KR100523758B1 - Efficient method of Agrobacterium-mediated transformation in Chinese cabbage - Google Patents

Abstract

The present invention relates to a method for transforming cabbage using Agrobacterium tumefaciens .

More specifically, the present invention is to adjust the pH, agar concentration, and additives of the medium to increase the efficiency of Chinese cabbage transformation from the pre-inoculation stage of Agrobacterium tumafaciene to the stage of chute induction The present invention relates to an improved method for transforming cabbage, which greatly increases the transformation efficiency of cabbage, which has been extremely low.

Description

Efficient method of Agrobacterium-mediated transformation in Chinese cabbage}

The present invention relates to a method for transforming cabbage using Agrobacterium tumefaciens , which can introduce useful genes to crops.

Recently, the most commonly used plant transformation technique is to use the plant pathogen Agrobacterium tumefaciens as a vector. These bacteria have the ability to transfer T-DNA, which is part of their Ti (tumor-inducing) or Ri (root-inducing) plasmid DNA, into infected plant cells. Therefore, when the target gene is recombined in the T-DNA and co-cultured with the bacteria and the plant having the same, plant cells can be transformed (Binns et al., Ann Rev Microbiol. 42: 575-606,1988).

Chinese cabbage is a vegetable mainly used for kimchi, and is one of the important domestic horticultural crops with annual output of 2.4 million tons. Nevertheless, Chinese cabbage is rarely studied, so it is generally difficult to transform, and sometimes its efficiency and stability are extremely low. Existing patents related to the transformation method of Chinese cabbage include Korean Patent No. 10-0375674 which suggests a method of inducing callus by placing the embryonic axis of Agrobacterium-inoculated cabbage on MS medium containing silver nitrate. .

In view of the above problems, the present invention provides a method for stably transferring foreign genes to Chinese cabbage, thereby obtaining a number of transformed cabbages. The purpose.

The technical problem of the present invention as described above, using the Agrobacterium tumafacien to introduce a gene useful for crops in the laboratory transforms the cabbage axis, induce callus (callus) in callus induction medium from the axis Then, after inducing the shoot (shoot) in the chute induced medium from the induced callus, the transformed cabbage is obtained through a three-step induction process of inducing the root in the root induced medium from the induced chute again, At this time, it was achieved by adjusting the pH and agar concentration of the medium, and the additives (AgNO _3, acetosyringone) in order to increase the efficiency of Chinese cabbage transformation from the pre-inoculation of Agrobacterium tumafaciene to the stage of chute induction. Genetic stability was confirmed by PCR analysis from progeny obtained through self-correction of cabbage plants transformed by the method of the present invention.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a method for transforming cabbage using Agrobacterium tumefaciens .

More specifically, the present invention, by adjusting the pH and agar concentration of the medium, and additives in order to increase the efficiency of cabbage transformation from the pre-inoculation of Agrobacterium tumafaciene to the stage of chute induction, The present invention relates to an improved method for transforming cabbage, which greatly increases the transformation efficiency of extremely low cabbage.

The present invention comprises the steps of culturing Agrobacterium tumafaciene used as a medium for transformation of Chinese cabbage in YEP medium; Transfecting the cabbage of the cabbage with Agrobacterium tumafacien to transform the gene; Culturing the transformed cabbage embryos in a callus induction medium so that callus is induced only in the transformed tissue; Cutting the induced callus to induce the chute by culturing in a chute induction medium having the same composition as the callus induction medium; And cutting the induced chute to differentiate into normal young plants in root induction medium and to purify in vermiculite vessels.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Transformation Carrier

The transformation carrier used in the present invention is a hygromycin resistance gene-containing carrier (pRCV2), a plant transformation vector pRCV2 for rescue cloning comprising a hygromycin resistance gene, filed on November 20, 2001. Application No. 10-2001-0072265, inventor: Park Young-Doo, Kim Hyung-Seok) was used as the same (see Fig. 1). pRCV2 was produced by inserting pBluescriptII KS (+), which has an ampicillin resistance gene and bacterial origin, into pCAMBIA1301, which carries a hygromycin resistance gene (hpt) among plant transformation vectors. It is disclosed in detail in the patent publication.

Example 2 Cultivation of Agrobacterium Tomafaciene Containing a Transforming Carrier (pRCV2)

Agrobacterium tumafacien LBA4404 / pRCV2 containing the plant transformation vector pRCV2 of Example 1 was used as a transformation medium inoculated to cabbage. The microorganism was deposited on November 19, 2001, with the accession number KFCC 11283 to the Korean Culture Center of Microorganisms. Hereinafter, Agrobacterium tumafacien refers to Agrobacterium tumafacien LBA4404 / pRCV2.

