KR20070073715A - A method for producing chinese cabbage transformant using tissues of flower stalk and a transformant with promoted soft rot resistance obtained from the method - Google Patents

Abstract

A method for preparing a Chinese cabbage transformant using a flower stalk tissue is provided to obtain the high efficient transformation rate and provide the Chinese cabbage transformant with increased soft rot resistance. The method for preparing a Chinese cabbage transformant comprises a step of transfecting Agrobacterium tumefaciens such as Agrobacterium tumefaciens LBA4404 strain into flower stalk tissues of the Chinese cabbage which is a J3-11M-44 family, and then tissue-culturing it. A foreign gene, BcPGIP(polygalacturonase- inhibiting protein), or npt II gene is introduced in the Agrobacterium tumefaciens.

Description

Method for producing Chinese cabbage transformant using tissues of flower stalk and a transformant with promoted soft rot resistance obtained from the method

1 shows the T-DNA site of the binary vector pCAMBIA2300- BcPGIP2 .

FIG. 2 shows regeneration of adventitious shoots and transformed plants formed from cultured hard tissue sections of Chinese cabbage.

Figure 3 nptII in Chinese cabbage transformants It is a photograph showing the result of agarose gel electrophoresis of the PCR amplification product for gene identification.

4 is T ₀ NptII in transformant plants Southern blot analysis was performed using genomic DNA to trace genomic DNA.

Figure 5 nptII using RT-PCR in transformant plants The results of detecting gene expression are shown.

6 BcPGIP2 in transformant plants The results of detecting gene expression are shown.

Figure 7 shows the result of comparing the degree of bruising in wild-type and transformed cabbage.

The present invention relates to a method for producing a transformant of Chinese cabbage using hard tissue, and to a transformant having improved resistance to choking disease produced therefrom. More specifically, the present invention provides a method of obtaining a transformant by introducing a foreign gene into the hardened tissue of Chinese cabbage and tissue culture, and providing a transformant having enhanced resistance to the disease by introducing a gene related to the disease of the disease as a foreign gene. It is about.

Chinese cabbage is a cold-cooled vegetable that prefers a cool climate. It is the most important vegetable crop as the main ingredient of kimchi in Korea, and accounts for 25% of the average annual vegetable consumption (Ministry of Agriculture and Forest, 2002). .

Most of the components of Chinese cabbage are leafy vegetables with water and calcium and vitamin C-rich leaves, and they are cold-tolerant vegetables that prefer relatively cool climates with a growth temperature of 18-22 ° C. Growth is good and muscle groups are widely distributed. Morphologically, the leaf inflorescence is 2/5, spirally engrafted, the stem is shortened to rosette shape, and it has self-incompatibility.

Conventional cabbage breeding is mainly done by typical hybrid breeding using F1 hybrid accents, but there are limitations in broadening varieties because of limited genetic resources. Recent developments in tissue culture and genetic engineering techniques have been attempted to develop technology to overcome this.

Tissue culture technology aims to select good individuals or to maintain and propagate them when they have a particular trait. Recently, various plant transformation technologies have been developed, and new varieties are actively developed through plant transformation, but low regeneration rate remains a problem to be improved. Therefore, in order to effectively use plant transformation technology, the establishment of tissue culture technology with high regeneration rate is required.

Plant transformation was the first success in tobacco, and has been used since then as a very powerful method for determining the function of specific genes, and has been used as a method for developing new crops by introducing useful genes into plants. Transformation techniques that introduce useful genes into plants include electroporation, direct gene transfer, micro-injection, tissue injection, PEG, Ca ⁺² , Agro A method using bacterium ( Agrobacterium ) has been developed, and transgenic plants can be obtained after selection of cells into which useful genes have been introduced and plant regeneration.

