KR100496028B1 - A Method for Producing Herbicide-Resistant Chili Pepper Plant - Google Patents

Abstract

The present invention relates to a method of obtaining a herbicide resistant pepper plant by introducing a herbicide resistant gene into a pepper plant, and a transgenic pepper plant obtained by the method.

The present invention comprises the steps of preparing an expression vector for plant transformation comprising the herbicide Basta resistant bar gene, and transformed by transforming Agrobacterium tumefaciens with the prepared expression vector Obtaining Agrobacterium, co-culturing the transformed Agrobacterium with cotyledon of the pepper plant, selecting and extending transformed shoots from the cotyledon of the co-cultivated pepper plant, and selecting Organizing the roots in the transformed shoots grown.

By the production method of according to the present invention it is possible to easily obtain a new kind of pepper having herbicide resistance.

Description

A Method for Producing Herbicide-Resistant Chili Pepper Plant

The present invention relates to a method of obtaining a herbicide resistant pepper plant by introducing a herbicide resistant gene into a pepper plant, and a transgenic pepper plant obtained by the method. More specifically, a plant transformation carrier comprising a herbicide resistance gene is prepared, introduced into Agrobacterium, co-cultured with cotyledon of pepper plants, herbicide resistance through selection of transformed plant shoots, shoot regeneration and purifying. It relates to a pepper plant production method and a pepper plant obtained by the method.

In Korea, the area of cultivation of red pepper is 7.6 million ha and the output is 41.2MT, which is one of the largest market among vegetables ('01). The size of the seed market is 26% of the total market size and amounts to about 300 billion won. In addition, the quality of red pepper seeds is excellent, and the export amount is about 2 million dollars ('97), which is increasing every year.

Korean red pepper breeding has been progressing since the 1970s by developing many F1 hybrid varieties and improving the traits such as water quality, environmental adaptability, prematurity and disease resistance. However, due to the lack of genetic resources or lack of labor, breeding by conventional breeding methods has had many problems. Globally, the breeding of crops by biotechnological methods has been experimented since the 1980s, and now many varieties are grown and commercialized by these methods.

In addition, vegetables and crops grown like red peppers grow and compete with weeds throughout the entire growing season, and because weeds are not only reduced in yield but also difficult to manage crops, numerous herbicides have been developed for weed control in modern agriculture. The use of is increasing day by day.

Herbicides are divided into various types such as glyphosate, sulphonylurea, imidazolinine, atrazin and Phosphinothricin. Recently, many herbicide-resistant crops using plant genetic engineering techniques have been developed for weed control. For example, glyphosate (Fillatti et al ., Bio / Technology (1987) 5: 726-730), sulphonylurea and imidazolinine (Newhouse et al. , Imidazolinone herbicides (1991) 139-151), glutamine, which inhibit the synthesis of aromatic amino acids Phosphinothricins that inhibit synthesis (Rathore et al ., Plant Molecular Biology (1993) 21: 871-874), atrazine that inhibits photosynthetic mechanisms (Christy et al ., Theoretical and Applied Genetics (1991) 83: 201-208) The development of crops resistant to the same herbicides has been actively reported. In particular, phosphinothricin (PPT) is the main component of basta herbicide (Crop Science Korea), which inhibits the reduction process of glutamine by combining free amino group (-NH ₂ ) with glutamic acid in the respiratory and root of plants during amino acid metabolism of plants. . As a result, a potent non-selective herbicide that accumulates ammonia in the body, which inhibits plant photosynthesis and kills the plant (Tachibana et al ., J. Pesticide Sci. (1986) 11: 297-304). After a while, the medicinal effect appears, and when it falls to the ground, the degree of environmental pollution is very excellent compared to other herbicides.

Genes that degrade these PPT herbicides have recently been cloned from Streptomyces hygroscopicus (Thompson et al ., EMBO (1987) 6: 2519-2523), and research on the development of transgenic crops using this gene has been conducted. Resistant plants are being developed: potato (De Greef et al ., Biotechnology (1989) 10: 309-314), wheat (Vasil et al ., Bio / Technology (1992) 10: 667-674, rice (Rthore et al . , Plant Molecular Biology (1993) 21: 871-874), Corn (Spancer et al ., Theor. Appl. Genet. (1990) 79: 625-631; Fromn et al ., Bio / Technology (1990) 8: 833 Many crops have reported herbicide resistance genes introduced into plants.

