Disinfection method and application of soybean seeds for plant tissue culture

Abstract

The invention belongs to the technical field of biology, and discloses a disinfection method and application of soybean seeds for plant tissue culture. The method comprises the following steps: selecting a plurality of parts of seeds which are consistent in size, full and smooth in surface without spots, and soaking with ethanol; shaking while soaking, and pouring out ethanol; respectively adding sodium hypochlorite solutions with different percentages of available chlorine into the obtained soybean seeds, and oscillating a container containing the soybean seeds and the sodium hypochlorite solutions; and cleaning the soybean seeds with sterile water, and blow-drying the soybean seeds on sterilized filter paper to obtain the disinfected soybean seeds. According to the method, only the sodium hypochlorite solution with the low concentration is used for treating the seeds, the disinfection time can be shortened, pollution to the environment can be reduced, meanwhile, seed coats of the seeds treated in the treatment mode can be reserved, scratching treatment can be conveniently conducted after the seeds germinate for 12-16 h, in addition, the germination rate is high, and the pollution rate is low.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for sterilizing soybean seeds for plant tissue culture and application thereof. Background Art

[0002] Soybean seeds are the main production and application object and are an irreplaceable source of plant protein and oil. Seeds are the core of agricultural development. The use of biobreeding with transgenic technology as the core can increase crop yields and reduce the risks of agricultural production through genetic improvement and genetic engineering. Tissue transformation of different soybean materials, screening and identification of those with good callus induction ability, regeneration ability and transformation ability, lay the foundation for the subsequent transgenic and gene editing work. Soybean seed disinfection is the first step in tissue culture.

[0003] The main methods for disinfecting soybean seeds include chlorine disinfection, sodium hypochlorite disinfection, mercuric chloride disinfection and secondary disinfection. The more commonly used method is chlorine disinfection, but because chlorine is a highly toxic gas, the airtightness of the experimental device is very high, and it is impossible to unify the chlorine concentration and disinfection time, which may cause excessive disinfection and cause damage to seed activity and environmental pollution. Mercury is highly toxic and pollutes the environment, so the mercuric chloride disinfection method has been eliminated internationally. The secondary disinfection process is too cumbersome and time-consuming. There is currently no uniform standard for the concentration of sodium hypochlorite disinfection of soybeans. Therefore, a suitable sodium hypochlorite seed disinfection method is very important for solving soybean tissue culture problems. Summary of the invention

[0004] In order to overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a method and application of disinfecting soybean seeds for plant tissue culture.

[0005] The technical solution is as follows: a method for disinfecting soybean seeds for plant tissue culture, which screens the sodium hypochlorite concentration suitable for disinfection through gradient screening and optimizes the steps of the sodium hypochlorite disinfection method, specifically comprising the following steps:

[0006] S1, select multiple soybean seeds of uniform size, fullness, smooth surface and no stains, soak them in ethanol, shake them while soaking, and pour off the ethanol;

[0007] S2, adding sodium hypochlorite solutions with different percentages of available chlorine to the soybean seeds obtained in step S1, and shaking the container containing the soybean seeds and the sodium hypochlorite solution;

[0008] S3, washing the soybean seeds in step S2 with sterile water, and drying them on sterilized filter paper to obtain sterilized soybean seeds.

[0009] In step S1, soak in 75% ethanol for 3 min.

[0010] In step S2, the effective chlorine concentration of sodium hypochlorite is 0.6%.

[0011] In step S2, sodium hypochlorite solutions with effective chlorine content of 0.6%, 1.2%, 1.8%, 2.4% and 3% were added to the obtained soybean seeds, respectively.

[0012] In step S2, a container containing soybean seeds and sodium hypochlorite solution is placed in a shaker at 25°C and shaken at 100-120 rpm for 30 minutes.

[0013] In step S3, the soybean seeds were washed 6 times with sterile water, each time by shaking for 1 min and blowing on sterile filter paper for 1-2 min.

[0014] In step S3, after obtaining the sterilized soybean seeds, they are cultured in a germination medium.

[0015] In step S3, the components of the germination medium include: B5, B5 vitamins, 1.67 mg / L 6-BA, 2% sucrose, 0.25% plant gum, pH=5.8.

