-BA 1.5-1.8 mg / L, gibberellin 0.02-0.03 mg / L, surfactant Silwet L-77 100 ml / L, pH = 5.4.

[0008] Note: Both 6-BA and gibberellin are hormones.

[0009] As an improvement to the root-promoting agent of the present invention, its formulation is as follows: B5 medium 3.16 g / L, sucrose 30 g / L, MES 3.9 g / L, acetylsuccinone 40 mg / L, 6-BA 1.67 mg / L, gibberellin 0.025 mg / L, surfactant Silwet L-77 100 ml / L, pH = 5.4.

[0010] This invention also provides a method for Agrobacterium rhizogenes-mediated transformation of soybean hairy roots using the above-mentioned root-infecting agent, comprising the following steps:

[0011] 1) Disinfection and germination of soybean seeds;

[0012] 2) Resuspend the Agrobacterium rhizogenes containing the target expression plasmid in a Agrobacterium rhizogenes co-infection agent to obtain Agrobacterium rhizogenes co-infection solution;

[0013] 3) Infection: Soak soybean cotyledons in Agrobacterium rhizogenes infection solution for 10-15 minutes to obtain infected cotyledons;

[0014] 4) Co-culture: The infected cotyledons were first cultured on a co-culture medium (culture time approximately 3±0.5 days), and then cultured on a rooting medium (culture time approximately 14±2 days).

[0015] 5) Screen for positive roots.

[0016] Note: Steps 1), 4), and 5 above refer to existing technologies.

[0017] As an improvement to the soybean hairy root induction transformation method of the present invention, step 2) involves performing the following steps sequentially:

[0018] 2.1) Transform competent Agrobacterium rhizogenes strain Ar.Qual with a plasmid containing the target gene to obtain Agrobacterium rhizogenes expressing the target gene;

[0019] 2.2) Pick a single colony of Agrobacterium rhizogenes (Agrobacterium rhizogenes of the target expression plasmid) obtained from step 2.1) into LB medium containing 50 mg / ml kanamycin and 50 mg / ml streptomycin sulfate, and incubate in a shaker (220 rpm) at 28±1℃ until the OD 600 is 1±0.1 (incubation time is about 14 to 16 hours);

[0020] After centrifugation, the supernatant was discarded, and Agrobacterium rhizogenes precipitate was obtained;

[0021] 2.3) Resuspend the Agrobacterium tumefaciens precipitate obtained in step 2.2) with a rooting agent until the final bacterial solution OD600 is 1±0.1; thus obtaining the Agrobacterium tumefaciens infection solution.

[0022] In this step, the volume of the hair root infection aid is approximately the same as the volume of the supernatant in step 2.2).

[0023] As a further improvement to the soybean hairy root induction transformation method of the present invention, step 3) is as follows:

[0024] Take the sterile bean sprouts obtained in step 1), peel off the seed coat, cut off the apical bud and axillary bud, cut off the hypocotyl of 2-3 mm, and make wounds (3-4 wounds) from the middle to the end of the cotyledons; immerse the cotyledons obtained by the above treatment in Agrobacterium rhizogenes infection solution for 10-15 minutes.

[0025] As a further improvement to the soybean hairy root-induced transformation method of the present invention,

[0026] The germination in step 1) is as follows: cultivate until the soybean cotyledons turn green and just sprout true leaves (cultivation time is about 5 days).

[0027] The co-culture in step 4) is as follows: the infected cotyledons are transferred to a co-culture medium covered with sterile filter paper and cultured in the dark at 25°C for 3 days ± 0.5 days; the enlarged cotyledons obtained from the co-culture medium are transferred to a rooting medium and cultured in the dark at 28°C for 14 ± 2 days (at this time, the hairy roots of the cotyledons gradually elongate and cover the entire medium).

[0028] Step 5) is: detecting positive roots using GFP.

[0029] The technical advantages of this invention are:

[0030] 1. The use of the root-promoting agent specially designed in this invention significantly improves the positive conversion efficiency of hairy root production.

[0031] This invention utilizes a newly developed mixture containing acetylsyringone, plant hormones (6-BA, gibberellin), and SilwetL-77 infection-aiding agent in the traditional explant culture stage. The use of this infection-aiding agent significantly improved the transformation efficiency of soybean hairy roots.

