KR100871090B1 - Potato plant having multiple resistances against herbicides and process for the preparation thereof - Google Patents

Abstract

The present invention relates to an antibiotic marker-free herbicide complex resistant potato plant transformed with an expression vector comprising a herbicide resistant bar gene and a CP4-EPSPS gene, and which does not include an antibiotic marker gene. , Potato plant of the present invention has a complex resistance to two non-selective herbicides can improve the weed control effect to increase potato productivity, and because it is antibiotic marker-free, does not cause antibiotic resistance diffusion problem, the cultivation of the When the two non-selective preparations are mixed, synergistic herbicidal effect is shown, which reduces the final treatment dose (herbicide input) and thus reduces the environmental burden.

Potato, Agrobacterium, Herbicide Resistance, Antibiotic Marker-Free, Transformation, Bar, CP4-EPSPS

Description

Potato plants having complex resistance to herbicides and preparation method thereof {POTATO PLANT HAVING MULTIPLE RESISTANCES AGAINST HERBICIDES AND PROCESS FOR THE PREPARATION THEREOF}

1A and 1B are schematic views of plasmid pCP4-EPSPS and herbicide resistant gene plant transformation vector p35S-EPSPS-B.

Figure 2 is the result of PCR analysis showing the presence of herbicide resistance genes ( bar and CP4-EPSPS ) in the transgenic potato plants obtained by the method according to the present invention. M is the molecular weight marker, NC is the nontransformant, PC is the plasmid DNA, and E3-6 is the transformant potato strain.

3 is a result of Southern hybridization showing the presence of herbicide resistance genes in transgenic potato plants obtained by the method according to the present invention. NT refers to nontransformants and E3 refers to transformant potato strains. A is a bar gene as a probe and B is a CP4-EPSPS gene as a probe.

4 is a result of ELISA analysis showing normal expression of herbicide resistance genes ( bar and CP4-EPSPS ) in the transgenic potato plants of the present invention. N means non-transformant, P means plasmid and E3-6 means potato transformant.

Figure 5 is a photograph showing the resistance to glufosinate and glyphosate herbicide of the transformed potato plant of the present invention.

The present invention relates to antibiotic marker-free herbicide complex resistant potato plants and methods for their preparation.

Potatoes are grown as one of the world's four food crops because of their short growing period and high yield per unit area. In addition, many functional varieties have been cultivated globally since the 1980s, and their practical use is expected to increase the area of potato cultivation in the future (Mullins et al., 2006, Trends in Plant Sci ., 11). : 254-260).

In general crop cultivation, including potatoes, it is a weed problem that requires a lot of labor and has a great impact on production. Therefore, many herbicides have been developed and used for weed control (Kim et al., 2001, Han Weed , 21: 122-143). The most important point in herbicide use is to get the maximum weed control effect with less labor. To this end, two methods have been devised. First, two or more selective herbicides having different properties are mixed and processed once or twice. Currently, many items are developed and applied to weed control at home and abroad. Second, the development and cultivation of herbicide-tolerant crops using plant genetic engineering technology simplifies the weed control efforts, while increasing the weed control effect and ultimately increasing productivity. That is, by introducing a gene having a resistance to the non-selective herbicide in the crop, and then cultivating it, the non-selective herbicide is treated in the growing season to easily remove other weeds other than the transformed crop.

The development of herbicide-tolerant transgenic crops has begun in earnest since Monsanto launched roundup ready soybeans in the United States in 1996. Currently, herbicide-tolerant crops, mainly non-selective herbicides (glyphosate, glufosinate ammonium), have been developed and put into practical use in corn, soybean, cotton, rapeseed (or canola), rice, and other crops. Duke SO, 2005, Pest Manag. Sci. , 61: 211-2118) Almost all have one herbicide resistance gene and include antibiotic resistance genes as selection marker genes. Genes that have been studied for herbicide-tolerant crops include CP4-EPSPS, bar, nitrilease, HPPD, ALS, PPO, P450 monooxygenase, phytoene desaturase Although many (including Jung, 2000, not a weed, such as 20:.... 159-173; Li & Nicholl, 2005, Pest Manag Sci, 61: 277-285, Matringe et al, 2005, Pest Manag Sci. , 61: 269-276), and there are only four commercially successful genes as of 2005: CP4-EPSPS, GOX, mutated corn CP4-EPSPS , bar (Duke SO, 2005, Pest Manag. Sci. , 61: 211-218).

