Soybean genetic transformation method

Abstract

The invention provides a soybean genetic transformation method. The method comprises the following steps: seed disinfection and germination, explant preparation, infection, co-culture, recovery culture, bud induction, bud elongation, transplanting and seedling hardening. According to the co-culture method, explants are placed in a culture dish without a co-culture medium, and the infection efficiency of the explants and the genetic transformation efficiency of the soybeans can be improved.

Description

Technical Field

[0001] The invention belongs to the field of biology and modern agricultural technology, and relates to a soybean genetic transformation method, which specifically comprises the step of placing an explant in a culture dish without a co-culture medium for co-culture. Background Art

[0002] Soybean is one of the most important legume crops and an important source of plant protein and vegetable oil in human diet. Traditional hybrid breeding technology is highly random, inefficient, has a long breeding cycle, and lacks the source of some excellent traits. Gene editing breeding makes up for the shortcomings of conventional breeding methods and has triggered a revolution in agricultural breeding. Soybean genetic transformation is the core and key link of soybean gene editing breeding. However, soybean genetic transformation still faces technical difficulties such as low transformation efficiency, unstable transformation system, severe genotype restrictions and long transformation cycle.

[0003] Agrobacterium-mediated genetic transformation of soybean cotyledon nodes is the most commonly used soybean transformation system. Hinchee et al. first used this method in 1988 to obtain positive transformed plants. They germinated mature soybean seeds for 5 to 7 days, then divided the cotyledons of the seedlings into two along the hypocotyl, removed the epicotyl, and scratched the cotyledon nodes at the base of the cotyledons with a scalpel. After 30 minutes of Agrobacterium infection, the explants were placed on co-culture medium and cultured for 3 to 5 days. Transformed plants were obtained through bud induction, bud elongation, rooting, and transplanting. Paz et al. improved the soybean cotyledon node transformation system and called it the "half-seed" method. They used half cotyledons obtained by cutting mature soybean seeds along the hypocotyl after overnight imbibition germination as explants, and the average genetic transformation efficiency reached 3.8%.

[0004] However, the Agrobacterium-mediated transformation method also has many serious problems, including two main ones: first, soybeans are poorly sensitive to Agrobacterium tumefaciens and have low infection efficiency; second, there is a lack of an efficient regeneration system, making it difficult to extend multiple buds. These two factors have resulted in the transformation efficiency of this method being still very low, which has seriously restricted soybean gene function research and molecular breeding. Summary of the invention

[0005] In order to improve the genetic transformation efficiency of soybean, the present invention provides a soybean genetic transformation method.

[0006] In one aspect, the present invention provides a method for genetic transformation of soybean, comprising the step of placing an explant in a culture dish without co-cultivation medium for co-cultivation.

[0007] In another preferred embodiment, the culture dish containing no co-cultivation medium includes a blank culture dish and / or a culture dish only covered with a layer of dry filter paper.

[0008] In another preferred embodiment, the method comprises the step of placing the explant in a blank culture dish without co-cultivation medium, or in a culture dish without co-cultivation medium covered with filter paper for co-cultivation.

[0009] In another preferred embodiment, the method comprises the following steps:

[0010] (1) Seed disinfection and germination;

[0011] (2) preparing explants;

[0012] (3) Infection;

[0013] (4) co-cultivating using the above method;

[0014] (5) Recovery culture;

[0015] (6) bud induction;

[0016] (7) bud elongation;

[0017] (8) Transplanting and hardening of seedlings.

[0018] In another preferred embodiment, the seed disinfection and germination in step (1) are performed using conventional methods.

[0019] In another preferred embodiment, the method for preparing the explant in step (2) is to remove part of the embryonic axis, gently break the seed in the middle, and retain half of the cotyledon with a complete cotyledon node growth point as the explant.

[0020] In another preferred embodiment, the infection in step (3) is performed using conventional methods.

[0021] In another preferred embodiment, the culture medium used for infection in step (3) contains 0.8-1.2 mg / L zeatin.

