Genetic transformation method of soybean

Abstract

The invention belongs to the technical field of plant transgenosis, and particularly relates to a soybean genetic transformation method which comprises the following steps: removing terminal buds and all lateral buds of soybean seedlings, dripping agrobacterium containing target genes on growing points without the lateral buds to realize soybean genetic transformation. T0-generation transgenic seedlings can be obtained within 180 days or more, and the T0-generation transgenic seedlings can be obtained within 40 days, so that the genetic transformation period is greatly shortened; in terms of transformation efficiency, soybean genetic transformation is difficult, the traditional genetic transformation efficiency is about 5%-10%, and the genetic transformation efficiency of the method is stabilized to be 20% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of plant transgenic technology, and particularly relates to a soybean genetic transformation method. Background Art

[0002] Soybean is an important economic crop in the world and also the main source of edible oils and proteins for humans and animals. Due to the influence of biological and abiotic stress, the quality and yield of soybeans are difficult to meet people's needs. Conventional breeding technology plays an important role in quality improvement and new variety selection, but the long breeding cycle has always been one of the bottlenecks of this technology. Plant genetic engineering technology can improve the undesirable traits of plants in a targeted manner, greatly shorten the breeding period, and thus improve economic benefits. Genetic transformation technology is the most widely used technology in gene function research, genetic improvement, and biological breeding research, but in soybeans, genetic transformation technology is still relatively difficult and time-consuming, which also hinders the rapid development of high-throughput gene function analysis and biological breeding in soybeans.

[0003] Plant genetic transformation technology is an essential tool for plant genetic engineering and modern agricultural molecular breeding. However, the existing soybean genetic transformation methods have a long cycle and low transformation rate, which restricts the genetic transformation of most resource plants or crops and has become a technical barrier to the development and utilization of plant resources. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a soybean genetic transformation method.

[0005] A soybean genetic transformation method comprises the following steps: removing the stem top and all lateral buds of soybean seedlings, dripping Agrobacterium containing a target gene on the growth point where the lateral buds are removed, and culturing under non-dark conditions until the lateral buds grow out, thereby achieving soybean genetic transformation. After dripping Agrobacterium containing a target gene on the growth point where the lateral buds are removed, culturing under normal light can promote soybean absorption of Agrobacterium containing a target gene and promote the occurrence of genetic transformation. The method has a short operation cycle and high genetic transformation efficiency.

[0006] It should be noted that although there is a method in the prior art for transforming plants such as peanuts, potatoes, garlic, and freesia by injecting Agrobacterium containing the target gene at the growth point of the excised bud, when the Agrobacterium containing the target gene is injected at the growth point after the soybean bud is excised by the prior art method, the soybean stalk tissue structure is dense and cannot be directly injected into the corresponding tissue site, resulting in the inability to genetically transform it by injection; and the prior art cuts off the upper part of the peanut seed joint and cuts off other side buds, and injects Agrobacterium at the cut wound, and the upper part of the soybean seedling joint absorbs Agrobacterium, which is not only prone to false positives, but also affects the transformation rate, resulting in the genetic transformation method of peanuts not being suitable for the genetic transformation method of soybeans; in addition, the regeneration ability of soybean hypocotyls is weaker than that of peanut hypocotyls, and the genotype dependence is strong. The genetic transformation efficiency of different varieties is different, and the genetic transformation method between soybeans and peanuts is not universal, so the existing soybean genetic transformation method requires complicated operation processes such as callus tissue culture, propagation, subculture, and rooting induction. These soybean genetic transformation methods have a long cycle and low genetic transformation efficiency. The invention drips an Agrobacterium solution containing the target gene on the growth point of the lateral bud, and the soybean absorbs the Agrobacterium solution containing the target gene through absorption, and is cultured under non-dark conditions until the lateral bud grows, thereby realizing soybean genetic transformation with a high transformation rate.

[0007] Preferably, the soybean seedlings are soybean seedlings cultured under light / darkness = 14 hours / 10 hours until the fourth compound leaf expands.