Glufosinate ammonium (CropScience Korea), one of the non-selective herbicides frequently used in farms, is a phosphinothricin (PPT) whose main component is to inhibit plant glutamine synthase and accumulate ammonia in the body and after spraying 5 After a certain period of time, the plant is strongly killed (Tachibana et al., 1986, J. Pesticide Sci ., 11: 297-304), which is degraded as soon as it falls into the soil, resulting in less environmental pollution than other herbicides. Known. Phosphothricin acetyltransferase, an enzyme that acetylates and detoxifies PPT herbicides, has been identified from Streptomyces hygroscopicus , and its gene is termed bar (bialaphos resistance gene). (Thompson et al, 1987, EMBO J., 6:. 2519-2523) has been used in the development of many transgenic crops (Chung Jae-hun et al., 2000, if a weed, 20: 159-173).

On the other hand, glyphosate herbicides that are widely used in farms are non-selective, non-residual, broad-spectrum herbicides, foliage herbicides that have been used by Monsanto for more than 20 years and are easily absorbed and transferred to plants after treatment. The absorbed herbicide component slowly kills plants by inhibiting the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase) of the aromatic amino acid biosynthesis process (Dill GM, 2005, Pest Manag. Sci. , 61: 211-218). In nature, bacteria that break down glyphosate or are resistant to glyphosate have been found, and studies have been made to identify and utilize genes from them. The most widely used gene for producing glyphosate- resistant crops is CP4-EPSPS , which is isolated from Agrobacterium sp and overexpressed in crops for resistance (Dill GM, 2005, Pest Manag. Sci). ., 61: 211-218). CP4-EPSPS Most crops induce resistance to glyphosate by introducing a single gene, but glyphosate oxidoreductase (GOX), which degrades glyphosate, is introduced with CP4-EPSPS. (Canola) has also been developed (Duke et al., 2005, Outlooks on Pest Management- October: 208-211).

Potatoes vary depending on the species, but practically all weeds are controlled because they can be grown throughout the spring to fall season. Therefore, the transformation that has resistance to only one non-selective herbicide is thought to be more efficient for weed control if the method of using non-selective herbicide is devised rather than the technique of spraying the selective herbicide several times depending on the species and the growing season of the weed. Crops have been developed and used for over a decade. However, as the same herbicide continues to be used, problems such as accumulation of herbicides and degradation intermediates in living organisms, weeding and weed colonization that are poorly controlled by the herbicides, and the emergence of resistant weeds occur (Owed & Zelaya). , 2005, Pest Manag.Sci . , 61: 301-311; Arregui et al., 2003, Pest manag.Sci . , 60: 163-166; Sandermann H., 2006, Trends in Plant Sci. , 11: 324- 328). In addition, environmentally friendly agriculture requires spraying as low a herbicide as possible. However, even non-selective herbicides do not control all weeds at the same concentration, so uncontrolled weeds occur at low concentrations. Therefore, in order to solve these technical problems in terms of weed control, it is necessary to find a combination of additive and synergistic non-selective herbicides so that they can be mixed or systematically treated at low concentration. Eggplants are desirable to grow potatoes.

On the other hand, potatoes are more difficult to cross compared to other crops due to chromosomal characteristics, which are tetraploids, and because they are heterozygous, they have breeding difficulties that are separated from later generations after mating, so it takes about 10 years to select one variety. Therefore, a method of transferring certain characteristics of potatoes produced by the transformation method to other varieties through hybrid breeding is not efficient. Potatoes, however, are botanically grown as tubers, which are stems, which are very advantageous over other crops in terms of ensuring genetic stability of the transformants (Ghislain et al., 1997, Biotechnology and the Potato , International Potato Center). , Peru). Because of these characteristics, it is necessary to make transformants for each variety in order to introduce foreign genes into potatoes, and a transformation technology for each variety must be established. However, potato transformants developed in the past are limited to several foreign species such as Atlantic, Desire, Superior, Russet Burbank, and others (Choi et al., 1996, Kor.J Plant Tiss. Cult. , 23: 97-101; Youm et al., 2002, Kor.J Plant Biotech ., 29: 93-98). Therefore, there is a need for transgenic potatoes using new varieties of potatoes developed by domestic breeders.

On the other hand, as a plant transformation method for introducing foreign genes into the plant, a method of introducing DNA using a biological carrier such as Agrobacterium, a method of high-pressure spraying DNA-containing microparticles (particle bombardment), an electroporation method ( Although electroporation and microinjection are known (Hansen & Wright, 1999, Trends Plant Sci. , 4: 226-231), the most commonly used plant transformation method is Agrobacterium, a plant pathogen. Agrobacterium tumefaciens ) is used as a carrier. 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., 1988, Ann Rev Microbiol. , 42: 575-606). In this case, antibiotic resistance genes have been mainly used as markers to select cells or plants to which target genes have been introduced. However, cultivation of transgenic crops thus produced naturally exposes antibiotic resistance genes to the environment or human beings. There is a high possibility of causing the problem. Therefore, we developed a transformation technique that does not use antibiotic resistance marker genes (Scutt et al., 2002, Biochimie , 84: 1119-1126), and created the so-called transformant crops without the problem of antibiotic resistance ("Antibiotic marker- Free crops ”) is strongly required.