Widely (Bialaphos resistance gene) bar of resistance genes for herbicide Basta used is Streptomyces hygroscopicus himself a phosphino tree Shin (phosphinothricin) acetylated by a phosphino tree Shin-acetyl transferase (phosphinothricin acetyltransferase) non-toxic crystallized enzyme production The gene (EMBO 6, 2519-2523,1987) is widely used for the development of herbicide resistant plants containing a selection marker or phosphinothricin during transformation. Phosphinotrycin strongly inhibits glutamine synthesis, increasing the concentration of ammonia in plants, causing them to brown and die. Bailaphos produced by S. hygroscopicus or Basta containing phosphinothricin is known as a nonselective herbicide which is less potent than other herbicides due to residual environmental pollution.

On the other hand, as an effective plant cell transformation method for introducing foreign genes into a plant, a method of transferring foreign gene DNA to plant cells using a biological vector such as Agrobacterium, a method of projecting DNA-coated microparticles, There are methods for delivering foreign gene DNA directly into plant cells, such as electroporation, sonication and microinjection (Hansen and Wright, Trends Plant Sci . 4: 226-231, 1999). In particular, there are cases where the transformation of major crops such as corn, rice, wheat, barley and soybean by microparticle projection method, but the foreign gene DNA must be introduced into the cell that can be re-differentiated. The problem is that they are very diverse (De block M. Euphytica . 71: 1-14, 1993).

Recently, the most commonly used plant transformation method 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 tumor-inducing (Ti) or Ri (root-inducing) plasmid DNA, into infected plant cells. Therefore, plant cells can be transformed by recombining the target gene into T-DNA and then co-culturing the plant with the bacteria (Binns et al., Ann Rev Microbiol . 42: 575-606, 1988).

The invention regarding a method for producing red pepper and the red pepper produced therefrom among plants transformed using the Agrobacterium as a vector has been described in Application No. 10-1999-0007458. As can be seen from the above patent data, so far pepper transformation has used a process of obtaining plants through the root organic medium and root kidney medium for root development of transformed shoots. Since this multi-step process includes the steps of root organic medium and root kidney medium, it is not only cumbersome to work and requires a long time, but also has a low efficiency of regeneration.

An object of the present invention is to provide a method for producing a transformant pepper plant, which is easy to bioassay, such as herbicide resistance, and has high applicability, and a herbicide resistant pepper plant produced by the method.

In another aspect, the present invention is to provide a method of increasing the organic ratio of transformed shoots, trimming the stretched shoots close to the normal shoots and transferring them to artificial soil to simultaneously carry out rooting and purifying.

The present invention relates to a method for producing herbicide resistant pepper plant, comprising the steps of: (A) preparing an expression vector for plant transformation comprising herbicide Basta resistant bar gene; (B) transforming Agrobacterium tumefaciens with the prepared expression vector to obtain a transformed Agrobacterium; (C) co-culturing the transformed Agrobacterium with the cotyledon of the pepper plant; (D) selecting and extending the transformed shoots from the cotyledons of the co-cultured pepper plants; And (E) organically spraying roots from the selected and expanded transformed shoots.

The present invention relates to a method for producing a new kind of pepper by introducing a bar gene into the pepper plant. The herbicide resistance gene used for transformation in the present invention is not only a useful target gene itself, but also can be used as a selection gene, so that the concept of simultaneously expressing the target gene and the selection gene becomes possible. In the present invention, since the expression vector will be introduced into the pepper plant together with Agrobacterium, any vector configured to enable replication, expression and selection in Agrobacterium is possible. Specifically, in the present invention, the expression vector pCK-Bar for plant transformation having plasmid pCAMBIA 2300, herbicide resistance gene (Bar) as a backbone, CaMV 35S as a promoter, and a kanamycin resistance gene (NPTII gene) as a plant selection factor were prepared. Used. The gene sequence of the major part of the pCK-Bar plasmid is shown in FIG. 1.