[0008] Preferably, the method for obtaining Agrobacterium containing the target gene is as follows: the Agrobacterium to be transformed with the target gene is applied to an LB plate containing antibiotics, and after the colonies are grown, a single clone is picked up and placed in an LB liquid culture medium containing antibiotics, and the culture is carried out at 28°C, 200rpm, and the Agrobacterium is collected by centrifugation after cultivation, and the collected Agrobacterium is resuspended in an injection solution to form a suspension, and the concentration of the suspension is adjusted to OD600 of 0.6-0.8, and the suspension with an OD600 of 0.6-0.8 is continuously cultured for 1 hour to 2 hours.

[0009] Preferably, the dosage of the Agrobacterium containing the target gene is 1 drop to 2 drops.

[0010] Preferably, after the lateral buds grow, the soybean plants are cultured under the conditions of light / darkness = 14 hours / 10 hours for 20 to 30 days, and the lateral buds grow new leaves or stems.

[0011] Preferably, the soybean is Williams82.

[0012] Preferably, the target gene is GmSPL13 .

[0013] Compared with the prior art, the present invention has the following beneficial effects: The invention is simple to operate, and in terms of process, does not require complicated operation procedures such as callus culture, propagation, subculture, and root induction of traditional tissue culture; in terms of cost, because the traditional tissue culture, propagation, subculture and other processes and the high reagent costs of culture medium, antibiotics, high-standard tissue culture rooms and the like involved in the process are not required, the method only requires conventional culture, which greatly reduces the cost; in terms of environment, traditional tissue culture requires a sterile environment, such as a sterile laboratory, a sterile culture room and other high-standard experimental environments, and the method is operated in an ordinary culture room, or the outdoor environment has no effect on the experiment, and has no special requirements for the environment; in terms of time, traditional tissue culture usually undergoes a complex culture process, and it takes more than 180 days to obtain T0 generation transgenic seedlings, and the method only takes 40 days to obtain T0 generation seedlings, which greatly shortens the genetic transformation cycle; in terms of transformation efficiency, soybean genetic transformation is difficult, the traditional genetic transformation efficiency is about 5% to 10%, and the genetic transformation efficiency of the method is stable at more than 20%.

[0014] In terms of genetic stability, both T0 and T1 generations of the present invention can obtain transgenic positive seedlings, which can be stably inherited. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 1 is a diagram of the soybean genetic transformation process, where A is soybean seedling culture, B is soybean lateral buds, C is soybean seedlings with lateral buds removed, D is Agrobacterium solution containing the target gene dripped onto the damaged lateral buds, E is the soybean seedlings after the target gene is dripped into them, F is the position of the lateral buds where the target gene can be dripped into, G is the growth of new lateral buds, and H is the growth of new lateral buds.

[0016] Figure 2 is a transformation example, wherein A is a soybean seedling with side buds removed, B is a soybean seedling with new side buds growing, and C is an enlarged view of the new side buds.

[0017] Figure 3 The results of BAR test strips. Note: 1 is the positive control, 2 is the negative control, 3-25 are the test seedlings, and 26-27 are the injection blank controls.

[0018] Figure 4 The results of BAR marker PCR detection, among which, lane M in A is a marker, lanes 1 to 24 are test seedlings 1 to 24, lanes 25 to 26 are empty vector injection controls, and lanes 27 to 28 are non-injection controls; lane M in B is a marker, lanes 1 to 13 are transgenic seedlings that have been sequenced and verified. The PCR results are completely consistent with the sequencing and test strip test results.

[0019] Figure 5 is the phenotype of the overexpressed transgenic T1 gene, where A is the wild-type Williams82 and B is GmSPL13 Overexpression positive strains, GmSPL13The overexpression positive strain was recorded as OE-GmSPL13, C was the wild type and GmSPL13 Plant height statistics of overexpression positive strains.

[0020] Figure 6 for GmSPL13 Comparison of gene sequences of mutant materials.

[0021] Figure 7 It is the phenotype of gene-edited mutants, where A is a comparison chart of wild-type and mutant strains, and B is the plant height statistics of wild-type and mutant strains. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0023] The soybean genetic transformation method of the present invention is achieved by the following steps: 1. Soybean seedling cultivation Soybean seeds with intact seed coats were selected and sown in pots with a diameter of 15 cm and a depth of 16 cm, with 3 seeds planted in each pot. The seeds were placed under a light condition of light / darkness = 14 hours / 10 hours until the fourth compound leaf expanded. This light condition can be achieved by natural light or a light-controlled greenhouse.