[0016] Another object of the present invention is to provide an application of a method for disinfecting soybean seeds for plant tissue culture in soybean rooting transformation, wherein a sodium hypochlorite solution containing 0.6% effective chlorine is added to the obtained soybean seeds and a container containing the soybean seeds and the sodium hypochlorite solution is shaken for 30 minutes.

[0017] In combination with all the above technical solutions, the beneficial effects of the present invention are as follows: the method for disinfecting soybean seeds for plant tissue culture provided by the present invention is first disinfected with 75% ethanol for 3 minutes, then treated with 1.2% sodium hypochlorite solution at 25°C and 100rpm shaking table for 30 minutes, then washed with sterile water 6 times, each time shaking and washing for 1 minute, blowing on sterilized filter paper for 1-2 minutes, and finally cultured on germination medium at 25°C. The present invention only uses a relatively low concentration of sodium hypochlorite solution to treat seeds, which can reduce disinfection time and environmental pollution. At the same time, the seeds treated in this way can retain the seed coat, which is convenient for scratching after 12-16 hours of seed germination. In addition, the germination rate of this treatment is high and the pollution rate is low.

[0018] The present invention uses a low-concentration sodium hypochlorite solution disinfection method to replace the chlorine gas disinfection method, which can reduce the use of hydrochloric acid, reduce costs and reduce environmental pollution. The present invention uses the effective chlorine concentration to screen and calibrate the optimal disinfection concentration of sodium hypochlorite. The present invention uses the effective chlorine concentration index to screen and determine the optimal disinfection concentration, solving the problem that there is no unified standard for disinfecting soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings herein are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the description, serve to explain the principles of the present disclosure;

[0020] Figure 1 is a flow chart of a method for disinfecting soybean seeds for plant tissue culture provided by an embodiment of the present invention;

[0021] Figure 2 Schematic diagram of sterile seedlings obtained after Williams82 soybean seeds of the present invention are treated by the treatment method of Example 2-6;

[0022] Figure 3 A schematic diagram of sterile seedlings obtained after the Suinong 53 soybean seeds of the present invention are treated by the treatment method of implementation case 2-6;

[0023] Figure 4 A schematic diagram of sterile seedlings obtained after the Nannong 66 soybean seeds of the present invention are treated by the treatment method of implementation case 2-6;

[0024] Figure 5 It is a schematic diagram of the sterile seedlings obtained after the Williams82, Suinong53, and Nannong66 soybean seeds of the present invention are treated by the method of implementation case 7;

[0025] Figure 6 The attached figure is a schematic diagram of the rooting of soybeans successfully transformed from the Suinong 53 soybean seeds of the present invention after implementation case 8. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0027] The innovation of the present invention is that the present invention only uses a relatively low concentration of sodium hypochlorite solution to treat seeds, which can reduce disinfection time and environmental pollution. At the same time, the seeds treated in this way can retain the seed coat, which is convenient for scratching treatment after the seeds germinate for 12-16 hours. In addition, the treatment has a high germination rate and a low pollution rate.

[0028] Example 1: The method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention screens the sodium hypochlorite concentration suitable for disinfection through gradient screening, and optimizes the steps of the sodium hypochlorite disinfection method.

[0029] like Figure 1 As shown, the specific steps include:

[0030] S1, select multiple soybean seeds of uniform size, fullness, smooth surface and no stains, soak them in ethanol, shake them while soaking, and pour off the ethanol;

[0031] Soak in 75% ethanol for 3 minutes;

[0032] S2, adding sodium hypochlorite solutions with different percentages of available chlorine to the soybean seeds obtained in step S1, and shaking the container containing the soybean seeds and the sodium hypochlorite solution;

[0033] Sodium hypochlorite solutions with effective chlorine content of 0.6%, 1.2%, 1.8%, 2.4% and 3% are added to the obtained soybean seeds respectively, and the container containing the soybean seeds and the sodium hypochlorite solution is placed in a shaker at 25° C. and shaken at 100-120 rpm for 30 minutes; sodium hypochlorite is highly safe;

[0034] S3, washing the soybean seeds in step S2 with sterile water, and drying them on sterilized filter paper to obtain sterilized soybean seeds;

[0035] The soybean seeds were washed 6 times with sterile water, shaken for 1 min each time, and blown on sterile filter paper for 1-2 min to obtain sterilized soybean seeds, which were then cultured in germination medium.