Accordingly, it is an object of the present invention to provide an expression vector for plant transformation comprising resistance genes for two non-selective herbicides and microorganisms for plant transformation transformed with the vector.

It is another object of the present invention to provide an antibiotic marker-free potato plant which is transformed with the expression vector and shows complex resistance to two non-selective herbicides.

Still another object of the present invention is to provide a method for producing the potato plant.

Still another object of the present invention is to provide a method of treating herbicides which enhances weed control effect by mixing or systematically treating glufosinate and glyphosate on the plantation of the potato plant.

In accordance with the above object, the present invention provides an expression vector for plant transformation comprising a bar gene and a CP4-EPSPS gene.

According to another object of the present invention, the present invention provides Agrobacterium transformed with the expression vector.

According to another object of the present invention, the present invention provides an antibiotic marker-free herbicide complex resistant potato plant, which is transformed with the expression vector and does not include an antibiotic marker gene.

According to another object of the present invention, (1) preparing an expression vector for plant transformation comprising a bar gene and CP4-EPSPS gene, (2) transforming Agrobacterium ( Agrobacterium ) with the expression vector (3) co-culturing the transformed Agrobacterium with the tissue of the potato plant, and (4) selecting transformed shoots from the co-cultivated potato plant tissue. It provides a method for producing herbicide complex resistant potato plant, comprising the step of stretching and organically rooting.

According to still another object, the present invention provides a method for controlling weeds by mixing or systematically treating glufosinate and glyphosate on a plantation of the herbicide complex resistant potato plant.

Hereinafter, the present invention will be described in more detail.

The herbicide complex-resistant potato plant of the present invention is transformed with a plant transformation expression vector comprising a glufosinate ammonium resistant bar gene and a herbicide glyphozate resistant CP4-EPSPS gene to express all of the genes. It exhibits complex resistance to glufosinate ammonium and glyphosate. In addition, the potato plant of the present invention does not include the antibiotic marker gene, there is an advantage that does not cause antibiotic resistance diffusion problem. Potatoes that can be used for the production of herbicide complex resistant potato plants in the present invention include Taedong Valley, Early Valley, Atlantic, Desiree, Superior, Russet (Russet), Burbank (Burbank) and the like can be exemplified, the Taedong Valley is more preferred.

Since the expression vector of the present invention will be introduced to the potato-type object with Agrobacterium ( Agrobacterium ), any vector configured to enable replication, expression and selection in Agrobacterium can be used in the present invention. In a preferred embodiment of the invention, the herbicide resistance gene CP4-EPSPS is inserted into the plasmid pCAMBIA 3300 (Center for Application of Molecular Biology to International Agriculture, Australia) containing the bar gene as a plant selector to regulate expression by the CaMV 35S promoter. Expression vector p35S-EPSPS-B for plant transformation was prepared. The gene sequence of plasmid p35S-EPSPS-B is shown in Figure 1b.

Agrobacterium strain for plant transformation in the present invention can be used any strain commonly used. Specifically, in the present invention, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), which is known to have high pathogenicity necessary to penetrate plant samples and transfer foreign genes, and more preferably Agrobacterium tumefaciens EHA 105 ( A. tumefaciens EHA) 105). The Agrobacterium tumefaciens EHA 105 (Agrobacterium tumefaciens EHA 105 / p35E-Bar) transformed with the plant transformant expression carrier p35S-EPSPS-B was October 25, 2006. The gene bank was deposited with the accession number KCTC 11008BP.

The Agrobacterium transformed with the vector of the present invention is co-cultured with potato plant tissues such as leaves, stems, or leaf diseases of potatoes, and then organically elongated shoots, and then through a two-step culture method of inducing root development and elongation. Obtain a transformant. In the culturing step of organic elongation of shoots to select a transformant, phosphinothricin is added to a conventional culture medium, for example, MS solid medium (Murashige & Skoog, Physiol. Plant 15: 473-497, 1962). The transformant may be selected using a medium. At this time, phosphinothricin may be added to the medium in an amount of 0.1 to 10 mg / L, preferably 0.5 to 5 mg / L. The transformant selection medium may also contain plant growth hormones such as Zeatin and Gibbelleic acid 3 in amounts of 1.0 to 5.0 mg / L, and 0.05 to 0.5 mg / L, respectively.

Subsequently, the obtained transformants were subjected to polymerase chain reaction (PCR) amplification, real-time PCR (RT-PCR), Southern hybridization, enzyme immunoassay (ELISA), and the like to introduce and stably express genes. You can check whether or not. The herbicide resistance of the obtained transgenic plants can be examined by bioassay such as chlorophyll localization reaction of glufosinate ammonium and confirmation of shikimate accumulation in glyphosate-treated leaf sections. Final treatment of herbicide resistance can be achieved through single or mixed treatment of glufosinate ammonium and glyphosate.