[0024] 2. Agrobacterium Preparation Take out Agrobacterium GV3101 containing the target gene or gene editing vector from -80℃, spread it on an LB plate containing 50μg / mL rifampicin and 50μg / mL kanamycin, and culture it at 28℃ for 1 to 2 days. After the colonies grow, pick out a single clone and transfer it to 10mL LB liquid culture medium containing antibiotics, and culture it at 28℃, 200rpm, overnight.

[0025] At room temperature, centrifuge at 5000g for 10 minutes to collect the above Agrobacterium, resuspend the collected Agrobacterium in the injection solution to form a suspension, measure the concentration of the suspension until the OD600 is 0.6~0.8, and continue to culture the suspension with an OD600 of 0.6~0.8 for 1 hour to 2 hours. The injection solution contains MgCl2 10 mM, MES 10 mM, and acetosyringone 200 μM.

[0026] 3. Genetic transformation Use a sharp scalpel to remove the soybean terminal bud and all lateral buds in step 1, use a pipette to absorb the Agrobacterium prepared in step 2, use a 200μL pipette to drop 1~2 drops above the growth point where the lateral buds were removed, and culture under natural light conditions until lateral buds grow, and then culture after the plants are established.

[0027] 4. Post-plant cultivation The treated soybean plants are moved to medium culture conditions for further culture. If Agrobacterium is dripped into each lateral bud during the treatment, it is not necessary to remove the newly grown lateral buds. If only one or several lateral bud growth points are treated, the lateral buds at the growth points where Agrobacterium is not dripped are removed every 2 to 3 days. After 20 to 30 days of culture, the lateral buds grow new leaves or stems.

[0028] 5. Positive test Take new leaves for herbicide smear experiment or test strip test, screen and determine the transgenic positive plants, keep them for further cultivation until maturity, and harvest the seeds.

[0029] Example A soybean genetic transformation method, characterized in that it comprises the following steps: 1. Soybean seedling cultivation Soybean seeds with intact seed coats were selected and sown in pots with a diameter of 15 cm and a depth of 16 cm, with 3 seeds planted in each pot. The seeds were placed under a light condition of light / darkness = 14 hours / 10 hours until the fourth compound leaf was extended. Figure 1 A.

[0030] 2. Agrobacterium Preparation Take out Agrobacterium GV3101 containing the target gene or gene editing vector from -80℃, spread it on an LB plate containing 50μg / mL rifampicin and 50μg / mL kana, and culture it at 28℃ for 2 days. After the colonies grow, pick out a single clone and transfer it to 10mL LB liquid culture medium containing antibiotics, and culture it at 28℃, 200rpm, overnight.

[0031] At room temperature, the Agrobacterium was collected by centrifugation at 5000g for 10 minutes, and the collected Agrobacterium was resuspended in the injection solution to form a suspension, and the concentration of the suspension was measured until the OD600 was 0.6, and the suspension with OD600 of 0.6 was continued to be cultured for 1 hour. The injection solution contained MgCl2 10 mM, MES 10 mM, and acetosyringone 200 μM.

[0032] 3. Genetic transformation Use a sharp scalpel to remove the soybean top bud and all side buds in step 1. Figure 1 Use a pipette to draw the Agrobacterium prepared in step 2. Figure 1For C~F, use a 200μL pipette to drop 1 drop above the growth point where the lateral buds have been removed, and culture under natural light until lateral buds grow, and then culture after the plants have established.

[0033] 4. Post-plant cultivation The treated soybean plants were transferred to medium culture conditions for further culture. If Agrobacterium was dripped into each lateral bud during the treatment, the newly grown lateral buds do not need to be removed. If only one or several lateral bud growth points were treated, the lateral buds that were not dripped with Agrobacterium were removed every 2 days. After 25 days of culture, the lateral buds grew new leaves or stems. Figure 1 G~H.