In particular, Agrobacterium tumerfaciens ( Agrobacterium) plant transformation method using a tumefaciens) is Agrobacterium (Agrobacterium) is known as a method for the stable transition of the useful gene by using the mechanism to transition their DNA part of the plant, genetic, biochemical, genetic It is widely used throughout the field of plant research, such as physiological basic research and application to practical agriculture. Agrobacterium tyumeo Pacifico Enschede (Agrobacterium tumefaciens ) And Agrobacterium tumefaciens, such as Rizzo's Nes (A. rhizogenes) is transformed by their genes to infected wounds the plant tissue with Gram-negative soil microorganisms to plants Agrobacterium tumefaciens (crown gall) or hairy roots (hairy root) It is known to cause cancer tissue. Agrobacterium tyumeo Pacific Enschede (A. tumefaciens) and Agrobacterium separation tank to Ness (A. rhizogenes) in transgenic systems to the Agrobacterium tyumeo Pacific Enschede due to the plasmid with the microorganism are induced by (A. for tumefaciens) is due to the case of uihamyeo, Agrobacterium separation tank to Ness (A. rhizogenes) to the Ti (tumor inducing) plasmid, the Ri (root inducing) plasmid. The induction of cancer tissue by the Ti or Ri plasmid is caused by the transfer of specific DNA fragments (T-DNA) present in the Ti plasmid of the microorganism to plant cells, and the transfer of T-DNA (transfer DNA) is caused by the Ti plasmid. It is known to be regulated by the expression of vir genes located at.

In order to stably transfer useful genes into plants, it is important to establish a relatively efficient transformation method. In the case of transformation using Agrobacterium (Agrobacterium) is known as one of the factors, exert the greatest influence on conversion efficiency, improve the establishment of a high regeneration capacity tissue culture systems transformed from tissue sections of the plant.

Among Brassica plants, transformation using Agrobacterium vector is the most studied in rape ( B. napus ). Plant tissues used for transformation are stem, section, epidermis, hypocotyl, pollen origin. Various tissues such as pear and cotyledon are used. In addition, cabbage (B. oleracea), mustard (B. juncea), Brassica Carry appear (B. carinata), Brassica you Gras (B. nigra), but also a report that was obtained transgenic bars, etc., cabbage (B . transformation of campestris) is the research situation and the relatively very low compared to other Brassica spp. Brassica cam Fest Variety lease Farah (B. campestris var. Para) Agrobacterium to the root of the Leeum's Wellness Resorts (Agrobacterium Tanaka et al. (1985) and Paszkowski et al. (1986) reported that B. campestris var. rapa was the first observer of hairy roots to infect rhizogenes . It has been reported that the cauliflower mosaic virus gene was directly transformed into the leaf protoplasts to obtain a plant in which the gene was inserted, and Olsson and Eriksson (1988) reported Brassica. Fast cam-less variety Olay Blow by infecting Agrobacterium derived from the hypocotyl protoplasts of (B. campestris var. oleifera) did not get the conversion plant traits, but reported that to obtain a kanamycin-resistant callus. Examples of obtaining transgenic plants from Chinese cabbage ( B. campestris ) include Mukhopadhyay et al. (1992), Neomycin phosphotransferase II in the axis of B. campestris var. Sarson Agrobacterium infected with the gene (neomycin phosphotransferase II gene, NPTII gene) and beta glucuronidase-intron gene succeeded in obtaining cabbage transformants containing the above genes was, gave (June), such as a Brassica cam paste-less sub-Made cis peki norbornene outer sheath protein genes by infecting the cotyledon (B. campestris ssp. pekinensis) into Agrobacterium tobacco mosaic viruses (tobacco mosaic virus) (coat Obtaining seven transgenic plants containing protein genes and NPTII genes is an example of successful transformation. As such, the case of obtaining a regeneration plant having a new trait by transforming a foreign gene into cabbage is very poor in research results compared to other Brassica plants.

Tissue cultures of Chinese cabbage have been reported to have been obtained from replanted plants through tissue culture using cotyledons, embryos, medicines, and leaf slices of Chinese cabbage, but they have been known to be less efficient than other Brassica plants. It is insignificant. Recently, studies have been made to improve the culture efficiency, such as the composition of the medium, the composition of the plant growth regulator, the addition of AgNO _{3 in the} medium.