The transgenic potato plants of the present invention have complex resistance to glufosinate ammonium and glyphosate, which are non-selective herbicides, so that they are elevated to weeds by treating glufosinate ammonium and glyphosate on the plantation of the plant. Control effect can be obtained.

Accordingly, the present invention also provides a method for controlling weeds by mixing or systematically treating glufosinate ammonium and glyphosate in the plantation of the transgenic potato plant of the present invention.

Cultivation of potatoes resistant to the two non-selective herbicides of the present invention allows the weed control system (mixing and system treatment) to be more smoothly established since the drug selection for weed control is expanded from one to two. As we use herbicides with different properties, weed control effects can be improved, ultimately increasing the productivity of potatoes with less labor input.

In addition, when cultivating potatoes made to use only one non-selective herbicide, the continuous use of herbicides may cause the problem of early emergence of resistant weeds, the potato of the present invention alternately alternately herbicides of different mechanisms The treatment can prevent or minimize the problem of early emergence of resistant weeds. In addition, glyphosate herbicides have already been reported in nature with some resistant weeds (Sandermann H. 2006, Trends in Plant Sci ., 11: 324-328). These can be easily controlled by treating them (Duke et al., 2005, Outlooks on Pest Management-October: 208-211).

In addition, when glufosinate and glyphosate herbicides are mixed in order to control the weeds of the transgenic potato plantation of the present invention, the herbicides exhibit a synergistic effect, thereby lowering the final treatment amount (herbicide dosage). It has the effect of reducing the burden.

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 will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples.

Example 1: Preparation of p35S-EPSPS-B, an expression carrier of herbicide tolerance gene

Plant expression vector p35S-EPSPS-B was constructed by introducing the CP4-EPSPS gene between the CaMV 35S promoter and the NOS terminator of pCAMBIA3300 (Center for Application of Molecular Biology to International Agriculture, Australia) containing the Bar gene (Fig. 1b).

Specifically, in order to first separate the CP4-EPSPS (CP4 5-enol-pyruvylshikimate-3-phosphate synthase) gene, primers of SEQ ID NO: 1 and SEQ ID NO: 2 to which BglII and SacI restriction enzyme positions were introduced, respectively. Furthermore transgenic kongin Roundup - separating the total RNA in the ready (Roundup-Ready ^®, Monsanto) in the conventional manner beans, and cDNA was prepared using reverse transcriptase therefrom. After amplifying CP4-EPSPS gene by performing 30 cycles of PCR for 1 minute at 94 ° C, 1 minute at 52 ° C, and 2 minutes at 72 ° C using the above primer and synthetic cDNA, a protocol provided by a supplier PCR products amplified according to the cloned in pGEM-T Easy and then the nucleotide sequence was determined, and the resulting plasmid was named pCP4-EPSPS.

Finally, p35S-EPSPS-B was prepared by introducing CP4-EPSPS between the CaMV 35S promoter and the NOS terminator of pCAMBIA3300 including the bar gene (FIG. 1B). Specifically, p35S-EPSPS-B was obtained by inserting a CP4-EPSPS fragment obtained by cleaving pCP4-EPSPS with BglII and SacI restriction enzyme at a position where pCAMBIA3300 was cleaved with BamHI and SacI upstream of the CaMV 35S promoter upstream of NOS terminator. Produced.

p35S-EPSPS-B was converted to Agrobacterium tumefaciens EHA105 (Hood, et al., Trans. Res. , 2) according to the method of An (An, Meth. Enzymol. , 153: 292-305, 1987) . 208-218, 1993), and the resulting Agrobacterium strain was named Agrobacterium tumefaciens EHA 105 / p35E-Bar, and was assigned to the Korea Biotechnology Research Institute (KCTC) on October 25, 2006. It was deposited as accession number KCTC 11008BP.

Example 2: Efficient Regeneration of Potatoes

Potato ( Solanum tuberosum ) was used as a new varieties of Taedong Valley and Early Valley, and Atlantic was used as a control. Taedong Valley was developed by Korea Breeding Material Bank of Kangwon National University as a cultivar of varieties, and it was listed on the national breed list in 2002 as a potato varieties grown by artificial breeding with W870 as a model and A88431-1 as a copy (registered number 202002000007). ). Early Valley is a short-lived varieties with short dormancy, which allows two operations, and is a breed bred by artificial breeding using Suncrisp as a model and A87109-10 as a copy (breed protection application number 2002-06, varietal name application). No. 2002-24).

First, the degree of shoot formation was investigated using five differentiation media to identify efficient regeneration conditions in these potato slices. Based on MS medium (Murashige & Skoog, Physiol. Plant , 15: 473-497, 1962), 3% sucrose, 0.8% agar was added and the pH was adjusted to 5.7. In addition, various hormones were added to the medium as described in Table 1.