[0022] In another preferred embodiment, the culture medium used for infection in step (3) contains 1 mg / L zeatin.

[0023] In another preferred embodiment, the culture medium used for infection in step (3) comprises the following components: 0.215 g / L MS basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamin, 3.9 g / L MES sodium salt buffer, 80 mg / L acetosyringone, 200 mg / L dithiothreitol, 0.8-1.2 mg / L zeatin, and the pH is adjusted to 5.4.

[0024] In another preferred embodiment, the culture medium used for infection in step (3) comprises the following components: 0.215 g / L MS basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamins, 3.9 g / L MES sodium buffer, 80 mg / L acetosyringone, 200 mg / L dithiothreitol, and 1 mg / L zeatin, and the pH is adjusted to 5.4.

[0025] In another preferred embodiment, the infection method in step (3) comprises the step of infecting soybean explants with Agrobacterium containing the target gene.

[0026] In another preferred embodiment, the target gene includes the GUS gene.

[0027] In another preferred embodiment, the co-cultivation method in step (4) is to place the explant in a blank culture dish without co-cultivation culture medium and culture it in the dark at 22° C. for 3 to 5 days.

[0028] In another preferred embodiment, the step (5) recovery culture is performed using conventional methods.

[0029] In another preferred embodiment, the culture medium used for bud induction in step (6) contains 12.6±1 mg / L of glyphosate.

[0030] In another preferred embodiment, the culture medium used for bud induction in step (6) contains 12.6 mg / L of glyphosate.

[0031] In another preferred embodiment, the culture medium used for bud induction in step (6) comprises the following components: 3.1 g / L B5 basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamins, 0.6 g / L MES sodium buffer, 1.5 mg / L BAP, 200 mg / L timentin, 150 mg / L ceftriaxone sodium, 5.4 g / L agar powder, 12.6±1 mg / L glyphosate, and the pH is adjusted to 5.8.

[0032] In another preferred embodiment, the culture medium used for bud induction in step (6) comprises the following components: 3.1 g / L B5 basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamins, 0.6 g / L MES sodium buffer, 1.5 mg / L BAP, 200 mg / L timentin, 150 mg / L ceftriaxone sodium, 5.4 g / L agar powder, 12.6 mg / L glyphosate, and the pH is adjusted to 5.8.

[0033] In another preferred embodiment, the culture medium used for bud elongation in step (7) contains 8.5±1 mg / L of glyphosate.

[0034] In another preferred embodiment, the culture medium used for bud elongation in step (7) contains 8.5 mg / L of glyphosate.

[0035] In another preferred embodiment, the culture medium used for bud elongation in step (7) comprises the following components: 4.3 g / L MS basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamin, 0.6 g / L MES sodium buffer, 0.1 mg / L IAA, 0.5 mg / L GA3, 1 mg / L zeatin, 200 mg / L timentin, 150 mg / L ceftriaxone sodium, 5.4 g / L agar powder, 8.5±1 mg / L glyphosate, and the pH is adjusted to 5.8.

[0036] In another preferred embodiment, the culture medium used for bud elongation in step (7) comprises the following components: 4.3 g / L MS basal medium, 30 g / L sucrose, (1000×) 1 mL / L B5 vitamin, 0.6 g / L MES sodium buffer, 0.1 mg / L IAA, 0.5 mg / L GA3, 1 mg / L zeatin, 200 mg / L timentin, 150 mg / L ceftriaxone sodium, 5.4 g / L agar powder, 8.5 mg / L glyphosate, and the pH is adjusted to 5.8.

[0037] In another preferred embodiment, the method of transplanting and hardening the seedlings in step (8) is that the elongated buds are transplanted into a plug tray filled with vermiculite for rooting, and the plug tray filled with vermiculite does not contain a rooting medium.

[0038] In another preferred embodiment, the soybean is any one or more of the following: Jack, Williams 82.

[0039] In another preferred embodiment, the method can improve the infection efficiency and / or genetic transformation efficiency of soybean.