In order to increase the efficiency of transformation when the cabbage of the cabbage is inoculated with Agrobacterium tumafaciene, first, Agrobacterium tumefaciens LBA4404 / pRCV2 containing the transforming carrier (pRCV2) is used as kanamycin ( kanamycin) was plated in solid YEP medium containing 50 mg / L and incubated for 48 hours at 28 ° C., and several colonies of cultured Agrobacterium tumafaciene cells were taken to give 5 mg / L of kanamycin 5 Inoculated in mL liquid YEP medium and incubated for 48 hours at 28 ℃. 700 μl of the cultured suspension was extracted and then added to 50 ml liquid YEP medium with 0.02 mM Acetosyringone added and pH adjusted to 5.6 and suspended incubated at 28 ° C. until OD ₆₀₀ became 1.0. Finally, the incubated suspension was centrifuged at 4000 rpm for 15 minutes and resuspended in 25 ml liquid YEP medium to be used for inoculation of Chinese cabbage embryos.

Example 3 Chinese Cabbage Transformation and Callus Induction

Chinese cabbage [Seoul, Donghan Hannong Co., Ltd.] was germinated under aseptic conditions, cut into 5-10 mm of axial axis, and inoculated by dipping for 10 minutes in Agrobacterium tumafacien suspension prepared according to the method described in Example 2. . Inoculated sections were cocultured (MS base medium, 3% sucrose, 0.8% plant agar, 4 mg / L BA, 1 mg / L NAA, 4 mg / L AgNO ₃ , 5 mg / L acetosyringone, pH 5.6) Co-culture for 3 days. The cabbage embryos are then washed with a liquid co-culture medium to wash out Agrobacterium tumafaciene and then callus-induced medium (MS base medium, 3% sucrose, 0.8% plant agar, 4 mg / L BA, 1 mg / L NAA). , 4 mg / L AgNO ₃ , 5 mg / L acetosyringone, 10 mg / L hygromycin, 200 mg / L cefotaxime, pH 5.6). 2).

Example 4 Chute and Root Induction

The induced transformed callus was cut and transferred to a chute induction medium (same as a callus induction medium) to induce a chute. The induced transformed chute was a root induction medium (1/2 MS base medium, 3% sucrose, 0.7% plant agar, 200 mg / L cefotaxime, pH 5.8) to induce root development. The transformants with roots were transplanted into sterile vermiculite and purified and then transplanted into pots and grown in greenhouses (see FIG. 2).

Example 5 Gene Insertion and Stability Confirmation in Transformants and Subsequents

In order to confirm that the hygromycin resistance gene used in the invention was successfully inserted into these transgenic individuals, genomic DNA was isolated from the cabbage transformant and Southern blot analysis was performed. In this case, a probe for confirming whether the hygromycin gene has been inserted is a primer pair 3'-CCTCTACGTTATCCAGTCCGA-5 'and 3'-GGTTGTTACAGGACTGCCTGT-5' which can be amplified inside the hygromycin gene. It was used as a product amplified by PCR in the template. As a result, a 0.6 kb band was generated in the transgenic cabbage, and it was confirmed that the hygromycin gene was stably inserted into the cabbage genome (see FIG. 3). In order to confirm whether the transformed gene is inherited stably after the cabbage transformant, the seeds obtained by self-correcting the cabbage transformant are germinated, and then genomic DNA is extracted from the subsequent object, and the inside of the hygromycin gene As a result of PCR reaction with the primer pair 3'-CCTCTACGTTATCCAGTCCGA-5 'and 3'-GGTTGTTACAGGACTGCCTGT-5', which can be amplified in, it was confirmed that the gene was stably inherited even after the cabbage transformant (see FIG. 4).

As is apparent from the above examples, the present invention provides the pH and agar concentration of the medium, and additives (AgNO) in order to increase the efficiency of transformation from the pre-inoculation step of Agrobacterium tumafaciene to the step of induction of the chute. _3, acetosyringone) to control by was able to significantly increase the transfection efficiency of Chinese cabbage showed the extremely low efficiency of the existing, strong antibiotic genes to be introduced by using a selection of the high thereby transformants transfected with neomycin or not properly inserted unstable transformants The number of converters could be minimized. This not only enabled us to stably introduce useful genes in Chinese cabbage, which is a very important horticultural crop, but also to secure a large number of Chinese cabbage transformants. Since the scaffold is provided, the present invention is a very useful invention for the agricultural and food industries.