Ten to twenty sections of tissue per petri dish were wounded and subcultured, and at least six petri dishes were examined to evaluate the regeneration rate. The tissue culture conditions of potatoes were 2,000 lux, 16/8 hours photoperiod, and culture temperature 24 ℃.

The results are shown in Table 2. Compared to the Great Ocean and Taedong Valley, Early Valley had a lower regeneration rate using any medium. Daedae and Taedong Valley showed high regeneration rate over 90% in M3 medium. Based on these results, in the present invention, Taedong Valley, which has high regeneration rate, was used as a transformation material.

Example 3: Transformation of Potatoes

A single colony of Agrobacterium transformed with the p35S-EPSPS-B vector prepared in Example 1 was taken and inoculated into LB medium to which 50 mg / L of kanamycin was added and incubated overnight at a speed of 180 rpm in a 28 ° C. agitator. Then, the culture was incubated so as to have an absorbance of 0.8 at 600 nm, and the Agrobacterium obtained by centrifuging the culture solution at 3000 rpm was diluted in the same amount in MS liquid medium.

Cut potato slices (leaves, stems, and leaf diseases) into a certain size and incubated for 24 hours in a pre-culture medium (PPM) (Table 3), and then put in the Agrobacterium dilution prepared above 10 Infected for a minute. Thereafter, it was dried for 10 minutes in sterile filter paper, and the dried sections were incubated for 48 hours in a potato co-culture media (PCM), and then transferred to a selection medium (PSA) for regeneration. The shoots were organic through. The subdivided shoots Survivors were selected for transfer to sub-culture media (SUM) containing 5 mg / L of phosphinothricin, respectively. Selected shoots were rooted in basic MS solid medium (PRMM medium) containing 500 mg / L of antibiotic carbenicillin without hormones. Basic MS solid medium containing 500 mg / L of antibiotic carbenicillin was also used for proliferation of selected shoots.

Explants of leaves, stems, and leaf diseases were collected from incubated plants of the Taedong Valley and the Great Atlantic, and co-cultured with Agrobacterium containing the bar and CP4 - EPSPS genes, respectively, phosphinothricin 0.5 mg With / L Callus began to form on the cut surface of the explants after 10 days in PSM medium, and shoots began to develop after about 6-8 weeks. Transgenic plants were selected after transferring various shoots from primary selection medium to SUM medium containing 5 mg / L of phosphinothricin. Shooting incidence and rooting rates of individuals selected from shoot-induced selection medium (PSM) and rooting-induced selection medium (PRMM) during transformation were shown in Table 4.

The above procedure was performed several times to obtain a large number of transformed individuals, and the soil was purified as follows. In other words, carefully remove the rooted objects, rinse them well in water, and transplant them into pots containing horticulture soil No. 5. Adapt slowly to natural conditions.

Example 4 Confirmation of Gene Introduction of Potato Transformants

The plant obtained in Example 3 was confirmed whether the gene was introduced through PCR, Southern analysis, enzyme immunoassay analysis.

1) Assay by PCR

In order to confirm the transformation of the selected transformed dongdong Valley potato plant (E3-6), the bar gene specific primers SEQ ID NO: 4 and SEQ ID NO: 5, CP4-EPSPS gene specific primers SEQ ID NO: 6 and SEQ ID NO: 7 PCR was performed using the primers described. PCR amplification reaction was performed in Perkin Elmer GeneAmp PCR system 9700, after denaturation reaction at 94 ℃ for 3 minutes, 30 seconds denaturation at 94 ℃, 60 ℃ ( bar ) / 65.6 ℃ ( CP4-EPSPS ) 30 seconds of polymerization annealing at 30 ° C., and extension reaction at 35 ° C. for 30 seconds, followed by further extension at 72 ° C. for 10 minutes. The enzyme used was Taq polymerase (Takara) and the results of PCR amplification were confirmed by electrophoresis on 1.0% (w / v) agarose gel. The same was done using potato nontransformer (NC) and plasmid p35S-EPSPS-B (PC) for comparison.

As a result, bar and CP4-EPSPS bands were observed in the selected potato plant samples, confirming that the genes were inserted into the potato plants (FIG. 2).

2) Identification of transformants through Southern hybridization analysis

Diene not from leaves of transformed potato plants gestation valley and non-transformed potato plants are grown in the greenhouse conversion ^® Plant Maxi Kit, to extract the genomic DNA using the (DNeasy Plant Maxi ^® kit, QIAGEN Inc.), respectively. 30 μg of each of the obtained genomic DNAs was digested with restriction enzymes EcoRI and HindIII and electrophoresed on an agarose gel. After transferring genomic DNA on the gel to Zeta Probe membrane (Bio-Rad), a 0.4 kb fragment, which is part of the DNA and bar genes delivered to the membrane, and a 1.0 kb fragment, which is part of the CP4-EPSPS gene, are transferred. ^A hybridization reaction was performed using a ³² P-labeled gene fragment as a probe. After the hybridization reaction was complete, the membrane was washed and exposed to an X-ray film to identify the bands.