[0040] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0041] The main advantages of the present invention are:

[0042] The present invention has found through research that, in the soybean genetic transformation process, placing the explant in a blank culture dish without co-cultivation medium for co-cultivation can improve the soybean genetic transformation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 , the degree of staining of GUS dye at the cotyledon node of the explant.

[0044] Figure 2 , infection efficiency and transformation efficiency of different soybean varieties. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0046] Example 1: Effect of co-cultivation mode on soybean transient infection efficiency and stable transformation efficiency

[0047] In this example, two soybean varieties, Jack and Williams 82, were selected and experiments were carried out according to the following experimental methods to test their infection efficiency and stable transformation efficiency.

[0048] 1. Vector construction

[0049] The binary vector is used as the basic vector for transformation to construct the expression vector. The recombinant expression vector contains two expression elements, one is the GUS expression element for transient expression analysis, which is composed of pr35S-GUS-tNOS, and the other is the screening marker gene for screening: pr35S-CP4.EPSPS-tNOS. The binary vector is introduced into Agrobacterium EHA101 using the electroporation transformation method to obtain recombinant Agrobacterium, which is stored in a -80℃ refrigerator for use.

[0050] 2. Seed disinfection

[0051] Select smooth and plump soybean seeds and place them in a petri dish, then place the petri dish in a closed desiccator in a fume hood with the lid open. First, take 120 ml of sodium hypochlorite and 8 ml of concentrated hydrochloric acid and mix them in a beaker in the desiccator, quickly seal the desiccator, and use the chlorine generated by the reaction of hydrochloric acid and sodium hypochlorite to sterilize the soybean seeds for 18 hours, and place the sterilized soybean seeds in a sterile clean bench for about 2 hours to blow away the chlorine.

[0052] 3. Seed germination

[0053] Insert the disinfected seeds with the hilum downwards into the solid germination medium, the insertion depth is about 2 / 3 of the seeds, 16 seeds / dish, and germinate overnight at 22°C in the dark. The composition of the solid germination medium is: B5 basal medium 3.1g / L, sucrose 20g / L, B5 vitamin (1000×) 1mL / L, plant gel phytagel 4g / L, and the pH is adjusted to 5.6.

[0054] 4. Explant Preparation

[0055] The conventional method for preparing explants is to cut off part of the hypocotyl after the seeds germinate, and use a scalpel to divide the cotyledons together with the embryo tip growth point into two, so as to obtain a half-seed explant. In this embodiment, after cutting off part of the hypocotyl, the seeds are gently broken apart from the middle, and half of the cotyledons with complete cotyledon node growth points are retained as explants. This method can maximize the retention of meristem cells with strong division and differentiation ability in the growth point, which is beneficial to the subsequent bud induction and extension.

[0056] 5. Infection

[0057] The above Agrobacterium was taken out from the -80℃ refrigerator and activated, and then collected and resuspended in liquid infection medium. The final OD 660 The pH value is controlled at 0.6-0.8 to obtain the infection solution, and then the soybean cotyledon node explants are placed in the infection solution for infection for 1-2 hours. The composition of the infection medium is: MS basic medium 0.215g / L, sucrose 30g / L, B5 vitamin (1000×) 1mL / L, MES sodium salt buffer 3.9g / L, zeatin 1mg / L, acetosyringone 80mg / L, dithiothreitol 200mg / L, and the pH is adjusted to 5.4.

[0058] The concentration of zeatin used in the infection solution is preferably 0.8-1.2 mg / L. The inventors have found that adding zeatin in this concentration range to the infection solution is more conducive to the infection of Agrobacterium and does not affect the subsequent bud induction effect.

[0059] 6. Co-cultivation

[0060] The conventional co-culture method is to place the infected explants on the surface of a solid co-culture medium covered with a layer of filter paper or a filter paper containing a certain volume of liquid co-culture medium, and co-culture for 3 to 5 days under dark conditions at 22°C. The commonly used liquid co-culture medium is composed of: MS basal medium 0.215g / L, sucrose 30g / L, B5 vitamin (1000×) 1mL / L, MES sodium salt buffer 3.9g / L, zeatin 1mg / L, and the pH value is adjusted to 5.4.