[0032] 2. This invention simplifies the operation steps and reduces human-caused pollution:

[0033] Existing technologies require approximately 15-30 minutes of incubation in a shaker at 100-200 rpm before Agrobacterium infects the cotyledons. After incubation, the infected soybean cotyledons need to be washed with a solution (generally, the cotyledon surface needs to be washed 3 times). Due to these two steps, the transformation process is time-consuming and easily contaminated due to the extra steps.

[0034] The present invention adds a root-infection aid to the bacterial solution, which reduces the time required for co-culturing the bacterial solution with soybean cotyledons (only 10-15 minutes), thus eliminating the need for the steps described in the prior art.

[0035] Actual results show that by using a root-infection aid and eliminating the steps of shaking cotyledons with engineered bacterial solution and washing cotyledons, the present invention can significantly shorten the conversion time, reduce contamination during operation, and significantly improve the conversion efficiency. Attached Figure Description

[0036] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Figure 1 The process of soybean hairy root transformation using cotyledons as explants;

[0038] Figure 1 middle:

[0039] 1: Soybean sterilization with chlorine gas; 2: Soybean germination in germination medium; 3: Soaking treated soybean cotyledons in Agrobacterium-infected solution; 4: Co-culturing cotyledons with Agrobacterium; 5: Transferring cotyledons to rooting medium for culture; 6: Cotyledon status after 5 days in rooting medium; 7: Cotyledon status after 8 days in rooting medium; 8: Cotyledon status after 14 days in rooting medium.

[0040] Figure 2 Schematic diagram of soybean cotyledon removed;

[0041] Figure 3 Comparison of cotyledon size before and after co-cultivation;

[0042] Figure 3In the image, the left image represents the cotyledon state after 0 days of co-cultivation, and the right image represents the cotyledon state after 3 days of co-cultivation.

[0043] Figure 4 Hairy root reporter gene detection;

[0044] Figure 4 The left image shows a microscopic photograph of soybean hairy roots under white light, while the right image shows a microscopic photograph of soybean hairy roots under fluorescence.

[0045] Figure 5 Hairy root reporter gene detection;

[0046] Figure 5 From left to right: Microscopic photograph of positive roots under white light; Microscopic photograph of positive roots under fluorescence; Photograph taken by camera under white light.

[0047] Figure 6 The effect of root-promoting agents on the transformation efficiency of hairy roots in soybeans of different genotypes.

[0048] Figure 7 The effect of different components in root-promoting agents on the conversion efficiency of soybean hairy roots. Detailed Implementation

[0049] To further illustrate this method and its effects, the following detailed description is provided with examples and accompanying figures.

[0050] Example 1: A method for genetic transformation of soybean mediated by Agrobacterium rhizogenes and assisted by a root-promoting agent, mainly including the following steps:

[0051] 1. Preparation before the experiment (sterilization):

[0052] Select healthy soybean seeds harvested that year that are smooth, plump, free of disease spots, and placed in petri dishes. Then, place the petri dishes in a sealed desiccator within a fume hood, leaving the lids open. First, mix 96 ml of sodium hypochlorite and 6 ml of concentrated hydrochloric acid in an Erlenmeyer flask within the desiccator. Quickly seal the desiccator and sterilize with chlorine for 8 hours. After sterilization, place the soybean seeds in a sterile laminar flow hood under ultraviolet light to remove the chlorine (approximately 1 hour). Figure 1-1 As stated above.

[0053] Step 1 above is a conventional technique.

[0054] 2. Germination:

[0055] The sterilized seeds were cultured on germination medium for 5 days, such as... Figure 1-2The cultivation conditions were: temperature 25℃, darkness, with 16 hours of light (3000 lux) starting on the fourth day, followed by 8 hours of darkness at 25℃. Cultivation continued until the soybean cotyledons turned green and true leaves just emerged; the total cultivation time at this point was 5 days.

[0056] Step 2 above is a standard technique.

[0057] 3. Preparation of engineered Agrobacterium rhizogenes culture:

[0058] 3.1) Transform competent Agrobacterium rhizogenes strain Ar.Qual with a plasmid containing the target gene to obtain Agrobacterium rhizogenes expressing the target gene. The target gene contains a GFP fluorescent transgenic plant selection marker for rapid screening of successfully transformed soybean plants. This step is a standard technique and can be performed, for example, by referring to the published "A Method for Cultivating Marker-Free Transgenic Plants and Its Dedicated Expression Vector".

[0059] The target gene is, for example, a disease-resistant gene, a stress-resistant gene, or an insect-resistant gene.