As a result, no band was found in the untransformed control plant (NT), but the transformed plant (E3) was found to be stably introduced into the potato genome because the bar and CP4-EPSPS genes exist in three bands ( 3: A: bar gene, B: CP4-EPSPS gene). CP4-EPSPS hybridizes with the plant's appearance and appears to have more than three bands.

3) ELISA assay

ELISA is a quantitative assay using antibody proteins that specifically recognize proteins produced by genes introduced into GM crops. In order to observe the expression level of EPSPS protein of the E. coli transformant (E3-6), protein samples were obtained from potato transformants in a general manner, and then enzyme-immunized reactions using EPSPS antibodies were performed. In order to detect EPSPS, it was investigated by ELISA-DAS method (Clark & Adams, 1977, Journal of General Virology , 34: 475-483) using Agdia PSP 74000/0096 PathoScreen Kit. For comparison, the same was done using Agrobacterium transformed with potato non-transformer (N) and plasmid p35S-EPSPS-B.

As a result, the potato transformant showed an absorbance of 1.332, which showed a 34-fold increase in absorbance compared to the non-transformant (N), and showed a higher protein expression than the control plasmid (P) having an absorbance of 1.230 ( 4).

Example 5: Herbicide Resistance Assay of Potato Transformants

Two stages were investigated for herbicide resistant bioassay of regenerated plants. The first step is a simple assay. In the case of bar- resistance, glufosinate ammonium is applied to a part of the leaves to determine cell necrosis. Investigate and evaluate. In the second step, when the plant reaches normal size through normal growth, non-selective herbicides are applied to the whole plant to check whether they show actual herbicide resistance.

1) Simple Test of Herbicide Resistance

As a simple assay for determining whether the Bar gene is active, glufosinate ammonium solution was prepared at a concentration of 250-1000 µg / ml (containing 0.1% Tween 20), and 10 µl was applied to about 1 cm ² of purified young plant leaves. As a result, no cell necrosis was observed in the Taedong Valley potato transformant, but cell necrosis occurred with the sulfidation symptom.

On the other hand, as a simple test for determining the CP4-EPSPS gene activity, it was examined whether Shichimate accumulated in plant tissues grafted in glyphosate solution (Kim et al., 2006, vegetation announcement , 33: 57-63). The accumulation of shchimate due to EPSPS inhibition of glyphosate increases the absorbance at 380 nm as in nontransformers. As a result, potato transgenic plants were found to be resistant to glyphozate because no increase in absorbance was observed (Table 5).

2) Herbicide Resistance Assay by Whole Plant Foliage Treatment

Root-produced embryos of the native plant and transformants transformed on board were transplanted into a circular pot of 13 cm in diameter and 12 cm in height containing No. 5, and then purified. 2 months (20-30 cm above ground). Herbicide Glyphozate Raw Material (62% Purity, Light Co., Ltd.) and Glufosinate Ammonium (50% Purity, Bayer CropScience) were added to a solution of Tween 20 0.1% + Acetone 43% in 750 μg / ml and 500 μg /, respectively. After dissolving at the ml concentration, the herbicide solutions obtained were sprayed at 50 ml per pot soaking the leaves of potato plants, either alone or mixed, respectively.

Herbicide-treated potato plants were transferred to greenhouse conditions, and the herbicidal response was examined by measuring the chlorophyll content of leaves and the photon yield change of photosystem II on the second and seventh days after treatment. The chlorophyll content of the leaves was investigated non-destructively using the chlorophyllometer (SPAD 502, Minolta, Japan), and the photon yield value was determined using the Pulse Amplitude Modulation Fluorometer (PAM 2000, Walz, Germany). It was investigated according to equation 1:

Photon yield = (FmF-Ft) / Fm '

In the above formula, Fm 'means maximum fluorescence yield in bright conditions, and Ft means minimum fluorescence yield in bright conditions.

As a result, when glufosinate ammonium alone was treated with the non-transformant, necrosis began to appear on the second day after the treatment. At this time, the photon yield was 41% of the untreated group, but no difference was observed in the chlorophyll content. On the third day, the leaves began to dry and almost died on the fifth day, and on the seventh day after treatment, both the chlorophyll content and the photon yield were 0.0. However, the transformant (E3-6) showed no weakness at all, and all the values showed the same values as the untreated group (Tables 6 and 7). On the other hand, when the glyphosate alone was treated with the non-transformant, no herbicidal activity was observed on the second day after the treatment, so that the photon yield and the chlorophyll content were almost the same as those of the untreated group. At 7 days after treatment, growth inhibition and sulphide symptoms were observed. Photon yield was 0.647, which was 82.9% of untreated, and chlorophyll content was 52.7% of untreated. However, the transformant (E3-6) showed no damage at all, and all the values showed the same values as the untreated group (Table 6 and Table 7).