[0061] In this embodiment, the control group adopts the conventional co-cultivation method, and the experimental group places the explants in a blank culture dish without co-cultivation medium or in a culture dish covered with a dry filter paper, and the explants are evenly placed in each dish, and cultured for 3 to 5 days under 22°C dark culture conditions. The other steps are the same as the control group. In this embodiment, the explants are stained with GUS dye, and the infection efficiency is calculated according to the coloring degree of the cotyledon node.

[0062] The liquid or solid co-culture medium in the conventional co-culture method can provide moisture and nutrition for the explants during the co-culture period. However, the co-culture method of the present invention uses a blank culture dish without a co-culture medium, which does not provide moisture and nutrition for the explants, resulting in dryness and nutritional stress on the explants. A layer of dry filter paper can even be laid in the blank culture dish to absorb the infection liquid remaining on the surface of the explants after infection, further strengthening the dryness and nutritional stress. This stress may weaken the defense response of the plant, but increase the sensitivity of the plant cells to Agrobacterium, thereby improving the infection efficiency.

[0063] 7. Resume training

[0064] The explants after co-cultivation were transferred to the recovery medium and cultured at 26°C for 7 days, with a light condition of 16 hours light / 8 hours dark during the culture period. The composition of the recovery medium was: B5 basal medium 3.1 g / L, sucrose 30 g / L, B5 vitamin (1000×) 1 mL / L, MES sodium buffer 0.6 g / L, BAP 0.5 mg / L, timentin 200 mg / L, cefotaxime sodium 150 mg / L, agar powder 5.4 g / L, and the pH was adjusted to 5.8.

[0065] 8. Bud induction culture

[0066] The explants after recovery culture were transferred to a bud induction medium containing glyphosate and cultured at 26°C for 21 days. The light conditions during the culture period were 16 hours of light / 8 hours of darkness. The composition of the bud induction screening medium was: B5 basal medium 3.1 g / L, sucrose 30 g / L, B5 vitamin (1000×) 1 mL / L, MES sodium buffer 0.6 g / L, BAP 1.5 mg / L, glyphosate 12.6 mg / L, timentin 200 mg / L, cefotaxime sodium 150 mg / L, agar powder 5.4 g / L, and the pH was adjusted to 5.8.

[0067] The present invention preferably sets the screening concentration of glyphosate to 12.6±1 mg / L, and this concentration range can achieve effective screening of negative buds without affecting the bud induction effect.

[0068] 9. Bud elongation culture

[0069] The clustered buds obtained by screening were transferred to the bud elongation medium and continued to be cultured at 26°C for 21 days to obtain elongated buds. The lighting conditions were: 16 hours of light / 8 hours of darkness. The bud elongation medium consisted of: MS basal medium 4.3g / L, sucrose 30g / L, B5 vitamin (1000×) 1mL / L, MES sodium buffer 0.6g / L, IAA 0.1mg / L, GA3 0.5mg / L, zeatin 1mg / L, glyphosate 8.5mg / L, timentin 200mg / L, cefotaxime sodium 150mg / L, agar powder 5.4g / L, and the pH was adjusted to 5.8.

[0070] The present invention preferably sets the concentration of the screening agent glyphosate to 8.5±1 mg / L, which is a concentration range that can significantly increase the positive rate of extended buds while not affecting the efficiency of bud extension.

[0071] 10. Transplanting and hardening seedlings

[0072] The conventional transplanting and seedling hardening method is: transfer the extended buds to the rooting medium and culture for 21 days, and then transplant them to the greenhouse nutrient soil for further seedling hardening and harvesting seeds after rooting. The composition of the rooting medium is: MS basal medium 2.15g / L, sucrose 20g / L, B5 vitamin (1000×) 1mL / L, MES sodium buffer 0.6g / L, IBA 1mg / L, timentin 200mg / L, cefotaxime sodium 150mg / L, agar powder 5.4g / L, and the pH is adjusted to 5.8.