Agrobacterium strains for plant transformation in the present invention can be applied to any commonly used. Specifically, in the present invention, Agrobacterium tumefaciens EHA 105 ( Agrobacterium tumefaciens EHA 105), which is known to have great pathogenicity required to penetrate plant samples and transfer foreign genes, was used. That is, the expression vector pCK-Bar for plant transformation was introduced into Agrobacterium tumefaciens EHA 105, and the transformed Agrobacterium was introduced into cotyledon plant.

Plasmid pCK-Bar was introduced into Agrobacterium tumefaciens EHA 105 and deposited in the Genetic Bank of Agricultural and Microbiological Conservation Center, Agricultural Biotechnology Research Institute on March 5, 2003 (Accession Number: KACC 95012).

In the present invention, Agrobacterium tumefaciens EHA 105 was used to increase transformation efficiency.

In addition, after the co-culture of pepper and Agrobacterium co-culture, the second pre-culture system was used, and the root organic medium and root growth medium for pepper root development were omitted. In other words, the screened and stretched shoots were trimmed to approximate normal vegetation and moved to artificial soil mixed with peat moss and vermiculite. By the method of the present invention, it is possible to significantly increase the root organic efficiency and to shorten the period of acquiring the regenerated individual.

In the present invention, PCR amplification, Southern blot (Southern hybridization) by molecular biological methods to determine whether the transformed plants actually contain foreign genes (target genes) to the transformants produced by the above method As shown in FIG. 5 and FIG. 6, it was confirmed that the foreign bar gene was stably expressed in the transformant.

In this process, the inventors developed a new primer set (SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 3-SEQ ID NO: 4) that can be used to confirm the presence of the bar gene in a convenient manner.

Accordingly, the present invention provides a primer set consisting of a sense oligonucleotide of SEQ ID NO: 1 and an antisense oligonucleotide of SEQ ID NO: 2 or a primer set consisting of a sense oligonucleotide of SEQ ID NO: 3 and an antisense oligonucleotide of SEQ ID NO: 4. These two pairs of primer sets can be used to select plants that are herbicide resistant.

In addition, the present invention is to extract the total DNA from the plant to be investigated, and to perform the PCR reaction using the primer set on the extracted DNA, and if there is a PCR reaction product and if present how much molecular weight of the reaction product The present invention relates to a method of easily identifying a herbicide-resistant plant and selecting a resistant plant by investigating and analyzing it.

In the present invention, the herbicide resistance and susceptibility were confirmed by Mendel's law of the herbicide resistance gene from T1 plants by spraying the bath star solution and confirming the herbicide resistance with the herbicide-resistant GMO assay kit by spraying 0.6% bath star solution. The ratio of 3: 1 was confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident

Example 1 Preparation of Herbicide Resistant Gene Carrier and Introduction to Agrobacterium

A carrier for pepper transformation was prepared and introduced into Agrobacterium.

Plasmid pBCUBNK containing PAT (phosphinothricin acetyl transferase) gene from Streptomyces hygroscopicus was digested with restriction enzyme HindIII and electrophoresed on 1% agarose gel to insert 2 kb of 35S :: Bar :: Ti7 DNA fragment. Ready. Separately, pCAMBIA 2300, a binary vector, was cleaved with HindIII and treated with shrimp alkaline phophatase to dephosphorylate the DNA sock end to prepare a backbone vector DNA that was self-ligation resistant. The prepared insert DNA and the vector DNA were ligated at 16 ° C. for 30 minutes using T4 DNA ligase, and then transformed into E. coli DH5α strains. E. coli DH5α strains were cultured in kanamycin medium to select transformed E. coli DH5α strains.

Plasmid DNA was isolated from the selected colonies and sequencing of the -70 partial sequence of the 35S promoter was carried out to confirm that it was a Bar gene carrier.