Conventional cabbage transformation has been mainly used cotyledon and embryonic tissue method, but the low efficiency, the present inventors transformed cabbage using a simple tissue method and a simple tissue method to improve this .

Accordingly, it is an object of the present invention to provide a method for producing transformants of Chinese cabbage, which has a high transformation rate using the hardened tissue of Chinese cabbage.

It is another object of the present invention to provide a cabbage transformant having an increased resistance to elder disease prepared according to the above method.

The object of the present invention is to introduce a gene related to the disease resistance to the transforming vector Agrobacterium strain, to infect the strain in the hardened tissue of the Chinese cabbage in order to enhance the resistance of Chinese cabbage, and the hard tissue Tissue culture to re-differentiate to investigate the transformation rate, genomic DNA extracted from the leaf tissue of the re-differentiated transformant to confirm the transformation of Chinese cabbage through PCR, DNA gel blot and RT-PCR, transformed cabbage Achievement was achieved by examining the resistance of the disease to

The present invention comprises the steps of constructing a vector for transformation and introducing into the genus Agrobacterium strains; Infecting the strain to the hardened tissue of Chinese cabbage; Tissue culture step of the landscape tissue; Extracting genomic DNA from the leaf tissue of the re-differentiated transformant to confirm whether the Chinese cabbage is transformed through PCR, DNA gel blot and RT-PCR; And a step of investigating the resistance of the cabbage to the transformed cabbage.

* The present invention has a feature of providing a transformant production method of Chinese cabbage, comprising the step of infecting the microorganisms including a plant transformation vector introduced with a foreign gene into the hardened tissue of the Chinese cabbage.

The foreign gene used in the transformant production method of the Chinese cabbage of the present invention can be introduced into the BcPGIP2 (Polygalacturonase-inhibiting protein 2) gene or other genes according to the needs and purposes for enhancing the disease resistance.

Microorganism used in the embodiment of the present invention for introducing a foreign gene and the Agrobacterium genus Solarium (Agrobacterium) strain, can be used for other strains depending on the efficiency.

The present invention has a feature of providing a cabbage transformant having enhanced resistance to choking disease prepared according to the cabbage transformant production method by introducing a gene related to chamomile resistance as a foreign gene.

Hereinafter, the specific configuration and operation of the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLE

Example  One: Organization  Chinese Cabbage Transformant Preparation

In order to prepare a transformant of the cabbage using the hardened tissue of the cabbage, a vector for transformation is first constructed, introduced into the Agrobacterium strain, and then infected with the hardened tissue of the cabbage to culture the tissues to improve the transformation rate. Investigate.

* Vector construction for transformation

After introduction of the 54-J3-11M system PGIP2 gene of Chinese cabbage using a Bam HⅠ, Kpn Ⅰ site to the pCAMBIA2300 vector for transforming plants, this Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens ) was introduced into LBA4404 and used for transformation of Chinese cabbage.

After the J3-11M-54 strains of BcPGIP2 was introduced using the Bam HⅠ, Kpn Ⅰ site of pCAMBIA2300 vector for transforming plants, this Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens ) was introduced into LBA4404 and used to transform tobacco and cabbage (FIG. 1).

1 shows the T-DNA site of the binary vector pCAMBIA2300- BcPGIP2 . In this case, NPTII represents Neomycin phosphotransferase II, 35S Pro represents a CaMV 35S promoter, and NOS represents a nopaline synthase gene terminator.

Transformation of Cabbage (other than the existing evidence against J3 -11M-44 system is deleted, O and was an all please check).