1 is a gene structure of a site delivered to the plant genome of pRCV2, a plant transformation carrier developed for transforming cabbage.

Figure 2a shows the process of coculture with the Agrobacterium embryonic axis during the transformation process of Chinese cabbage by Agrobacterium.

Figure 2b shows a process of inducing transformed callus (callus) in callus induction medium during the transformation process of Chinese cabbage by Agrobacterium.

Figure 2c shows the process of the shoot (shoot) development in the chute differentiation medium during the transformation process of Chinese cabbage by Agrobacterium.

Figure 2d shows the process of the root (root) is generated from the chute during the transformation process of Chinese cabbage by Agrobacterium.

Figure 2e shows the Chinese cabbage transgenic plants being grown in the greenhouse during the transformation process of Chinese cabbage by Agrobacterium.

3 is a diagram confirming the hygromycin resistance gene inserted into the transformed cabbage by Southern hybridization method.

Figure 4 is a picture confirming the hygromycin resistance gene in the later obtained from the transgenic cabbage by nucleic acid amplification reaction (PCR).

Claims (4)

Culturing Agrobacterium tumafaciene containing pRCV2, a transforming carrier, and preparing a suspension; Inoculating the suspension into cabbage embryos and culturing in coculture medium to transform the embryonic segments; Inducing callus by culturing the transformed cabbage embryos in a callus induction medium; Differentiating the induced callus by culturing in a chute induction medium; In the method of producing a transgenic cabbage with agrobacterium tumafaciene consisting of inducing the root by culturing the induced chute in a root induction medium, Agrobacterium tumafaciene suspension preparation step for inoculating the cabbage section is plated in solid YEP medium containing 50 mg / L of kanamycin (kanamycin), kanamycin 50 mg / L by taking a colony of the cultured cells (colony) Suspension culture was inoculated in 5 ml liquid YEP medium containing and then 700 μl of the cultured suspension was added to 50 ml liquid YEP medium in which 0.02 mM Acetosyringone was added and the pH was adjusted to 5.6 and the OD ₆₀₀ became 1.0. Method for producing a transformed cabbage with Agrobacterium tumafaciene, characterized in that the suspension culture. Culturing Agrobacterium tumafaciene containing pRCV2, a transforming carrier, and preparing a suspension; Inoculating the suspension into cabbage embryos and culturing in coculture medium to transform the embryonic segments; Inducing callus by culturing the transformed cabbage embryos in a callus induction medium; Differentiating the induced callus by culturing in a chute induction medium; In the method of producing a transgenic cabbage with agrobacterium tumafaciene consisting of inducing the root by culturing the induced chute in a root induction medium, The composition of the co-culture medium used in the co-culture step is MS base medium of pH 5.6 containing 3% sucrose, 0.8% plant agar, 4 mg / L BA, 1 mg / L NAA 4 mg / L AgNO ₃ , 5 mg / L Acetosyringone (acetosyringone) is added, characterized in that the production method of transformed cabbage by Agrobacterium tumafaciene. Culturing Agrobacterium tumafaciene containing pRCV2, a transforming carrier, and preparing a suspension; Inoculating the suspension into cabbage embryos and culturing in coculture medium to transform the embryonic segments; Inducing callus by culturing the transformed cabbage embryos in a callus induction medium; Differentiating the induced callus by culturing in a chute induction medium; In the method of producing a transgenic cabbage with agrobacterium tumafaciene consisting of inducing the root by culturing the induced chute in a root induction medium, The composition of the medium used for the callus and chute induction step is 3% sucrose, 0.8% plant agar, 4 mg / L BA, 1 mg / L NAA, 10 mg / L hygromycin , Agrobacterium tumapa, characterized in that 4 mg / L AgNO ₃ , 5 mg / L acetosyringone was added to the MS base medium of pH 5.6 containing 200 mg / L cefotaxime Method for producing a transgenic cabbage by the yen. Culturing Agrobacterium tumafaciene containing pRCV2, a transforming carrier, and preparing a suspension; Inoculating the suspension into cabbage embryos and culturing in coculture medium to transform the embryonic segments; Inducing callus by culturing the transformed cabbage embryos in a callus induction medium; Differentiating the induced callus by culturing in a chute induction medium; In the method of producing a transgenic cabbage with agrobacterium tumafaciene consisting of inducing the root by culturing the induced chute in a root induction medium, The composition of the medium used for the root induction step is 1/2 MS base medium of pH 5.8 containing 3% sucrose, 0.7% plant agar and 200 mg / L cefotaxime. A method for producing transformed cabbage with Agrobacterium tumafaciene.