When glyphosate and glufosinate ammonium were mixed, the result was similar to that of glufosinate ammonium alone. In summary, the results showed that E3-6 transgenic potatoes showed the same resistance when both herbicides were treated alone or when they were mixed and treated simultaneously (FIG. 5).

Example 6 Enhancement of Weed Control Effect by Nonselective Herbicide Mixing Treatment

In order to examine whether synergistic effects of weed control were obtained when glufosinate and glyphosate were actually mixed, experiments were conducted under greenhouse conditions. The plant species used in the experiment were seven species, such as beans, cotton, crow, tabby, larvae, dokmari, and buckthorn.

The plants were sown in 350 cm ² square pots and placed in greenhouse conditions (day temperature 25-35 ° C./night temperature 20-25 ° C., 14 hours photoperiod) for 11 days. As the treatment compound, glufosinate ammonium (purity 51.0%, Bayer Gropsscience) and glyphosate isopropylamine (purity 62.0%, light concentrate) were used. In the case of pharmaceutical treatment, glufosinate sets 0, 20, 40, 80, 160 g / ha as the active ingredient (ai), and glyphosate is 0, 16, 32, 63, 125, 250 In addition, 500 g / ha treatment concentrations were made at each concentration to prepare a combination or a combination (combinatorial treatment). In addition to the herbicide component, the final treatment solution contained 50% and 0.1% of acetone and tween 20, respectively, and no harmful effects were observed.

The herbicidal activity was assessed by herbicide activity (0-100%) at 14 days after treatment with each herbicide while growing in greenhouse conditions for 2 weeks. After that, the average control value was obtained and the degree of herbicidal activity was displayed. The interaction evaluation by the mixing treatment and the degree were analyzed according to Colby's method (Colby, 1967, Weeds , 15: 20-22). In other words, if the observed value is equal to the expected value and subtracted from 0, the additive value is relatively high and the measured value is relatively high, indicating that the positive value is synergistic and the lower value is antagonistic. At this time, the rise and antagonistic acting degree refers to the size of the positive or negative of a value obtained by subtracting the expected value from the found value is a large value is interpreted as a large interactive level (Kim et al., 2005, if a weed, 25 (3 ): 171-178).

Experimental results showed that values of 0-14.4 were obtained for all combinations of treatment concentrations, so that both herbicides did not exhibit antagonistic activity and showed an additive or weak synergy (Table 8). This means that the herbicide effect is increased when mixed rather than the single treatment, so that a smaller amount of herbicide can be added.

The transgenic potato plants of the present invention have complex resistance to two non-selective herbicides, so that weed control effects can be improved, and ultimately, the productivity of potatoes is increased by less labor input. When the non-selective preparation is mixed, synergistic herbicidal effect is shown, which reduces the final treatment dose (herbicide input), thereby reducing the environmental burden. In addition, since the potato of the present invention is an antibiotic marker-free, it does not cause the problem of antibiotic resistance spreading, and can improve the quality of processed potato cultivation, thereby increasing farm income.