[0060] 3.2) Pick the transformed Agrobacterium rhizogenes single colony into LB medium containing the corresponding kanamycin (50 mg / ml) and streptomycin sulfate (50 mg / ml), and incubate overnight (about 16 hours) in a shaker at 28°C and 220 rpm.

[0061] When the overnight bacterial culture reaches an OD of 1 at 6000, centrifuge at 6000 rpm for 10 min, discard the supernatant, and obtain Agrobacterium rhizogenes precipitate.

[0062] 3.3) Prepare a root-infection aid with the following formula: B5 medium 3.16 g / L, sucrose 30 g / L, MES sodium salt 3.9 g / L, acetylsuccinone 40 mg / L, hormone mixture (6-BA 1.67 mg / L, gibberellin 0.025 mg / L), surfactant Silwet L-77 100 ml / L, pH = 5.4.

[0063] The specific preparation method of the infection solution is as follows: 3.16g of B5 medium, 30g of sucrose, 3.9g of sodium MES, 40mg of acetylsuccinone, 1.67mg of 6-BA, 0.025mg of gibberellin, 100ml of Silwet L-77, add water to make up to 1000ml, and then adjust the pH to 5.4.

[0064] All of the above components are commercially available. For example, B5 culture medium was purchased from PM1322 (product number) of Beijing Cooler Technology Co., Ltd.; sodium MES (2-(N-morpholino) ethanesulfonate sodium) was purchased from CM7192 of Beijing Cooler Technology Co., Ltd.; and surfactant Silwet L-77 was purchased from CS9791 of Beijing Cooler Technology Co., Ltd.

[0065] 3.4) Resuspend the Agrobacterium rhizogenes precipitate obtained after centrifugation in step 3.2 with an equal volume of the rooting-promoting agent prepared in step 3.3, ensuring that the final bacterial culture OD600 is 1.0. That is, the volume of the rooting-promoting agent used is approximately the volume of the supernatant in step 3.2.

[0066] 4. Wound infection:

[0067] Take the sterile soybean seedlings obtained in step 2, peel off the seed coat, remove the true leaves, and cut off 3-4 mm from the end of the hypocotyl of the cotyledons. Figure 2 Make 3-4 wounds from the middle to the tip of the cotyledons; immerse the treated cotyledons in the Agrobacterium rhizogenes infection solution obtained in step 3 for 10-15 minutes (e.g., ...). Figure 1-3 ).

[0068] 5. Co-cultivation:

[0069] Remove the infected cotyledons and transfer them to a co-culture medium covered with sterile filter paper. Incubate in the dark at 25°C for 3 days (e.g., Figure 1-4 , Figure 3 (As shown).

[0070] The co-culture medium was formulated as follows: B5 medium 3.16 g / L, sucrose 30 g / L, sodium MES 3.9 g / L, DTT (dithiothreitol) 0.154 g / L, cysteine ​​0.4 g / L, sodium thiosulfate 0.248 g / L, pH = 5.4.

[0071] 6. Inducing cotyledon root development:

[0072] After 3 days of culture on a co-culture medium, the swollen cotyledons were transferred to a rooting medium (e.g., ...). Figure 1-4 Cultured in darkness at 28°C until the cotyledons develop hairy roots. Figure 1-5 ~ Figure 1 -8). The optimal time to observe positive root growth is at least 14 days after transferring to the rooting medium (at this time, the cotyledon hairy roots gradually elongate and cover the entire medium).

[0073] The rooting medium formula is B5 medium 3.16 g / L, sucrose 20 g / L, MES 0.59 g / L, pH=5.6. The sources of B5, sucrose, and MES are the same as above.

[0074] 7. Positive root detection:

[0075] Under a fluorescence microscope, using a GFP fluorescence filter, observe the newly grown soybean hairy roots; if green fluorescence is observed in the roots under the fluorescence microscope, positive roots have been obtained. Figure 4 , Figure 5 As shown.

[0076] Experiment 1: Effects of root-promoting agents on hairy root transformation efficiency in soybeans of different genotypes

[0077] Experimental group (agent for infection): according to Example 1.

[0078] Control group (CK): The "rooting agent" was not used for resuspension, that is, steps 3.3 to 3.4 of Example 1 were omitted, and the overnight bacterial solution obtained in step 3.2 with an OD of 1 was directly used to replace the "rooting agent" of Agrobacterium tumefaciens obtained in step 3 for subsequent wound infection. The rest was the same as in Example 1.