In the J3-11M-44 strains, which were cultivated at the Chung-Ang University Plant Applied Science and Horticultural Crop Quality Laboratories, Chinese cabbage was transformed to enhance the resistance of B.C. Tissue culture for transformation was used for the hardened tissue of the Chinese cabbage. After seeding of J3-11M-44 strains, 4 weeks seedlings were cold-treated at 4 ° C for 4-6 weeks and grown in greenhouses. The oily tissue of the large flower garden was cut into 5 mm cylindrical sections, sterilized with 70% EtOH for 30 seconds, washed once with sterile water, immersed in 1% sodium hypochloride solution for 10 minutes, and then washed three times with sterile water. . The sterilized hard tissue sections were immersed in Agrobacterium culture cultured for 36 hours for 10 minutes, and then the culture solution was removed from sterilized filter papers. The inoculated sections were BI culture medium (4.4g / L MS medium, 1.0mg / L NAA, 4.0mg / L BAP, 4.0mg / L AgNO ₃ , 8g / L Phyto-agar), pH 5.7) and then co-cultured for 72 hours under dark conditions of 28 ℃. After co-culture for 72 hours, section tissue was washed for 5 minutes with washing medium (4.4 g / L MS medium, 1.0 mg / L NAA, 4.0 mg / L BAP, 200 mg / L Cefotaxime, pH 5.7) After washing three times and three times with sterile water, sterile water was removed from the filter paper. The washed sections were selected as medium (4.4g / L MS medium, 30g / L Sucrose, 8g / L Phyto-Agar, AgNO _3). 4.0mg / L, NAA 1.0mg / L, BAP 4.0mg / L, KM 25mg / L, Cytoxim 200mg / L, pH 5.7) until buds are formed, temperature condition of 23 ° C, 16 hours light condition, 8 hours It was cultured in the dark condition. Subcultures were carried out at 15-day intervals, and after cutting the callus tissue formed in J3-11M-44 and performing 4 to 6 passages, the selective medium having lowered the concentration of kanamycin (4.4 g / L MS medium, 30 g / L Sucrose) , Phyto-agar 8g / L, AgNO ₃ 4.0mg / L, NAA 1.0mg / L, BAP 4.0mg / L, KM 10mg / L, Celltaxim 200mg / L, pH 5.7) It was. When shoots were formed, rooting medium (MS medium 4.4 g / L, sucrose 30 g / L, phyto-agar 8 g / L, AgNO ₃ Rooting was induced by transplantation at 4.0 mg / L, Cytoxim 200 mg / L, pH 5.7) and differentiated to complete seedlings. After 2 ~ 3 weeks, the rooted plant is transplanted into a 7cm diameter vinyl pot containing aseptically sterilized soil, and then covered with a plastic wrap to maintain humidity. Placed in dark conditions for 5-7 days. After confirming that the growth of the above-ground part proceeds normally, the root spoilage is checked and planted in a pot of 25cm in diameter and cultivated in a greenhouse and used for experiments.

After inoculation of 782 tissues with three times of flower tissue sections of Chinese cabbage J3-11M-44 strain using transformed Agrobacterium, callus formed from J3-11M-44 strains was 4-6 times at 15-day intervals. Volumetric growth was continued while light green during subculture in selective medium, but shoots were not induced. The shoots were then induced from callus during the passage of the kanamycin concentration of the selected medium to 10 mg / L at 4 to 5 passages at 15-day intervals. Roots were transplanted into rooting media to induce rooting and to differentiate into complete seedlings. After 2 ~ 3 weeks, the seedlings rooted were named T ₀ -1, T ₀ -2, T ₀ -3, T ₀ -4, T ₀ -6, T ₀ -8, T ₀ -9, T ₀ -10, T ₀ -11, T ₀ -12, T ₀ -15, T ₀ -16, T ₀ -17, T ₀ -19, T ₀ -20, T ₀ -21, T ₀ -31, T ₀ -34, T ₀ -36, T ₀ -37, T ₀ -38, T ₀ -39 were purified and grown in a greenhouse and used for the experiment. The results are shown in Table 1 and FIG. In particular, FIG. 2 illustrates regeneration of adventitious shoots and transformed plants formed from cultured hard tissue sections of Chinese cabbage. At this time, A represents shoots formed in sections after 5 to 6 weeks of culture, B represents shoots transferred to medium during rooting, C represents transformed plants, and D represents transformed plants in which flower buds are formed.

As shown in Table 1 and FIG. 2, 52 shoots were induced, of which 39 were found and 22 were purified. In other words, the transformation rate was 2.8%, and the efficiency was more than doubled compared to 0.4-0.8% of the conventional cotyledons and embryos.