The final binary carrier for red pepper transformation, named pCK-Bar, used a CaMV 35S promoter to induce the systemic expression of herbicide resistance gene ( Bar ). The plant selection factor was kanamycin resistance (NPTII gene), and kanamycin was also used as a microbial selection antibiotic. The construction of the pCK-Bar plasmid and the gene sequence of the main part are shown in FIG. 1. The abbreviations of the symbols in the figure are as follows: BL (left border), BR (right border), P35S ( CaMV 35S promoter), nptII (neomycin phosphotransferase II gene), T35S ( CaMV 35S terminator), Bar (Bialapos resistance gene) ), Ti7 (tumor inducible transcription factor 7 terminator).

pCK-Bar was introduced into Agrobacterium tumefaciens EHA105 competent cells by conventional methods, and then cultured in selection medium containing rifampicin (100 µg / ml) and kanamycin (100 µg / ml) and transformed with pCK-Bar Agrobacterium tumefaciens EHA105 strain was selected. The colonies of the selected EHA105 were cultured to isolate DNA, and then introduced into the strain, the presence of pCK-Bar plasmid DNA and the presence of Ti plasmid DNA was used as Agrobacterium strain for pepper transformation.

Example 2 Pepper Transformation

Seeds of capsicum annuum (L. var. Subicho), inbred-line, fed from nutritional pepper test site are germinated aseptically for 7-8 days to collect cotyledons and cut off both ends and pre-culture medium (with antibiotics). The first pre-culture was carried out on the untreated direct-shoot organic medium. After 30 hours of incubation, the transformed Agrobacterium culture solution obtained in Example 1 was diluted about 20 times with MSO liquid medium, and added to 10 ml of each culture dish, and left for 1 hour while shaking every 10 minutes. The remaining cultures in the coculture dish were removed by pipette and co-cultured for 48 hours (22 ° C.) (see A in FIG. 2). Cocultured cotyledons were collected and placed on sterilized paper to remove external moisture and incubated on new pre-culture medium for 3 to 4 days.

Cultivated cotyledons were prepared with carbenicillin (400 mg / l) to remove Agrobacterium and kanamycin (200 mg / l) for selection of transformed shoots (MS base medium, 3% sucrose, 0.4% gelrite, 5 mg / L zeatin, 0.1 mg / L IAA, pH 5.6) were incubated for 3-4 weeks in the culture room (16 hours (27 ° C), 8 hours (23 ° C) for cancer) to induce kanamycin-resistant shoots (Fig. 2, see B, C and D).

When shoots were abandoned, young shoots with potential for regeneration were cut to include some cotyledon sections and incubated for two to three weeks on the same medium (see E and F in FIG. 2). In this process, agglutination of randomly differentiated cotyledon tissues and malformation and selection by callus occurring around the shoots were prevented.

In Figure 2, A is a co-culture, B is cultured in antibiotic-resistant medium, C and D are cultured in shoot starter and shoot kidney medium, E and F is before and after trimming elongated shoots , G is to purify trimmed shoot, H is to plant roots and I is to plant in pot.

Example 3 Antibiotic Resistant Shoot Regeneration

In order to quickly elongate the shoots selected in Example 2, trim the unnecessary parts to be close to the normal form of kidney kidney medium (MS base medium, 3% sucrose, 0.4% gelrite, 3mg / l zeatin, 0.05mg / l NAA, 200 kanamycin, 200 mg / L carbenicillin, pH 5.6). After 1 week of incubation, shoots start to elongate. At this time, the lid of the culture vessel was removed from the sterile workbench, and then the lower part of the same size plate was attached with the lid to enlarge the space required for the shoot extension (see D in FIG. 2). ).

Example 4 Root Organic and Purified Shoots

Shoots grown to 2 ~ 3㎝ in length were taken out of the culture vessel and soaked in tap water at 25 ~ 28 ℃, and each shoot was trimmed to a normal plant shape with growth point and leaves with fine scissors. F).

They were planted 5-8 mm in 50-hole seedling pots made of artificial soil mixed with peat moss and allel vermiculite 7: 3. During the planting process, water was sprayed continuously to prevent drying of shoots.