[0036] The disinfection process in step S2 is: soaking the soybean seeds in 0.6% sodium hypochlorite solution.

[0037] The shaking time in step S3 is 30 min.

[0038] The method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention can be applied to the soybean rooting transformation process.

[0039] It can be seen from the above embodiments that the method of treating seeds with a relatively low concentration of sodium hypochlorite solution provided by the present invention can reduce the disinfection time and the pollution to the environment, and the operation is simple and easy. At the same time, the seeds treated in this way can retain the seed coat, which is convenient for scratching the seeds after 12-16 hours of germination. In addition, the germination rate of the treatment is high and the pollution rate is low.

[0040] Example 2, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0041] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0042] 2) Adding a sodium hypochlorite solution containing 0.6% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaker at 25° C. and shaking at 100-120 rpm for 30 minutes.

[0043] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0044] Example 3, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0045] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0046] 2) Adding a sodium hypochlorite solution containing 1.2% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaking incubator at 25° C. and shaking at 100-120 rpm for 30 minutes.

[0047] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0048] Example 4, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0049] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0050] 2) Adding a sodium hypochlorite solution containing 1.8% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaker at 25° C. and shaking at 100-120 rpm for 30 minutes.

[0051] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0052] Example 5, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0053] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0054] 2) Adding a sodium hypochlorite solution containing 2.4% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaking incubator at 25° C. and shaking at 100-120 rpm for 30 minutes.

[0055] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0056] Example 6, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0057] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0058] 2) Adding a sodium hypochlorite solution containing 3% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaking incubator at 25° C. and shaking at 100-120 rpm for 30 minutes.

[0059] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0060] Germination test: Three soybean varieties, Williams 82, Suinong 53 and Nannong 66, were selected and tested according to the treatment method in Examples 2-6. About 100 disinfected soybean seeds were transferred to the same germination medium for culture. The components of the germination medium included: B5, B5 vitamins, 1.67 mg / L 6-BA, 2% sucrose, 0.25% plant gum, pH = 5.8. The number of germinations and the number of contaminations after the seeds germinated and grew for 4 days were counted, and the germination and contamination conditions were recorded by taking photos. Figure 2 The results of the sterile seedling test obtained by Williams82 according to the treatment scheme in Example 2-6 are as follows: Figure 3 This is the test result of sterile seedlings obtained after the soybean seeds of Suinong No. 53 were treated by the treatment method of implementation case 1-5. Figure 4 The results of the sterile seedling test of Nannong 66 soybean seeds after being treated by the treatment methods of implementation cases 1-5 were repeated 3 times. The results are shown in Table 1:

[0061] Table 1 Sterile seedling test results

[0062]

[0063] It can be seen from the results in Table 1 that according to the treatment method of Example 2, the germination rates of the three varieties, Williams82, Suinong 53 and Nannong 66, decreased with the increase of disinfection time, and the contamination rate of Nannong 66 soybean decreased with the increase of disinfection time. Considering the two factors of germination rate and contamination rate, it was found that the effective chlorine concentration of sodium hypochlorite was more suitable at 0.6%.

[0064] Example 7, as another preferred embodiment, the method for disinfecting soybean seeds for plant tissue culture provided in the embodiment of the present invention comprises:

[0065] 1) Select seeds of uniform size, fullness, smooth surface and no stains, soak them in 75% ethanol for 3 minutes, shake them while soaking, and then pour out the ethanol.

[0066] 2) Adding a sodium hypochlorite solution containing 3% effective chlorine to the soybean seeds obtained in step 1), placing the container containing the soybean seeds and the sodium hypochlorite solution in a shaking incubator at 25° C. at 100-120 rpm for 20, 30 and 40 minutes respectively.

[0067] 3) Wash the soybean seeds in step 2) with sterile water for 6 times, shaking for 1 minute each time, and blowing on sterile filter paper for 1-2 minutes to obtain sterilized soybean seeds.

[0068] The number of seeds germinated and contaminated after 4 days of growth was counted. The results are shown in Table 2.