Callus Induction and Regeneration Rate in Chinese Cabbage Lines system Number of Infection Sections (A) Number of callus forming segments (B) Number of neoplasia sections (C) Number of transgenic plants (D) frequency(%) B / A C / A D / A J3-11M-44 782 375 52 22 48.0 6.6 2.8

Example  2: Confirm whether foreign genes are introduced into the transformants of Chinese cabbage

PCR and DNA gel blots were performed to confirm the introduction of npt II and BcPGIP2 into the cabbage transformants prepared in Example 1.

Experimental Example  One: PCR  Through analysis nptII  Check whether a gene is introduced

Agrobacterium was inoculated into the slices of J3-11M-44 line, and the differentiated individuals were rooted, and then 0.1g of the leaf tissues cut off when transplanted into a vinyl pollen were ground using liquid nitrogen and a mortar and pestle. Genomic DNA was extracted using a DNeasy Plant mini kit (Qiagen). The extracted genomic DNA was quantified by spectrophotometer and stored and used for PCR analysis.

PCR was performed to verify the introduction of npt II gene, a kanamycin resistance gene introduced with BcPGIP2 into the transformed cabbage. 100 ng of the genomic DNA extracted above was used as a template, and PCR was performed using AccuPower PCR premix (manufactured by Bioneer). npt Ⅱ (forward) 5'-ATGGGGATTGAACAAGATGGATTGC-3 'and (reverse) 5'-TCAGAAGAACTCGTCAAGAAGGCGATAG-3' primers were denatured for 5 minutes at 94 ° C, followed by 1 minute at 94 ° C, 1 minute at 60 ° C, 72 30 cycles were repeated at 1 ° C. and finally reacted at 72 ° C. for 10 minutes. The completed solution was subjected to electrophoresis on 1.2% agarose gel to confirm the DNA fragment of 798bp size and the results are shown in FIG. 3. In FIG. 3, M represents 1 kb plus DNA ladder, lane 1 represents amplification products obtained from control plants, and lanes 2 to 15 represent amplification products obtained from transformants. In particular, lane 2; T ₀ -1, lane 3; T ₀ -2, lane 4; T ₀ -3, lane 5; T ₀ -4, lane 6; T ₀ -6, lane 7; T ₀ -8, lane 8; T ₀ -9, lane 9; T ₀ -10, lane 10; T ₀ -11, lane 11; T ₀ -12, lane 12; T0-15, lane 13; T ₀ -16, lane 14; T ₀ -17, lane 15; T ₀ -19, and P represents the positive control group (vector pCAMBIA 2300- BcPGIP2 ).

As a result of PCR, introduction of npt II was confirmed (FIG. 3).

Experimental Example  2: DNA  Gel Blot  Used nptII Check the introduction of

After extracting the leaves of the individuals grown in the greenhouse for DNA gel blot analysis of the re-divided transgenic individuals from the J3-11M-44 strain, each 200 mg of the sample was ground using a liquid nitrogen and mortar and pestle. 700 μl of extraction buffer (1.25% SDS, 0.1M Tris-HCl, 0.5M NaCl, 0.05M EDTA, 0.38% sodium bisufite, pH 8.0) preheated to ℃ was mixed for 20 minutes at 65 ℃. Reacted. After the reaction, 700 μl of phenol: chloroform (1: 1 = v: v) solution was added thereto, mixed slowly for 10 minutes, and then centrifuged at 15 ° C. and 13,000 rpm for 10 minutes. The supernatant was separated and 2 μl of RNase (10 mg / mL) was added, followed by reaction at room temperature for 10 minutes. The reaction solution and the same amount of -20 ℃ EtOH was added and slowly mixed, and then precipitated at -20 ℃. The precipitated DNA was transferred to a new 1.5 mL tube using Pasteur pipette and washed twice with 70% EtOH. After washing, dried and suspended in TE (10mM Tris-Cl pH 8.0, 1mM EDTA) buffer, electrophoresis with control DNA was quantitated and stored and used for DNA gel blot.