[0079] The target gene plasmid is pSM101-pCambia1300:35s-eGFP; this is an existing plasmid.

[0080] Three soybean varieties, “Zhonghuang 39,” “Williams 82,” and “Forrest,” were selected and tested according to the experimental and control groups described above. The positive conversion rates of the three varieties were statistically analyzed, including the proportion of positive roots produced by cotyledons, the proportion of hairy roots produced by cotyledons, and the positive root rate for each variety. The experiment was independently repeated three times before statistical analysis. The positive conversion rate was calculated using the formulas: positive roots / total roots * 100% (Statistic 1); cotyledons with positive roots / total cotyledons * 100% (Statistic 3); and the total rooting rate was calculated using the formula: cotyledons with hairy roots / total cotyledons * 100% (Statistic 2). Statistics 1 and 3 reflect the positive root conversion rate among all hairy roots, while Statistics 2 reflects the overall rooting rate in the experimental batch. The results are as follows (…). Figure 6 ):

[0081] The transformation cycle of the hairy root variety "Zhonghuang 39" was 22 days (the sum of the days for step 2 being 5 days, step 5 being 3 days, and step 6 being 14 days). The positive root transformation rates in the experimental group were 52.8% (Statistic 1) and 83.3% (Statistic 3), while the positive root transformation rates in the control group were 13.4% (Statistic 1) and 19.6% (Statistic 3). The total rooting rate in the experimental group was 95.5% (Statistic 2), and the total rooting rate in the control group was 85.8% (Statistic 2).

[0082] The "Williams 82" variety had a hairy root transformation cycle of 22 days. The positive root transformation rates in the experimental group were 27.4% (Statistic 1) and 71.5% (Statistic 3), while those in the control group were 7.5% (Statistic 1) and 21.4% (Statistic 3). The total rooting rate in the experimental group was 93.8% (Statistic 2), and in the control group it was 81.5% (Statistic 2).

[0083] Forrest variety: The hairy root transformation cycle was 22 days. The positive root transformation rates in the experimental group were 57.7% (Statistic 1) and 77.4% (Statistic 3), while the positive root transformation rates in the control group were 10.2% (Statistic 1) and 19.8% (Statistic 3). The total rooting rate in the experimental group was 85.8% (Statistic 2), and the total rooting rate in the control group was 60.2% (Statistic 2).

[0084] In summary, this invention introduces a root-forming Agrobacterium-associated infection agent during the Agrobacterium wound infection process. Compared to the control group without the added root-forming infection agent, it does not affect the overall rooting rate (Statistical 2 results). However, the positive transformation efficiency of different soybean varieties with the added root-forming infection agent is significantly improved, increasing to 3-4 times the original efficiency (based on Statistical 1 and 3 results). Figure 6 ).

[0085] Experiment 2: Effect of the addition of a mixture of surfactants and hormones to a root-promoting agent on the conversion efficiency of soybean hairy roots.

[0086] Experimental Group 1: The formula of the hair root infection aid was the same as in Example 1.

[0087] Experimental Group 2: The surfactant in the hair root dyeing agent formulation of Example 1 was removed, and the rest was the same as in Example 1.

[0088] That is, the formula for the root-infecting agent was changed to: B5 medium 3.16g / L, sucrose 30g / L, MES sodium salt 3.9g / L, acetylsuccinone 40mg / L, hormone mixture (6-BA 1.67mg / L, gibberellin 0.025mg / L), pH=5.4.

[0089] Experimental Group 3: The surfactant and hormone mixture in the hair root dyeing agent formulation of Example 1 were removed, and the rest was the same as in Example 1.

[0090] That is, the formula for the root-infecting agent was changed to: B5 medium 3.16g / L, sucrose 30g / L, MES sodium salt 3.9g / L, acetylsuccinone 40mg / L, pH=5.4.

[0091] The experiment was conducted using the "Wan Dou 28" variety according to the above experimental groups, and the results were as follows:

[0092] In experimental groups 1, 2, and 3, the hairy root transformation cycle was 22 days (the sum of the number of days in step 2 being 5 days, step 5 being 3 days, and step 6 being 14 days).

[0093] Experimental Group 1: The conversion rate of Statistical 1 was 53.7%, the conversion rate of Statistical 2 was 96.1%, and the conversion rate of Statistical 3 was 89.5%.