<110> Korea Research Institute of Chemical Technology          Korea Research Institute of Bioscience and Biotechnology          KNU-Industry Cooperation Foundation <120> POTATO PLANT HAVING MULTIPLE RESISTANCES AGAINST HERBICIDES AND          PROCESS FOR THE PREPARATION THEREOF <130> FPD / 200611-0243 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for CP4-EPSPS gene <220> <221> misc_feature (222) (5) .. (10) <223> BglII restriction site <400> 1 tagcagatct ttcaagaatg g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for CP4-EPSPS gene <220> <221> misc_feature (222) (4) .. (9) <223> SacI restriction site <400> 2 ttcgagctca tcaggcagcc t 21 <210> 3 <211> 1604 <212> DNA <213> Artificial Sequence <220> <223> CP4-EPSPS gene <400> 3 agatctttca agaatggcac aaattaacaa catggcacaa gggatacaaa cccttaatcc 60 caattccaat ttccataaac cccaagttcc taaatcttca agttttcttg tttttggatc 120 taaaaaactg aaaaattcag caaattctat gttggttttg aaaaaagatt caatttttat 180 gcaaaagttt tgttccttta ggatttcagc atcagtggct acagcctgca tgcttcacgg 240 tgcaagcagc cggcccgcaa ccgcccgcaa atcctctggc ctttccggaa ccgtccgcat 300 tcccggcgac aagtcgatct cccaccggtc cttcatgttc ggcggtctcg cgagcggtga 360 aacgcgcatc accggccttc tggaaggcga ggacgtcatc aatacgggca aggccatgca 420 ggccatgggc gccaggatcc gtaaggaagg cgacacctgg atcatcgatg gcgtcggcaa 480 tggcggcctc ctggcgcctg aggcgccgct cgatttcggc aatgccgcca cgggctgccg 540 cctgaccatg ggcctcgtcg gggtctacga tttcgacagc accttcatcg gcgacgcctc 600 gctcacaaag cgcccgatgg gccgcgtgtt gaacccgctg cgcgaaatgg gcgtgcaggt 660 gaaatcggaa gacggtgacc gtcttcccgt taccttgcgc gggccgaaga cgccgacgcc 720 gatcacctac cgcgtgccga tggcctccgc acaggtgaag tccgccgtgc tgctcgccgg 780 cctcaacacg cccggcatca cgacggtcat cgagccgatc atgacgtgcg atcatacgga 840 aaagatgctg cagggctttg gcgccaacct taccgtcgag acggatgcgg acggcgtgcg 900 caccatccgc ctggaaggcc gcggcaagct caccggccaa gtcatcgacg tgccgggcga 960 cccgtcctcg acggccttcc cgctggttgc ggccctgctt gttccgggct ccgacgtcac 1020 catcctcaac gtgctgatga accccacccg caccggcctc atcctgacgc tgcaggaaat 1080 gggcgccgac atcgaagtca tcaacctgcg ccttgccggc ggcgaagacg tggcggacct 1140 gcgcgttcgc tcctccacgc tgaagggcgt cacggtgccg gaagaccgcg cgcctccgat 1200 gatcgacgaa tatccgattc tcgctgtcgc cgccgccttc gcggaagggg cgaccgtgat 1260 gaacggtctg gaagaactcc gcgtcaagga aagcgaccgc ctctcggccg tcgccaatgg 1320 cctcaagctc aatggcgtgg attgcgatga gggcgagacg tcgctcgtcg tgcgtggccg 1380 ccctgacggc aaggggctcg gcaacgcctc gggcgccgcc gtcgccaccc atctcgatca 1440 ccgcatcgcc atgagcttcc tcgtcatggg cctcgtgtcg gaaaaccctg tcacggtgga 1500 cgatgccacg atgatcgcca cgagcttccc ggagttcatg gacctgatgg ccgggctggg 1560 cgcgaagatc gaactctccg atacgaaggc tgcctgatga gctc 1604 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for bar gene <400> 4 cggtctgcac catcgtcaac c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for bar gene <400> 5 gtccagctgc cagaaaccca c 21 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for CP4-EPSPS gene <400> 6 ccgtaaggaa ggcgacac 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for CP4-EPSPS gene <400> 7 aggaagctca tggcgatg 18

Claims (14)

Expression vector for potato plant transformation, comprising a plasmid p35S-EPSPS-B comprising a herbicide resistant bar gene and a CP4-EPSPS gene and having a cleavage map shown in FIG. delete Agrobacterium transformed with the expression vector of claim 1. The method of claim 3, wherein Agrobacterium is Agrobacterium characterized in that the Agrobacterium tumefaciens EHA 105 / p35E-Bar (Accession Number: KCTC 11008BP). An antibiotic marker free herbicide complex resistant potato plant, transformed with the expression vector of claim 1 and comprising no antibiotic marker gene. delete The method of claim 5, wherein Antimicrobial marker-free herbicide complex resistance, characterized in that the potato plant was prepared by transforming with Agrobacterium tumefaciens EHA 105 / p35E-Bar (Accession Number: KCTC 11008BP) transformed with the expression vector of claim 1 Potato plant. The method of claim 5, wherein Antimicrobial marker-free herbicide complex resistant potato plant, characterized in that the potato is a potato of the Taedong Valley variety. (1) preparing the expression vector of claim 1, (2) transforming Agrobacterium with the expression vector to obtain transformed Agrobacterium, (3) co-culturing the transformed Agrobacterium with the tissue of the potato plant, and (4) selecting and extending transformed shoots from the co-cultivated potato plant tissues and organically rooting them, Method for producing herbicide complex resistant potato plants. delete The method of claim 9, The Agrobacterium of step 2 is Agrobacterium tumefaciens EHA 105 / p35E-Bar (Accession Number: KCTC 11008BP). The method of claim 9, Characterized in that the potato of step 3 is a potato of the Taedong Valley variety. The method of claim 9, The selection of the shoots of step 4 is characterized in that the potato plant tissues are cultured in a medium containing phosphinothricin to select the transformed shoots. A method for controlling weeds by mixing or systematically treating glufosinate and glyphosate in a plantation of the antibiotic marker-free herbicide complex resistant potato plant of claim 5.

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