10 μg of genomic DNA was digested with Bam HI and Xho I , followed by electrophoresis for 16 hours at 30V voltage on 1.0% agarose gel. After confirming the developed DNA, depurination for 10 minutes with 0.25N HCl solution (depurination) and transferred to the nylon membrane (nylon membrane) overnight by alkaline transfer (alkaline transfer) method. DNA transfer was confirmed by staining the blotting gel with EtBr. npt Ⅱ probe the vector plasmid DNA as a template, was prepared a DNA fragment generated by doing a PCR according to the method of Experimental Example 1 by using Bca Best Labeling Kit (Bca Best labelling kit, manufactured by Takara Co.), 65 Hybridization was carried out for 24 hours at ℃. After the final wash at 65 ° C. with 0.1 × SSC, 1% SDS solution for 30 minutes, the X-ray film was sensitized, and the results are shown in FIG. 4. In Figure 4, M is lambda DNA / Hind III, C is a wild type control J3-11M-44, lanes 1 to 19 represent the transformant plants. In particular, lane 1; T ₀ -1, lane 2; T ₀ -2, lane 3; T ₀ -3, lane 4; T ₀ -6, lane 5; T ₀ -8, lane 6; T ₀ -9, lane 7; T ₀ -10, lane 8; T ₀ -11, lane 9; T ₀ -12, lane 10; T ₀ -15, lane 11; T ₀ -16, lane 12; T ₀ -17, lane 13 ; T ₀ -19, lane 14; T ₀ -20, lane 15; T ₀ -21, lane 16; T ₀ -34, lane 17; T ₀ -36, lane 18; T ₀ -38, lane 19; T ₀ -39 is represented. In addition, B is a sample digested with BamH Ⅰ, X represents a sample cut with Xba Ⅰ.

As shown in Figure 4, npt II gene introduction was confirmed, T ₀ -1, T ₀ -2, T ₀ -3, T ₀ -20 showed the same band pattern, T ₀ -6, T ₀ -8 , T ₀ -9, T ₀ -10, T ₀ -11, T ₀ -12, T ₀ -15, T ₀ -16, T ₀ -17, T ₀ -21, T ₀ -34, T ₀ -36 , T ₀ -38 and T ₀ -39 showed the same band pattern. These results suggest that two types of gene recombination occurred at the time of gene insertion, and the shoots formed afterwards were proliferated from these two types of callus.

Experimental Example  3: RT - PCR through npt Ⅱ  And BcPGIP2 Expression

Cabbage transgenic _{T 0 -6, T 0 -10,} T 0 -12, T 0 -15, T 0 -16, T 0 -17, T 0 -20, the npt Ⅱ BcPGIP2 and introduced into the T ₀ -21 The leaves of the obtained subjects were collected to confirm the expression of and the total RNA was isolated with an RNeasy Plant mini kit (Quagen Co., Ltd.), and then quantified by spectrophotometer. RT-PCR using a primary cDNA strand (1st strand cDNA) synthesized using a Superscript 1st-Strand Synthesis Kit (Invitrogen, USA) as a template in 5 µg total RNA Was performed. RT-PCR to confirm the expression of npt II was performed according to the method of Experimental Example 1 and the primers of RT-PCR used to confirm the expression of BcPGIP2 are as follows:

(forward): 5'-AGTCGGATCCATGGGTAAGACAACGACACTGCTTTTG-3 '

(reverse): 5'-GGCTGCGGTACCCTTTTACTTGCAACTATCAAGAG-3 '

After denature the template DNA for 5 minutes at 94 ℃ using the primer pair, 30 cycles were repeated for 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute conditions at 94 ℃, finally 10 minutes at 72 ℃ Reacted for a while. The primer BcActin used to amplify cabbage actin gene as a control is as follows:

(forward): 5'-ATGACATGGAGAAGATCTGG-3 '

(reverse): 5'-TGAGCTTGTTTTGGAAGTCT-3 '

RT-PCR was performed using a primary cDNA strand (1st strand cDNA) synthesized using the primer pair as a template. PCR denatured the template DNA at 94 ° C for 5 minutes, and then repeated 30 cycles at 94 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute, and finally reacted at 72 ° C for 10 minutes. DNA fragments were identified by electrophoresis on a 1.2% agarose gel.