The seedling pot was sealed in a transparent purifying box made of acrylic, and then the kidneys and roots of shoots were organically grown in a culture chamber under light conditions at 26 ° C. (see G in FIG. 2). After 3 weeks of incubation, new leaves appeared, gradually opening the lid of the purifying box little by little and spraying water twice a day to gradually purify the outside air.

Purified young plants in the culture room for 4 weeks or more were transferred to a greenhouse, left for 2 days under indirect light, and then induced in photosynthesis and planted in pots (see I in FIG. 2).

Finally, eight purified pepper plants were obtained.

Purified plants in greenhouses have some abnormal shape as re-divided individuals in tissue culture. The present inventors sequentially remove the negatively generated side branches for the normal growth of the purified plant to enhance the well-known growth.

Example 5: Herbicide Bioassay and Gene Conversion Selection

The herbicide resistance gene (Bar) was expressed in the purified pepper plants.

 First, in order to easily confirm the expression of the transgene, the GMO (Genetically Modified Organism) diagnostic kit (LL corn Grain test kit of Strategic Diagnostics Inc.), which can easily check the presence of herbicide resistance gene (Bar) expression protein, can be used. Utilized. As a result, it was confirmed that 2 out of 8 purified individuals showed positive herbicide resistance protein (FIG. 3).

For herbicide-resistance bioassay of re-differentiated plants, when the plants have reached 8-10 leaves with normal leaves, 0.6% bassta solution is sprayed twice over 2 days, and after 6-8 days Two individuals with complete resistance were selected (FIG. 4).

The test kit and herbicide resistance test results confirmed that the same two individuals (T0) were transformed herbicide-resistant pepper plants and named BP4 and BP5, respectively.

Example 6: Confirmation of insertion of herbicide resistance gene by PCR

The transgenic individuals BP4 and BP5 were directly confirmed whether the gene is actually inserted.

To this end, PCR was performed using the following two sets of PCR primers to determine whether the herbicide resistance gene was inserted into the genomic DNA isolated from the leaves of the T0 generation of red peppers. In the case of these two bar identification primer sets and 35S bar identification primer sets, DNA PCR (version 3.82) PrimerSelect program was used to perform dual PCR if necessary from the 1424bp base sequence connecting the 35S promoter and the Bar gene. The Tm values were similarly designed so that the DNA detection size between the two sets of primers was 279bp and 533bp, respectively, and the PCR reaction was possible in one tube.

1) Bar check primer set

5'-CCGTACCGAGCCGCAGGAACC-3 '(21mer) (SEQ ID NO: 1)

5'-GGCAGCCCGATGACAGCGACCAC-3 '(23mer) (SEQ ID NO: 2)

2) 35S Bar Check Primer Set

5'-GACCCCCACCCACGAGGAGCATC-3 '(23mer) (SEQ ID NO: 3)

5'-AGCAGGTGGGTGTAGAGCGTGGAG-3 '(24mer) (SEQ ID NO: 4)

PCR using the primer for identifying the bar and the primer for designing the 35S Bar (designed over the 35S promoter and the Bar gene region) showed that the non-transformant (N.S.) used as a negative control (NC; control) There were no amplified bands, but in the analyzed BP4 and BP5 transformants, bands (279bp and 533bp, respectively) at the same positions as pCK-Bar (CKB) and pCK-RTBar (RTB), the plasmid DNA of positive control (PC), were detected. It could be confirmed (Fig. 5).

Further PCR was performed using the following two sets of PCR primers to determine whether insertion of the kanamycin resistance gene, a selection marker, was used as a template of leaf genomic DNA of the same red pepper differentiation individual T0 generation.