[0094] Experimental Group 2: The conversion rate of Statistic 1 was 26.6%, the conversion rate of Statistic 2 was 74.8%, and the conversion rate of Statistic 3 was 55.6%.

[0095] Experimental Group 3: The conversion rate of Statistical 1 was 14.2%, the conversion rate of Statistical 2 was 68.3%, and the conversion rate of Statistical 3 was 22.4%.

[0096] In summary, the results for pea variety 28 showed that: compared with experimental group 3, the conversion efficiency of root-promoting agent with added plant hormone mixture (experimental group 2) was increased by 2.48 times; the conversion efficiency of experimental group 1 with both hormone mixture and surfactant was increased by approximately 4.0 times compared with experimental group 3; and the conversion efficiency of experimental group 1 was increased by 1.6 times by adding surfactant to the root-promoting agent with added plant hormone mixture. Figure 7 Different statistical methods for positive roots showed similar increases in conversion efficiency (Statistics 1 and 3). Both the hormone mixture and surfactant in the hair root growth promoter formulation significantly improved conversion efficiency, with the highest increase observed when both were added simultaneously.

[0097] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for inducing and transforming soybean hairy roots using Agrobacterium tumefaciens-mediated root hairy rooting agents, characterized in that... Includes the following steps: 1) Disinfection and germination of soybean seeds; germination refers to cultivating the soybean seeds until the cotyledons turn green and the true leaves just emerge. 2) Resuspend the Agrobacterium rhizogenes containing the target expression plasmid in a Agrobacterium rhizogenes infection aid to obtain an Agrobacterium rhizogenes infection aid solution; The formula for the root-promoting infection agent is as follows: B5 medium 3.0-3.5 g / L, sucrose 28-32 g / L, MES 3.8-4.0 g / L, acetylsuccinone 38-42 mg / L, 6-BA 1.5-1.8 mg / L, gibberellin 0.02-0.03 mg / L, surfactant Silwet L-77 100 ml / L, pH=5.4; Specifically, the following steps are performed in sequence: 2.1) Transform competent Agrobacterium rhizogenes ArQua strain with a plasmid containing the target gene to obtain Agrobacterium rhizogenes expressing the target plasmid; 2.2) Pick the single colony of Agrobacterium rhizogenes obtained from step 2.1) into LB medium containing 50 mg / ml kanamycin and 50 mg / ml streptomycin sulfate, and incubate in a shaker at 28±1℃ until the OD 600 is 1±0.1; After centrifugation, the supernatant was discarded, and Agrobacterium rhizogenes precipitate was obtained; 2.3) Resuspend the Agrobacterium rhizogenes precipitate obtained in step 2.2) with a rooting agent until the final bacterial solution has an OD 600 of 1 ± 0.1; thus obtaining the Agrobacterium rhizogenes infection solution. 3) Infection: Soak soybean cotyledons in Agrobacterium rhizogenes infection solution for 10-15 minutes to obtain infected cotyledons; 4) Co-culture: The infected cotyledons are first cultured on a co-culture medium, and then cultured on a rooting medium. 5) Screen for positive roots.

2. The soybean hairy root induction transformation method according to claim 1, characterized in that... Step 3) is as follows: Take sterile bean sprouts, peel off the seed coat, cut off the apical bud and axillary bud, cut off the hypocotyl of 2-3 mm, and make wounds from the middle to the end of the cotyledons; immerse the treated cotyledons in Agrobacterium rhizogenes infection solution for 10-15 minutes.

3. The soybean hairy root induction transformation method according to claim 2, characterized in that: The co-culture in step 4) is as follows: the infected cotyledons are transferred to a co-culture medium covered with sterile filter paper and incubated in the dark at 25°C for 3 days ± 0.5 days. The enlarged cotyledons obtained from co-culture were transferred to rooting medium and cultured in the dark at 28℃ for 14±2 days.

4. The soybean hairy root induction transformation method according to claim 3, characterized in that... Step 5) is: detecting positive roots using GFP.

5. The soybean hairy root induction transformation method according to any one of claims 1 to 4, characterized in that: The formula for the hair root infection aid is as follows: B5 medium 3.16 g / L, sucrose 30 g / L, MES 3.9 g / L, acetylsuccinone 40 mg / L, 6-BA 1.67 mg / L, gibberellin 0.025 mg / L, surfactant Silwet L-77 100 ml / L, pH = 5.4.

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