[0069] Table 2 Germination number and contamination number

[0070]

[0071] From Table 2 and Figure 5 From the experimental results of sterile seedlings obtained after the soybean seeds of Williams82, Suinong 53 and Nannong 66 of the present invention were treated by the method of implementation case 6, it can be seen that according to the treatment method of Example 2, the germination rates of the three varieties of Williams82, Suinong 53 and Nannong 66 decreased with the increase of disinfection time, and the contamination rate of Nannong 66 soybean decreased with the increase of disinfection time. Considering the two factors of germination rate and contamination rate, it was found that the sodium hypochlorite disinfection time of 30 minutes was more appropriate.

[0072] Implementation Case 8, after the seeds are disinfected according to the method of Implementation Case 2, they are inoculated on the germination medium (GM), and after inoculation, the medium is placed in a 26°C constant temperature box for dark culture for 16 hours. Take out the imbibed soybean seeds from the GM medium, cut the seeds in half along the hilum, pick out the embryo on the seed petal with the embryo with a scalpel, scratch the embryo axis under the embryo, and then put the scratched bean petals into the root-generating Agrobacterium infection solution for infection. After the infection is completed, pour out the Agrobacterium bacterial solution, pour the bean petals on the sterilized filter paper, absorb the bacterial solution, blow it in the clean bench for about 30 minutes, and then place the bean petals with the inner surface facing up on the co-culture medium covered with sterile filter paper, and co-culture at 23°C for 3 days. After 3 days of dark culture, cut the embryo, wash it with sterile water 4-5 times, and then wash it with liquid induction medium with hormones added 4-5 times to ensure that the Agrobacterium is washed clean. Spread it on sterile filter paper and let it dry for about 2 minutes. Then, insert the embryo obliquely into the solid induction medium with the wound side facing downward and upward, and check the rooting condition after 15-20 days of light culture.

[0073] Figure 6 The schematic diagram of soybean rooting successfully obtained from the Suinong 53 soybean seeds of the present invention after implementation case 7 shows that after the soybean seeds are disinfected with a sodium hypochlorite solution with an effective chlorine concentration of 0.6%, rooting transformation is carried out, and pollution-free transformed roots can be successfully obtained, indicating that this method can be applied to soybean rooting transformation to obtain pollution-free tissue culture explants.

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for sterilizing soybean seeds for plant tissue culture, characterized in that: The method screens the sodium hypochlorite concentration suitable for disinfection by gradient, and optimizes the steps of the sodium hypochlorite disinfection method, which specifically includes the following steps: S1, select multiple soybean seeds of uniform size, fullness, smooth surface and no stains, soak them in ethanol, shake them while soaking, and pour off the ethanol; S2, adding sodium hypochlorite solutions with different percentages of available chlorine to the soybean seeds obtained in step S1, and shaking the container containing the soybean seeds and the sodium hypochlorite solution; S3, washing the soybean seeds in step S2 with sterile water, and drying them on sterilized filter paper to obtain sterilized soybean seeds.

2. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S1, soak in 75% ethanol for 3 min.

3. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S2, the effective chlorine concentration of sodium hypochlorite is 0.6%.

4. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S2, sodium hypochlorite solutions with effective chlorine content of 0.6%, 1.2%, 1.8%, 2.4% and 3% were added to the obtained soybean seeds, respectively.

5. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S2, a container containing soybean seeds and sodium hypochlorite solution is placed in a shaker at 25°C and shaken at 100-120 rpm for 30 minutes.

6. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S3, the soybean seeds were washed 6 times with sterile water, each time by shaking for 1 min and blowing on sterile filter paper for 1-2 min.

7. The method for sterilizing soybean seeds for plant tissue culture according to claim 1, characterized in that: In step S3, after obtaining the sterilized soybean seeds, they are cultured in a germination medium.

8. The method for sterilizing soybean seeds for plant tissue culture according to claim 7, characterized in that: In step S3, the components of the germination medium include: B5, B5 vitamins, 1.67 mg / L 6-BA, 2% sucrose, 0.25% vegetable gum, pH=5.

8.

9. An application of the method for sterilizing soybean seeds for plant tissue culture according to any one of claims 1 to 8 in soybean rooting transformation, characterized in that: In this application, a sodium hypochlorite solution containing 0.6% effective chlorine is added to the obtained soybean seeds, and the container containing the soybean seeds and the sodium hypochlorite solution is shaken for 30 minutes.

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