3) Primer set for identifying kanamycin resistance gene (NPT II)

5'-GCGGGAAGGGACTGGCTGCTATTG-3 '(24mer) (SEQ ID NO: 5)

5'-GCATCGCCTTCTATCGCCTTCTTG-3 '(24mer) (SEQ ID NO: 6)

4) Primer set for 35S promoter confirmation

5'-CCTCGGATTCCATTGCCCAGCT-3 '(22mer) (SEQ ID NO: 7)

5'-GATAGTGGGATTGTGCGTCAT-3 '(21mer) (SEQ ID NO: 8)

In the results of dual PCR performed using the selection marker kanamycin resistance gene NPTII identification primer and 35S promoter identification primer, there was no amplified band for the nontransformant used as a negative control (control). In the analyzed BP4 and BP5 transformants, a band (500bp and 200bp, respectively) at the same position as the positive control plasmid DNA was confirmed, and it was confirmed that the target carrier was correctly inserted in the pepper chromosome (FIG. 5).

PCR was denatured at 95 ° C. for 5 minutes using 1 μl of red pepper genomic DNA (100 μg / μl) as a template and 1 μl of the primer (10 pmole / μl), respectively, followed by denaturation at 95 ° C. for 30 seconds. Polymerization at 60 ° C. for 30 seconds and extension reaction at 72 ° C. for 30 seconds were performed 35 times. The enzyme used was Ex-Taq polymerase (Takara) and was run on a GeneAmp PCR system 9700 manufactured by Perkin Elmer.

Example 7: Confirmation of insertion of gene through Southern hybridization analysis

The gene insertion was confirmed by Southern blot method.

Genomic DNA was extracted and quantified from red pepper redistributes according to the mCTAB method, and then digested with restriction enzymes EcoRI, EcoRV, HindIII, and ScaⅠ, electrophoresed on 0.8% Agarose gel, and transferred to nylon membrane. The hybridization was performed with a probe labeled P32 (FIG. 6). In the figure, PC is the gene carrier (pCK-Bar / HindIII) as a positive control, M is the DNA size marker at 1 kb interval, C is the non-transforming red pepper plant (Hortiflora) as negative control (control), 4 and 5 Means transformed pepper plants BP4 and BP5, respectively.

In the figure, 1x and 10x are obtained by dividing the DNA of the gene carrier (pCK-Bar) with the same enzyme (HindIII) as the one treated with the pepper chromosome, and diluting the amount of one and ten genes inserted into 20 ㎍ of pepper chromosome DNA. means control.

When digested with restriction enzyme HindIII, 2kb DNA bands of the same size as in positive control were detected in pepper plants BP4 and BP5. When treated with restriction enzyme EcoRI, between 35S promoter and Ti7 terminator of the gene carrier As a band of DNA of about 5 kb was formed by cleaving any part of the pepper chromosome DNA, it was confirmed that the Bar gene was inserted into the pepper chromosome by single copy. In the EcoRV site located on both sides of the Bar gene region in the gene carrier, a DNA band was detected at the expected position of about 1 kb.

As a result of genomic southern hybridization using Bar Probe, DNA band was not detected in C (nontransformant defenct) and DNA band was detected only in pepper regeneration plants BP4 and BP5. It was confirmed that it was inserted.

Example 8 Transgene Fixation and Subsequent Genetic Analysis

In order to cultivate the seed (T1 seed) for two individuals whose gene insertion was confirmed by the above method, put a 4 × 6 cm parchment paper (heritage paper) envelope on the flower before the fire was opened. Pollination was prevented. After the fire fell, the envelope was removed, and 40 days later, the red ripe peppers were harvested, and the pears were split and dried to harvest T1 seeds by line.

For genetic analysis of herbicide resistance genes of T1 plants, 73 seedlings (BP4 family T1) and 120 (BP5 T1) T1 plants were sown in seedling pots filled with horticultural soil.

When the main leaves of T1 plants reached 8 to 12 leaves, the Vast liquid (aesthetic pesticide) was prepared at 0.6%, twice the recommended amount, and sprayed twice a day for 5-7 days. The rate was investigated.

As a result, the ratio of resistance and susceptibility of the BP4 system was 56:17 and that of the BP5 system was 92:28. The separation ratio of the resistant and susceptible individuals was determined by the Δ2 assay (х2 value: 0.114, 0.177, 0.5 <P <0.95).

Therefore, it can be confirmed that the Bar gene is stably maintained in the transformed pepper plants and also stably inherited later.