Figure 5 nptII using RT-PCR in transformant plants The results of detecting gene expression are shown. M represents 1 kb plus DNA ladder, lane 1 represents wild type J3-11M-44, and lanes 2 to 9 represent transformant plants. In particular, lane 2; T ₀ -6, lane 3; T ₀ -10, lane 4; T ₀ -12, lane 5; T ₀ -15, lane 6; T ₀ -16, lane 7; T ₀ -17, lane 8; T ₀ -20, lane 9; represents a T ₀ -21.

6 also shows BcPGIP2 in transformant plants. The results of detecting gene expression are shown. At this time, M represents 1kb plus DNA ladder, lanes 1 to 3 represent wild type plants, and lanes 4 to 11 represent transformant plants. In particular, lane 4; T ₀ -6, lane 5; T ₀ -10, lane 6; T ₀ -12, lane 7; T ₀ -15, lane 8; T ₀ -16, lane 9; T ₀ -17, lane 10; T ₀ -20, lane 11; represents a T ₀ -21.

5 and 6, it was confirmed that npt II and BcPGIP2 are normally expressed.

Example  3: Confirming the Resistance to Soft Disease of Cabbage Transformants

In order to measure the resistance of the Chinese cabbage transformed in Example 1 to PCR, PCR and DNA gel blots and RT-PCR were carried out to confirm the introduction and expression of the BcPGIP2 gene of the 60 days old J3-11M-44 strain. Transgenic cabbage and wild-type leaves were harvested, and the pathogenicity against the disease was inoculated with 1.2x10 ⁵ cfu / μl of the fungal pathogen by the point inoculation method (Park et al., 2004). , 6, 12, 18, 24, 30, 36 hours to check the progress of the lesion was shown in Figure 7 the results. The results in FIG. 7 (A) are shown as mean ± standard deviation of four replicates. Figure 7 (B) is a photograph showing the appearance of the cabbage leaf 12 hours after inoculation.

As shown in Figure 7, the transformant T ₀ -21 compared to the control wild type was observed from 12 hours after inoculation, compared with the control group at 12 hours 54.6%, 18 hours 52.5%, 24 hours 29.1%, Disease resistance increased by 19.7% at 30 hours and 20.3% at 36 hours.

As described in the above Examples and Experimental Examples, the present invention relates to a method for producing a transformant of Chinese cabbage using hard tissues and a transformant having improved resistance to softening produced from the cabbage. As a result of the transformation, the transformation rate is 2.8%, which is an effect of improving the efficiency more than two times compared to 0.4 ~ 0.8% of the conventional method using cotyledons and hypocotyls. If the transformation method using the landscape tissue of the present invention is applied to the transformation of plants other than Chinese cabbage, it is expected to be able to construct a plant transformation system with higher efficiency than the conventional method. In addition, the present invention has an excellent effect of providing a Chinese cabbage transformant having improved resistance to chokeberry disease by transforming the Chinese cabbage according to the above transformation method. Therefore, the present invention is a very useful invention in the crop industry.

Claims (6)

Agrobacterium tyumeo Pacifico Enschede (Agrobacterium In the method for producing a transformant of Chinese cabbage using tumefaciens ), Transfecting method of the Chinese cabbage comprising the step of infecting the strain with the hardened tissue of the Chinese cabbage tissue culture. The method of claim 1, wherein the Agrobacterium tumerfaciens Agrobacterium tumerfaciens ( Agrobacterium) tumefaciens ) The method for producing a transformant of the cabbage, characterized in that the strain LBA4404. The method of claim 1, wherein the Chinese cabbage is J3-11M-44 strain. The method of claim 1, wherein the Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) is a transformant manufacturing method of the Chinese cabbage, characterized in that a foreign gene introduced. The method of claim 4, wherein the foreign gene is BcPGIP2 (Polygalacturonase-inhibiting protein) or npt II gene. A transformant of Chinese cabbage prepared by the method of any one of claims 1 to 5.

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