[0034] 5. Positive test Take new leaves for herbicide smear experiment or test strip test, screen and determine the transgenic positive plants, keep them for further cultivation until maturity, and harvest the seeds.

[0035] Effect verification 1. Gene overexpression The overexpression vector p35s:GmSPL13 was constructed and transferred into GV3101 Agrobacterium, and Williams82 was transformed using the above method. Figure 2 As shown in A. When the plant grows new shoots, Figure 2 B~C, and then use glyphosate to apply and BAR gene test strips to detect positive seedlings, such as Figure 3 , and PCR was used to detect BAR genes, such as Figure 4 The test results showed that transgenic positive seedlings were successfully obtained using the above transformation method.

[0036] The above method was used for 4 transformations, and transgenic positive seedlings were successfully obtained, with a transformation rate of 32.2%. GmSPL13 The height of the overexpression strain was significantly higher than that of the wild type, indicating GmSPL13 Gene overexpression has the function of promoting plant growth. Experiments have shown that this method can obtain stable genetic offspring and can be used in the genetic transformation of soybean transgenic, gene overexpression and genetic engineering.

[0037] Table 1 Genetic transformation rate

[0038] in, GmSPL13

[0039] 2. Gene Editing Build GmPRR7 Edit the vector. Use the online gRNA design tool GmSPL13 Two guidRNAs were designed in the gene coding region, namely gRNA1 and gRNA2. The online gRNA design tool address is http: / / crispor.tefor.net / . The two guidRNAs were constructed in series into the pRGEB31 vector. The gRNA was driven by the Arabidopsis U6 promoter and the cas9 was driven by 35S to form pRGEB31-SPL13. Williams82 was used as the genetic transformation receptor and the above method was also used for genetic transformation. The above detection method was used to screen and obtain 25 T0 generation transgenic positive strains in the offspring, and then the transgenic positive seedlings were sequenced and identified, and finally 11 mutant strains with successful editing were obtained, such as Figure 5~Figure 6 After obtaining the seeds, the phenotype of the T1 generation mutants was verified, and the results showed that SPL13 The mutant plant height is significantly lower than the wild type. Figure 7 ,and GmSPL13 The loss of gene function causes the plant to quickly switch from vegetative growth to reproductive growth and the plant height decreases.

[0040] The above experiments prove that this method can also be used for gene editing to obtain genetically improved soybean lines.

[0041] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0042] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0043] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for genetic transformation of soybean, characterized in that: The following steps are involved: Remove the stem top and all side buds of the soybean seedlings, drop the Agrobacterium containing the target gene on the growth point where the side buds have been removed, and culture under non-dark conditions until side buds grow, thereby achieving genetic transformation of soybeans.

2. The method for genetic transformation of soybean according to claim 1, characterized in that: The soybean seedlings are cultivated under light / dark conditions of 14 hours / 10 hours until the fourth compound leaf has expanded.

3. The method for genetic transformation of soybean according to claim 1, characterized in that: The method for obtaining Agrobacterium containing the target gene is as follows: the Agrobacterium to be transformed with the target gene is spread on an LB plate containing antibiotics, and after the colonies grow, a single clone is picked up and transferred to an LB liquid culture medium containing antibiotics, and the Agrobacterium is collected by centrifugation after cultivation, and the collected Agrobacterium is resuspended in an injection solution to form a suspension, and the concentration of the suspension is adjusted to OD600 of 0.6-0.8, and the suspension with an OD600 of 0.6-0.8 is continuously cultivated for 1 hour to 2 hours.

4. The soybean genetic transformation method according to claim 1, characterized in that: The dosage of the Agrobacterium containing the target gene is 1 drop to 2 drops.

5. The method for genetic transformation of soybean according to claim 1, characterized in that: After the lateral buds grow, the soybean plants are cultured under the condition of light / darkness = 14 hours / 10 hours for 20 to 30 days, and the lateral buds grow new leaves or stems.

6. The method for genetic transformation of soybean according to claim 1, characterized in that: The soybean is Williams82.

7. The method for genetic transformation of soybean according to claim 1, characterized in that: The target gene is GmSPL13 .

Images