The present invention is to establish a transformation system using the Agrobacterium tumefaciens EHA 105 which can introduce useful genes to the chromosome of a plant, using a herbaceous herb, a herbaceous plant, and developed a pepper with a herbicide resistance gene. Herbicide-resistant pepper has the effect of cultivation and increase yield, and can be used directly as breeding material to efficiently breed new herbicide-resistant varieties in a short period of time, and can develop various breeding resources by using herbicide as a selection marker.

1 is a diagram showing the main part of the herbicide resistance gene (Bar) plant transformation carrier.

Figure 2 is a photograph showing a pepper plant manufacturing process according to the method according to the present invention.

Figure 3 is a photograph showing the results of the assay by herbicide (vasta) resistant GMO assay strip.

Figure 4 is a photograph showing the herbicide resistance of the transgenic pepper plants obtained by the method according to the present invention.

Figure 5 is a PCR analysis picture showing the presence of herbicide resistance gene (Bar) in the transgenic pepper plants obtained by the method according to the present invention.

Figure 6 is a photograph of Southern Blot analysis showing the presence of herbicide resistance gene (Bar) in the transgenic pepper plants obtained by the method according to the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> A Method for Producing Herbicide-Resistant Chili Pepper Plant <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> Primer for identifying BAR sequence <400> 1 ccgtaccgag ccgcaggaac c 21 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> Primer for identifying BAR sequence <400> 2 ggcagcccga tgacagcgac cac 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for identifying 35S BAR sequence <400> 3 gacccccacc cacgaggagc atc 23 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for identifying 35S BAR sequence <400> 4 agcaggtggg tgtagagcgt ggag 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> Primer for identifying NPT 2 sequence <400> 5 gcgggaaggg actggctgct attg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> Primer for identifying NPT 2 sequence <400> 6 gcatcgcctt ctatcgcctt cttg 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> Primer for identifying 35S promoter sequence <400> 7 cctcggattc cattgcccag ct 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> Primer for identifying 35S promoter sequence <400> 8 gatagtggga ttgtgcgtca t 21

Claims (12)

(A) preparing a plant transformation expression vector comprising a herbicide Basta resistant bar gene; (B) transforming Agrobacterium tumefaciens with the prepared expression vector to obtain a transformed Agrobacterium; (C) co-culturing the transformed Agrobacterium with the cotyledon of the pepper plant; (D) selecting transformed shoots from cotyledons of the co-cultured pepper plants and subcultured by cutting and separating the young shoots to include some cotyledon fragments; (E) immediately before transplanting the selected and transformed transformed shoots into artificial soil, removing and removing the abnormal parts so that the shoots become a normal plant type including growth points and leaves, and organically purifying the roots. A method for producing herbicide-resistant pepper plants. The method of claim 1, Method for producing a herbicide-resistant pepper plant, characterized in that the expression vector for plant transformation comprising the herbicide Basta resistant bar gene is pCK-Bar. delete The method of claim 1, In the step (D), in order to induce the rapid growth of the selected pepper shoots, the method of producing a herbicide-resistant pepper plant, characterized in that by cutting the shoots during subculture to remove the abnormal portion close to the normal herb. delete Herbicide-resistant pepper plants prepared by the method according to claim 1 and seeds thereof. A primer set consisting of the sense oligonucleotide of SEQ ID NO: 1 and the antisense oligonucleotide of SEQ ID NO: 2. The method of claim 7, wherein The primer set is used to select a herbicide resistant plant. Total DNA is extracted from the plant, PCR reaction is performed on the extracted DNA using the primer set according to claim 7 or 8, and the presence of the reaction product and the molecular weight of the reaction product are analyzed to determine the herbicide-resistant plant. How to screen. A primer set consisting of the sense oligonucleotide of SEQ ID NO: 3 and the antisense oligonucleotide of SEQ ID NO: 4. The method of claim 10, The primer set is used to select a herbicide resistant plant. Total DNA is extracted from the plant, PCR reaction is performed on the extracted DNA using the primer set according to claim 10, and the presence of the reaction product and the molecular weight of the reaction product are analyzed to determine the herbicide-resistant plant. How to screen.