[0073] In this embodiment, the elongated buds are directly transplanted into a plug tray filled with vermiculite for rooting without rooting in a rooting medium. After the lid is covered and cultured for 4 to 5 days, the air holes on the lid are opened. After 8 to 12 days, the lid can be removed for normal growth. The culture temperature is 28° C. and the lighting conditions are 16 hours of light / 8 hours of darkness.

[0074] 11. Experimental Results

[0075] The calculation method of infection efficiency is: infection efficiency (%) = (number of successfully infected explants / total number of infected explants) × 100%.

[0076] Note: The coloring degree of GUS dye at the cotyledon node of the explant is divided into four levels: heavy, medium, light and no. Figure 1 As shown. Figure 1In the figure, the circled area is the cotyledon node of the explant. If the area is deeply stained after infection and co-culture, and the stained area is greater than 3 / 4, it is considered to be severely stained; if the staining is relatively deep and the stained area is greater than 1 / 2, it is considered to be moderately stained; if the staining is relatively light after infection and co-culture, and the stained area is less than 1 / 2, it is considered to be lightly stained; no staining means that there is no staining in the cotyledon node of the explant. Light staining and no staining indicate that there are fewer cells expressing the GUS gene in the cotyledon node and the infection effect is poor. Therefore, only heavy and moderate staining are defined as successful infection.

[0077] The results of statistical infection efficiency are as follows Figure 2 As shown: In the experimental group, the infection efficiency of Jack and Williams 82 were 70.2% and 61.1%, respectively; while in the control group, the infection efficiency of these two varieties were 36.8% and 45%, respectively.

[0078] The calculation method of transformation efficiency is: transformation efficiency (%) = (number of positive transgenic seedlings / number of infected explants) × 100%.

[0079] The results of statistical transformation efficiency are as follows Figure 2 As shown: In the experimental group, the transformation efficiency of Jack and Williams 82 were 25.3% and 15.7%, respectively; while in the control group, the transformation efficiency of these two soybean varieties was 15.6% and 10%, respectively.

[0080] In summary, compared with the control group, the infection efficiency of Jack and Williams 82 increased by 0.91 and 0.36 times, respectively, while their transformation efficiency increased by 0.62 and 0.57 times, respectively. Therefore, when performing soybean genetic transformation, replacing the co-cultivation medium with the blank medium can improve the infection efficiency and genetic transformation efficiency of soybean.

[0081] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for soybean genetic transformation, characterized in that: The method comprises the step of placing the explant in a culture dish without co-cultivation medium for co-cultivation.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Seed disinfection and germination; (2) preparing explants; (3) Infection; (4) co-cultivating using the method of claim 1; (5) Recovery culture; (6) bud induction; (7) bud elongation; (8) Transplanting and hardening of seedlings.

3. The method according to claim 2, characterized in that The method for preparing the explant in step (2) is to remove part of the embryonic axis, gently break the seed apart from the middle, and retain half of the cotyledon with a complete cotyledon node growth point as the explant.

4. The method according to claim 2, characterized in that: The culture medium used for infection in step (3) contains 0.8-1.2 mg / L zeatin.

5. The method according to claim 2, characterized in that: The culture medium used for bud induction in step (6) contains 12.6±1 mg / L of glyphosate.

6. The method according to claim 2, characterized in that The culture medium used for bud elongation in step (7) contains 8.5±1 mg / L of glyphosate.

7. The method according to claim 2, characterized in that The method of transplanting and hardening the seedlings in step (8) is that the elongated buds are transplanted into a plug tray filled with vermiculite to take roots, and the plug tray filled with vermiculite does not contain a rooting medium.

8. The method according to claim 2, characterized in that: The infection method in step (3) comprises the step of infecting soybean explants with Agrobacterium containing the target gene.

9. The method according to claim 8, characterized in that The target gene includes the GUS gene.

10. The method according to claim 1, characterized in that The soybean is any one or more of the following: Jack, Williams 82.

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